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Sample records for scanning confocal microscopy

  1. Confocal scanning microscopy

    DEFF Research Database (Denmark)

    Bariani, Paolo

    This report is based on a metrological investigation on confocal microscopy technique carried out by Uffe Rolf Arlø Theilade and Paolo Bariani. The purpose of the experimental activity was twofold a metrological instrument characterization and application to assessment of rough PP injection moulded...... replicated topography. Confocal microscopy is seen to be a promising technique in metrology of microstructures. Some limitations with respect to surface metrology were found during the experiments. The experiments were carried out using a Zeiss LSM 5 Pascal microscope owned by the Danish Polymer Centre...

  2. QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

    Directory of Open Access Journals (Sweden)

    Merete Krog Raarup

    2011-05-01

    Full Text Available This paper discusses recent advances in confocal laser scanning microscopy (CLSM for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer, FLIM (Fluorescence Lifetime Imaging Microscopy, FCS (Fluorescence Correlation Spectroscopy and FRAP (Fluorescence Recovery After Photobleaching are introduced and their applicability for quantitative imaging of biomolecular (co-localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

  3. Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity

    Science.gov (United States)

    De Luca, Giulia; Breedijk, Ronald; Hoebe, Ron; Stallinga, Sjoerd; Manders, Erik

    2017-03-01

    Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial resolution and signal-to-noise ratio, are characterized and compared with properties of standard confocal microscopy. The results show that the lateral resolution of RCM is ~170 nm compared to ~240 nm of confocal microscopy for 488 nm excitation and 1.49 NA. As the theory predicts, this improved lateral resolution is independent of the pinhole diameter. In standard confocal microscopy, the same lateral resolution can only be achieved with an almost closed pinhole and, consequently, with a major loss of signal. We show that the sectioning capabilities of the standard confocal microscope are preserved in RCM and that the axial resolution of RCM is slightly better (~15%) than the standard confocal microscope. Furthermore, the signal-to-noise ratio in RCM is a factor of 2 higher than in standard confocal microscopy, also due to the use of highly sensitive modern cameras. In case the pinhole of a confocal microscope is adjusted in such way that the lateral resolution is comparable to that of RCM, the signal-to-noise ratio in RCM is 4 times higher than standard confocal microscopy. Therefore, RCM offers a good alternative to standard confocal microscopy for higher lateral resolution with the main advantage of strongly improved sensitivity.

  4. Optomechatronics Design and Control for Confocal Laser Scanning Microscopy

    NARCIS (Netherlands)

    Yoo, H.W.

    2015-01-01

    Confocal laser scanning microscopy (CLSM) is considered as one of the major advancements in microscopy in the last century and is widely accepted as a 3D fluorescence imaging tool for biological studies. For the emerging biological questions CLSM requires fast imaging to detect rapid biological

  5. [Advances of in vivo confocal scanning laser microscopy].

    Science.gov (United States)

    Tian, Ke-bin; Zhou, Guo-yu

    2006-02-01

    In vivo confocal scanning laser microscopy is being widely established as a time-saving, non-invasive, investigative methods in the study of body surfaces. Skin can be observed in its native state in vivo without the fixing, sectioning and staining that is necessary for routine histology. It is a new technology that can provide detailed images of tissue architecture and cellular morphology of living tissue. This paper reviews the fundamentals of in vivo confocal imaging and its clinical applications.

  6. Integrated Confocal and Scanning Probe Microscopy for Biomedical Research

    Directory of Open Access Journals (Sweden)

    B.J. Haupt

    2006-01-01

    Full Text Available Atomic force microscopy (AFM continues to be developed, not only in design, but also in application. The new focus of using AFM is changing from pure material to biomedical studies. More frequently, it is being used in combination with other optical imaging methods, such as confocal laser scanning microscopy (CLSM and fluorescent imaging, to provide a more comprehensive understanding of biological systems. To date, AFM has been used increasingly as a precise micromanipulator, probing and altering the mechanobiological characteristics of living cells and tissues, in order to examine specific, receptor-ligand interactions, material properties, and cell behavior. In this review, we discuss the development of this new hybrid AFM, current research, and potential applications in diagnosis and the detection of disease.

  7. Quantitative single-molecule imaging by confocal laser scanning microscopy.

    Science.gov (United States)

    Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

    2008-11-25

    A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

  8. Reflection across plant cell boundaries in confocal laser scanning microscopy.

    Science.gov (United States)

    Liu, D Y T; Kuhlmey, B T; Smith, P M C; Day, D A; Faulkner, C R; Overall, R L

    2008-08-01

    The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.

  9. Confocal laser scanning microscopy-guided surgery for neurofibroma.

    Science.gov (United States)

    Koller, S; Horn, M; Weger, W; Massone, C; Smolle, J; Gerger, A

    2009-12-01

    The neurofibromatoses comprise at least two separate genetic disorders with variable clinical features and an unpredictable course. The most common type, neurofibromatosis 1, is characterized by > or = 6 café-au-lait spots and the occurrence of neurofibromas, which may present as cutaneous, subcutaneous or plexiform lesions. Normally, excision of neurofibromas is only indicated in the presence of neurological symptoms, suspicion of malignancy or for exceptional cosmetic reasons. For a good functional and aesthetic result with the least danger of recurrence, the surgeon's goal is to excise as much tissue as necessary and as little tissue as possible. One of the main issues during the surgical procedure is to distinguish between neurofibroma and surrounding tissue. We report for the first time the use of confocal laser scanning microscopy to differentiate between neurofibroma and healthy skin.

  10. Confocal laser scanning microscopy in study of bone calcification

    Science.gov (United States)

    Nishikawa, Tetsunari; Kokubu, Mayu; Kato, Hirohito; Imai, Koichi; Tanaka, Akio

    2012-12-01

    Bone regeneration in mandible and maxillae after extraction of teeth or tumor resection and the use of rough surface implants in bone induction must be investigated to elucidate the mechanism of calcification. The calcified tissues are subjected to chemical decalcification or physical grinding to observe their microscopic features with light microscopy and transmission electron microscopy where the microscopic tissue morphology is significantly altered. We investigated the usefulness of confocal laser scanning microscopy (CLSM) for this purpose. After staggering the time of administration of calcein and alizarin red to experimental rats and dogs, rat alveolar bone and dog femur grafted with coral as scaffold or dental implants were observed with CLSM. In rat alveolar bone, the calcification of newly-formed bone and net-like canaliculi was observed at the mesial bone from the roots progressed at the rate of 15 μm/day. In dog femur grafted with coral, newly-formed bones along the space of coral were observed in an orderly manner. In dog femur with dental implants, after 8 weeks, newly-formed bone proceeded along the rough surface of the implants. CLSM produced high-magnification images of newly-formed bone and thin sections were not needed.

  11. Confocal laser scanning microscopy in study of bone calcification

    Energy Technology Data Exchange (ETDEWEB)

    Nishikawa, Tetsunari, E-mail: tetsu-n@cc.osaka-dent.ac.jp [Department of Oral Pathology, Osaka Dental University, Osaka (Japan); Kokubu, Mayu; Kato, Hirohito [Department of Oral Pathology, Osaka Dental University, Osaka (Japan); Imai, Koichi [Department of Biomaterials, Osaka Dental University, Osaka (Japan); Tanaka, Akio [Department of Oral Pathology, Osaka Dental University, Osaka (Japan)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer High-magnification images with depth selection, and thin sections were observed using CLSM. Black-Right-Pointing-Pointer The direction and velocity of calcification of the bone was observed by administration of 2 fluorescent dyes. Black-Right-Pointing-Pointer In dog femora grafted with coral blocks, newly-formed bone was observed in the coral block space with a rough surface. Black-Right-Pointing-Pointer Twelve weeks after dental implant was grafted in dog femora, the space between screws was filled with newly-formed bones. - Abstract: Bone regeneration in mandible and maxillae after extraction of teeth or tumor resection and the use of rough surface implants in bone induction must be investigated to elucidate the mechanism of calcification. The calcified tissues are subjected to chemical decalcification or physical grinding to observe their microscopic features with light microscopy and transmission electron microscopy where the microscopic tissue morphology is significantly altered. We investigated the usefulness of confocal laser scanning microscopy (CLSM) for this purpose. After staggering the time of administration of calcein and alizarin red to experimental rats and dogs, rat alveolar bone and dog femur grafted with coral as scaffold or dental implants were observed with CLSM. In rat alveolar bone, the calcification of newly-formed bone and net-like canaliculi was observed at the mesial bone from the roots progressed at the rate of 15 {mu}m/day. In dog femur grafted with coral, newly-formed bones along the space of coral were observed in an orderly manner. In dog femur with dental implants, after 8 weeks, newly-formed bone proceeded along the rough surface of the implants. CLSM produced high-magnification images of newly-formed bone and thin sections were not needed.

  12. Confocal microscopy

    Indian Academy of Sciences (India)

    molecular aggregates in artificial light harvesting sys- tem it is important to elucidate the exciton dynamics of individual micro-rods which can be achieved by using confocal microscopy and polarization resolved single molecule fluorescence spectroscopy.30 41 In the present work, we have studied exciton dynamics of two.

  13. Confocal laser scanning microscopy. Using new technology to answer old questions in forensic investigations.

    Science.gov (United States)

    Turillazzi, Emanuela; Karch, Steven B; Neri, Margherita; Pomara, Cristoforo; Riezzo, Irene; Fineschi, Vittorio

    2008-03-01

    Confocal laser scanning microscopy (CLSM) is a relatively new technique for microscopic imaging. It has found a wide field of application in the general sphere of biological sciences. It has completely changed the study of cells and tissues by allowing greater resolution, optical sectioning of the sample and three-dimensional sanoke reconstruction. Confocal microscopy represents a valid, precious and useful tool capable of providing data (images) of unrivalled clearness and definition. This review discusses the possible applications of confocal microscopy in specific fields of forensic investigation, with specific regard to ballistics, forensic histopathology and toxicological pathology.

  14. Further observations on cerebellar climbing fibers. A study by means of light microscopy, confocal laser scanning microscopy and scanning and transmission electron microscopy.

    Science.gov (United States)

    Castejón, O J; Castejón, H V; Alvarado, M V

    2000-12-01

    The intracortical pathways of climbing fibers were traced in several vertebrate cerebella using light microscopy, confocal laser scanning microscopy, scanning and transmission electron microscopy. They were identified as fine fibers up to 1(micron thick, with a characteristic crossing-over bifurcation pattern. Climbing fiber collaterals were tridimensionally visualized forming thin climbing fiber glomeruli in the granular layer. Confocal laser scanning microscopy revealed three types of collateral processes at the interface between granular and Purkinje cell layers. Scanning electron microscopy showed climbing fiber retrograde collaterals in the molecular layer. Asymmetric synaptic contacts of climbing fibers with Purkinje dendritic spines and stellate neuron dendrites were characterized by transmission electron microscopy. Correlative microscopy allowed us to obtain the basic three-dimensional morphological features of climbing fibers in several vertebrates and to show with more accuracy a higher degree of lateral collateralization of these fibers within the cerebellar cortex. The correlative microscopy approach provides new views in the cerebellar cortex information processing.

  15. Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Peng; Behan, Gavin; Kirkland, Angus I. [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Nellist, Peter D., E-mail: peter.nellist@materials.ox.ac.uk [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Cosgriff, Eireann C.; D' Alfonso, Adrian J.; Morgan, Andrew J.; Allen, Leslie J. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Hashimoto, Ayako [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); Takeguchi, Masaki [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); High Voltage Electron Microscopy Station, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Mitsuishi, Kazutaka [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); Quantum Dot Research Center, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Shimojo, Masayuki [High Voltage Electron Microscopy Station, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Advanced Science Research Laboratory, Saitama Institute of Technology, 1690 Fusaiji, Fukaya 369-0293 (Japan)

    2011-06-15

    Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored. -- Research Highlights: {yields} The confocal probe image in a scanning confocal electron microscopy image reveals information about the thickness and height of the crystalline layer. {yields} The form of the contrast in a three-dimensional bright-field scanning confocal electron microscopy image can be explained in terms of the confocal probe image. {yields} Despite the complicated form of the contrast in bright-field scanning confocal electron microscopy, we see that depth information is transferred on a 10 nm scale.

  16. Superresolution upgrade for confocal spinning disk systems using image scanning microscopy (Conference Presentation)

    Science.gov (United States)

    Isbaner, Sebastian; Hähnel, Dirk; Gregor, Ingo; Enderlein, Jörg

    2017-02-01

    Confocal Spinning Disk Systems are widely used for 3D cell imaging because they offer the advantage of optical sectioning at high framerates and are easy to use. However, as in confocal microscopy, the imaging resolution is diffraction limited, which can be theoretically improved by a factor of 2 using the principle of Image Scanning Microscopy (ISM) [1]. ISM with a Confocal Spinning Disk setup (CSDISM) has been shown to improve contrast as well as lateral resolution (FWHM) from 201 +/- 20 nm to 130 +/- 10 nm at 488 nm excitation. A minimum total acquisition time of one second per ISM image makes this method highly suitable for 3D live cell imaging [2]. Here, we present a multicolor implementation of CSDISM for the popular Micro-Manager Open Source Microscopy platform. Since changes in the optical path are not necessary, this will allow any researcher to easily upgrade their standard Confocal Spinning Disk system at remarkable low cost ( 5000 USD) with an ISM superresolution option. [1]. Müller, C.B. and Enderlein, J. Image Scanning Microscopy. Physical Review Letters 104, (2010). [2]. Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy. Proceedings of the National Academy of Sciences of the United States of America 110, 21000-5 (2013).

  17. Emulation and design of terahertz reflection-mode confocal scanning microscopy based on virtual pinhole

    Science.gov (United States)

    Yang, Yong-fa; Li, Qi

    2014-12-01

    In the practical application of terahertz reflection-mode confocal scanning microscopy, the size of detector pinhole is an important factor that determines the performance of spatial resolution characteristic of the microscopic system. However, the use of physical pinhole brings some inconvenience to the experiment and the adjustment error has a great influence on the experiment result. Through reasonably selecting the parameter of matrix detector virtual pinhole (VPH), it can efficiently approximate the physical pinhole. By using this approach, the difficulty of experimental calibration is reduced significantly. In this article, an imaging scheme of terahertz reflection-mode confocal scanning microscopy that is based on the matrix detector VPH is put forward. The influence of detector pinhole size on the axial resolution of confocal scanning microscopy is emulated and analyzed. Then, the parameter of VPH is emulated when the best axial imaging performance is reached.

  18. Development of an add-on kit for scanning confocal microscopy (Conference Presentation)

    Science.gov (United States)

    Guo, Kaikai; Zheng, Guoan

    2017-03-01

    Scanning confocal microscopy is a standard choice for many fluorescence imaging applications in basic biomedical research. It is able to produce optically sectioned images and provide acquisition versatility to address many samples and application demands. However, scanning a focused point across the specimen limits the speed of image acquisition. As a result, scanning confocal microscope only works well with stationary samples. Researchers have performed parallel confocal scanning using digital-micromirror-device (DMD), which was used to project a scanning multi-point pattern across the sample. The DMD based parallel confocal systems increase the imaging speed while maintaining the optical sectioning ability. In this paper, we report the development of an add-on kit for high-speed and low-cost confocal microscopy. By adapting this add-on kit to an existing regular microscope, one can convert it into a confocal microscope without significant hardware modifications. Compared with current DMD-based implementations, the reported approach is able to recover multiple layers along the z axis simultaneously. It may find applications in wafer inspection and 3D metrology of semiconductor circuit. The dissemination of the proposed add-on kit under $1000 budget could also lead to new types of experimental designs for biological research labs, e.g., cytology analysis in cell culture experiments, genetic studies on multicellular organisms, pharmaceutical drug profiling, RNA interference studies, investigation of microbial communities in environmental systems, and etc.

  19. Preliminary Study of In Vivo Formed Dental Plaque Using Confocal Microscopy and Scanning Electron Microscopy

    Directory of Open Access Journals (Sweden)

    KA. Al-Salihi

    2009-12-01

    Full Text Available Objective: Confocal laser scanning microscopy (CLSM is relatively a new light microscopical imaging technique with a wide range of applications in biological sciences. The primary value of CLSM for the biologist is its ability to provide optical sections from athree-dimensional specimen. The present study was designed to assess the thickness and content of in vivo accumulated dental plaque using CLSM and scanning electron microscopy (SEM.Materials and Methods: Acroflat lower arch splints (acrylic appliance were worn by five participants for three days without any disturbance. The formed plaques were assessed using CLSM combined with vital fluorescence technique and SEM.Results: In this study accumulated dental plaque revealed varied plaque microflora vitality and thickness according to participant’s oral hygiene. The thickness of plaque smears ranged from 40.32 to 140.72 μm and 65.00 to 128.88 μm for live (vital and dead accumulated microorganisms, respectively. Meanwhile, the thickness of plaque on the appliance ranged from 101 μm to 653 μm. CLSM revealed both dead and vital bacteria on the surface of the dental plaque. In addition, SEM revealed layers of various bacterial aggregations in all dental plaques.Conclusion: This study offers a potent non-invasive tool to evaluate and assess the dental plaque biofilm, which is a very important factor in the development of dental caries.

  20. Comparison between optical-resolution photoacoustic microscopy and confocal laser scanning microscopy for turbid sample imaging.

    Science.gov (United States)

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

    Optical-resolution photoacoustic microscopy (ORPAM) in theory provides lateral resolution equivalent to the optical diffraction limit. Scattering media, such as biological turbid media, attenuates the optical signal and also alters the diffraction-limited spot size of the focused beam. The ORPAM signal is generated only from a small voxel in scattering media with dimensions equivalent to the laser spot size after passing through scattering layers and is detected by an acoustic transducer, which is not affected by optical scattering. Thus, both ORPAM and confocal laser scanning microscopy (CLSM) reject scattered light. A multimodal optical microscopy platform that includes ORPAM and CLSM was constructed, and the lateral resolution of both modes was measured using patterned thin metal film with and without a scattering barrier. The effect of scattering media on the lateral resolution was studied using different scattering coefficients and was compared to computational results based on Monte Carlo simulations. It was found that degradation of lateral resolution due to optical scattering was not significant for either ORPAM or CLSM. The depth discrimination capability of ORPAM and CLSM was measured using microfiber embedded in a light scattering phantom material. ORPAM images demonstrated higher contrast compared to CLSM images partly due to reduced acoustic signal scattering.

  1. Plastic-to-Elastic Transition in Aggregated Emulsion Networks, Studied with Atomic Force Microscopy-Confocal Scanning Laser Microscopy Microrheology

    NARCIS (Netherlands)

    Filip, D.; Duits, Michael H.G.; Uricanu, V.I.; Mellema, J.

    2006-01-01

    In this paper, we demonstrate how the simultaneous application of atomic force microscopy (AFM) and confocal scanning laser microscopy (CSLM) can be used to characterize the (local) rheological properties of soft condensed matter at micrometer length scales. Measurement of AFM force curves as a

  2. DTAF: an efficient probe to study cyanobacterial-plant interaction using confocal laser scanning microscopy (CLSM)

    NARCIS (Netherlands)

    Ahmed, M.; Stal, L.J.; Hasnain, S.

    2011-01-01

    A variety of microscopic techniques have been utilized to study cyanobacterial associations with plant roots, but confocal laser scanning microscopy (CLSM) is the least used due to the unavailability of a suitable fluorescent dye. Commonly used lectins have problems with their binding ability with

  3. DTAF: an efficient probe to study cyanobacterial-plant interaction using confocal laser scanning microscopy (CLSM).

    NARCIS (Netherlands)

    Ahmed, M.; Stal, L.J.; Hasnain, S.

    2011-01-01

    A variety of microscopic techniques have been utilized to study cyanobacterial associations with plant roots, but confocal laser scanning microscopy (CLSM) is the least used due to the unavailability of a suitable fluorescent dye. Commonly used lectins have problems with their binding ability with

  4. Imaging inclusion complex formation in starch granules using confocal laser scanning microscopy

    NARCIS (Netherlands)

    Manca, Marianna; Woortman, Albert J. J.; Loos, Katja; Loi, Maria A.

    The tendency of amylose to form inclusion complexes with guest molecules has been an object of wide interest due to its fundamental role in food processing. Here we investigated the features of starch granules from several botanical sources using confocal laser scanning microscopy (CLSM) and

  5. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis

    DEFF Research Database (Denmark)

    Skytte, Jacob Lercke; Ghita, Ovidiu; Whelan, Paul F.

    2015-01-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented...... to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis...

  6. Fluorescence (Multiwave) Confocal Microscopy.

    Science.gov (United States)

    Welzel, J; Kästle, Raphaela; Sattler, Elke C

    2016-10-01

    In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Scanning a microhabitat: plant-microbe interactions revealed by confocal laser microscopy.

    Science.gov (United States)

    Cardinale, Massimiliano

    2014-01-01

    No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology.

  8. Scanning a microhabitat: plant-microbe interactions revealed by confocal laser microscopy

    Directory of Open Access Journals (Sweden)

    Massimiliano eCardinale

    2014-03-01

    Full Text Available No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology.

  9. The application of dermal papillary rings in dermatology by in vivo confocal laser scanning microscopy

    Science.gov (United States)

    Xiang, W. Z.; Xu, A. E.; Xu, J.; Bi, Z. G.; Shang, Y. B.; Ren, Q. S.

    2010-08-01

    Confocal laser scanning microscopy (CLSM) allows noninvasive visualization of human skin in vivo, without needing to fix or section the tissue. Melanocytes and pigmented keratinocytes at the level of the basal layer form bright dermal papillary rings which are readily amenable to identify in confocal images. Our purpose was to explore the role of dermal papillary rings in assessment of lesion location, the diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. Seventy-one patients were imaged with the VivaScope 1500 reflectance confocal microscope provided by Lucid, Inc. The results indicate that dermal papillary rings can assess the location of lesion; the application of dermal papillary rings can provide diagnostic support and differential diagnosis for vitiligo, nevus depigmentosus, tinea versicolor, halo nevus, common nevi, and assess the therapeutic efficacy of NBUVB phototherapy plus topical 0.1 percent tacrolimus ointment for vitiligo. In conclusion, our findings indicate that the dermal papillary rings play an important role in the assessment the location of lesion, diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. CLSM may be a promising tool for noninvasive examination in dermatology. However, larger studies are needed to expand the application of dermal papillary rings in dermatology.

  10. Fluorescence confocal laser scanning microscopy for in vivo imaging of epidermal reactions to two experimental irritants

    DEFF Research Database (Denmark)

    Suihko, C.; Serup, J.

    2008-01-01

    Background: Fibre-optic fluorescence confocal laser scanning microscopy (CLSM) is a novel non-invasive technique for in vivo imaging of skin. The cellular structure of the epidermis can be studied. A fluorophore, e.g. fluorescein sodium, is introduced by an intradermal injection or applied...... dermatitis reactions caused by established model irritants, e.g. sodium lauryl sulphate (SLS) and pelargonic acid (PA). Methods: Twelve healthy individuals volunteered. The flexor aspect of the right and the left forearm was exposed to SLS in water and PA in isopropanol and occluded under Finn Chambers...... for 24 h. The reactions were rated clinically and, following epicutaneous and intra-dermal application of fluorescein sodium, studied by fluorescence CLSM, magnification x 1000. Results: Both irritants disturbed the epidermal intercellular borders, which became blurred, thickened and variably altered...

  11. Trypan blue as a fluorochrome for confocal laser scanning microscopy of arbuscular mycorrhizae in three mangroves.

    Science.gov (United States)

    Kumar, T; Majumdar, A; Das, P; Sarafis, V; Ghose, M

    2008-06-01

    Roots of three mangroves, Acanthus ilicifolius, Ceriops tagal and Excoecaria agallocha, collected from forests of the Sundarbans of India were stained with trypan blue to observe arbuscular mycorrhizal colonization. Spores of arbuscular mycorrhizal fungi isolated from rhizospheric soil, collected together with the root samples, also were stained for testing the suitability of the dye as a fluorochrome. Confocal laser scanning microscopy images were constructed. A. ilicifolius and E. agallocha exhibited "Arum" type colonization with highly branched arbuscules, whereas C. tagal showed "Paris" type association with clumped and collapsed arbuscules. We demonstrated that trypan blue is a suitable fluorochrome for staining arbuscular mycorrhizal fungal spores, fungal hyphae, arbuscules and vesicles, which presumably have a considerable amount of surface chitin. It appears that as the integration of chitin into the fungal cell wall changes, its accessibility to trypan blue dye also changes.

  12. Experimental setup for energy-filtered scanning confocal electron microscopy (EFSCEM) in a double aberration-corrected transmission electron microscope

    Energy Technology Data Exchange (ETDEWEB)

    Wang, P; Behan, G; Kirkland, A I; Nellist, P D, E-mail: peng.wang@materials.ox.ac.u [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom)

    2010-07-01

    Scanning confocal electron microscopy (SCEM) is a new imaging mode in electron microscopy. Spherical aberration corrected electron microscope instruments fitted with two aberration correctors can be used in this mode which provides improved depth resolution and selectivity compared to optical sectioning in a conventional scanning transmission geometry. In this article, we consider a confocal optical configuration for SCEM using inelastically scattered electrons. We lay out the necessary steps for achieving this new operational mode in a double aberration-corrected instrument with uncorrected chromatic aberration and present preliminary experimental results in such mode.

  13. Imaging hyphal growth of Physisporinus vitreus in Norway spruce wood by means of confocal laser scanning microscopy (CLSM)

    OpenAIRE

    Schubert, Mark; Stührk, Chris; Fuhr, Matthias J.; Schwarze, Francis W.M.R.

    2017-01-01

    Light microscopy and electron microscopy are the most common methods for analyzing wood-decay fungi. However, the 3D visualization and quantification of the filamentous structure of fungi in wood is difficult to realize by means of these traditional techniques. In the present work, confocal laser scanning microscopy (CLSM) was further developed for the quantitative imaging of the 3D microscopic hyphal growth of Physisporinus vitreus, a versatile fungus for engineering value-added wood product...

  14. Three-dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy.

    Science.gov (United States)

    Buda, Gregory J; Isaacson, Tal; Matas, Antonio J; Paolillo, Dominick J; Rose, Jocelyn K C

    2009-10-01

    Full appreciation of the roles of the plant cuticle in numerous aspects of physiology and development requires a comprehensive understanding of its biosynthesis and deposition; however, much is still not known about cuticle structure, trafficking and assembly. To date, assessment of cuticle organization has been dominated by 2D imaging, using histochemical stains in conjunction with light and fluorescence microscopy. This strategy, while providing valuable information, has limitations because it attempts to describe a complex 3D structure in 2D. An imaging technique that could accurately resolve 3D architecture would provide valuable additions to the growing body of information on cuticle molecular biology and biochemistry. We present a novel application of 3D confocal scanning laser microscopy for visualizing the architecture, deposition patterns and micro-structure of plant cuticles, using the fluorescent stain auramine O. We demonstrate the utility of this technique by contrasting the fruit cuticle of wild-type tomato (Solanum lycopersicum cv. M82) with those of cutin-deficient mutants. We also introduce 3D cuticle modeling based on reconstruction of serial optical sections, and describe its use in identification of several previously unreported features of the tomato fruit cuticle.

  15. Noninvasive in vivo detection and quantification of Demodex mites by confocal laser scanning microscopy.

    Science.gov (United States)

    Sattler, E C; Maier, T; Hoffmann, V S; Hegyi, J; Ruzicka, T; Berking, C

    2012-11-01

    In many Demodex-associated skin diseases Demodex mites are present in abundance and seem to be at least partially pathogenic. So far all diagnostic approaches such as scraping or standardized superficial skin biopsy are (semi-)invasive and may cause discomfort to the patient. To see whether confocal laser scanning microscopy (CLSM) - a noninvasive method for the visualization of superficial skin layers - is able to detect and quantify D. folliculorum in facial skin of patients with rosacea. Twenty-five patients (34-72 years of age) with facial rosacea and 25 age- and sex-matched normal controls were examined by CLSM. Mosaics of 8 × 8 mm and 5 × 5 mm were created by scanning horizontal layers of lesional skin and quantification of mites per follicle and per area as well as follicles per area was performed. In all patients D. folliculorum could be detected by CLSM and presented as roundish or lengthy cone-shaped structures. CLSM allowed the quantification of Demodex mites and revealed significant differences (P Demodex mites noninvasively in facial skin of patients with rosacea. © 2012 The Authors. BJD © 2012 British Association of Dermatologists.

  16. Analysis of a marine phototrophic biofilm by confocal laser scanning microscopy using the new image quantification software PHLIP

    NARCIS (Netherlands)

    Müller, L.N.; de Brouwer, J.F.C.; Almeida, J.S.; Stal, L.J.; Xavier, J.B.

    2006-01-01

    Background Confocal laser scanning microscopy (CLSM) is the method of choice to study interfacial biofilms and acquires time-resolved three-dimensional data of the biofilm structure. CLSM can be used in a multi-channel modus where the different channels map individual biofilm components. This

  17. Microradiography and confocal laser scanning microscopy applied to enamel lesions formed in vivo with and without fluoride varnish treatment

    NARCIS (Netherlands)

    Ogaard, B; Duschner, H; Ruben, J; Arends, J

    The aim of the present investigation was to combine 2 techniques suitable for lesion characterization: quantitative microradiography (TMR) and confocal laser scanning microscopy (CLSM) on in vivo induced lesions with and without a fluoride varnish (Duraphat(R)) treatment. Orthodontic bands were

  18. Enumeration of leukocyte infiltration in solid tumors by confocal laser scanning microscopy

    Directory of Open Access Journals (Sweden)

    Amirkhosravi A

    2006-07-01

    Full Text Available Abstract Background Leukocytes commonly infiltrate solid tumors, and have been implicated in the mechanism of spontaneous regression in some cancers. Conventional techniques for the quantitative estimation of leukocyte infiltrates in tumors rely on light microscopy of immunostained thin tissue sections, in which an arbitrary assessment (based on low, medium or high levels of infiltration of antigen density is made by the pathologist. These estimates are relatively subjective and often require the opinion of a second pathologist. In addition, since thin tissue sections are cut, no data regarding the three-dimensional distribution of antigen can be obtained. Results To overcome these problems, we have designed a method to enumerate leukocyte infiltration into tumors, using confocal laser scanning microscopy of fluorescently immunostained leukocytes in thick tissue sections. Using image analysis software, a threshold was applied to eliminate unstained tissue and residual noise. The total antigen volume in the scanned tissue was calculated and divided by the mean cell volume (calculated by "seeding" ten individual cells to obtain the cell count. Using this method, we compared the calculated leukocyte counts with those obtained manually by ten laboratory personnel. There was no significant difference (P > 0.05 between the cell counts obtained by either method. We then compared leukocyte infiltration into seven tumors and matched non-malignant tissue obtained from the periphery of the resected tissue. There was a significant increase in the infiltration of all leukocyte subsets into the tumors compared to minimal numbers in the non-malignant tissue. Conclusion From these results we conclude that this method may be of considerable use for the enumeration of cells in tissues. Furthermore, since it can be performed by laboratory technical staff, less time input is required by the pathologist in assessing the degree of leukocyte infiltration into tumors.

  19. Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

    Science.gov (United States)

    Hackley, Paul C.; Kus, Jolanta

    2015-01-01

    We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

  20. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide

    KAUST Repository

    Rodighiero, Simona

    2015-03-22

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. © 2015 Wiley Periodicals, Inc.

  1. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide.

    Science.gov (United States)

    Rodighiero, Simona; Torre, Bruno; Sogne, Elisa; Ruffilli, Roberta; Cagnoli, Cinzia; Francolini, Maura; Di Fabrizio, Enzo; Falqui, Andrea

    2015-06-01

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. © 2015 Wiley Periodicals, Inc.

  2. Studies of the microstructure of polymer-modified bitumen emulsions using confocal laser scanning microscopy.

    Science.gov (United States)

    Forbes, A; Haverkamp, R G; Robertson, T; Bryant, J; Bearsley, S

    2001-12-01

    Polymer-modified bitumen emulsions present a safer and more environmentally friendly binder for enhancing the properties of roads. Cationic bitumen emulsion binders containing polymer latex were investigated using confocal laser scanning microscopy. The latex was incorporated into the bitumen emulsion by using four different addition methods and all emulsions were processed with a conventional colloid mill. The emulsion binder films were studied after evaporation of the emulsion aqueous phase. We show how the microstructure and distribution of the polymer varies within the bitumen binder depending on latex addition method, and that the microstructure of the binder remains intact when exposed to elevated temperature. It was found that a distinctly fine dispersion of polymer results when the polymer is blended into the bitumen before the emulsifying process (a monophase emulsion). In contrast, bi-phase emulsion binders produced by either post-adding the latex to the bitumen emulsion, or by adding the latex into the emulsifier solution phase before processing, or by comilling the latex with the bitumen, water and emulsifier all resulted in a network formation of bitumen particles surrounded by a continuous polymer film. The use of emulsified binders appears to result in a more evenly distributed polymer network compared to the use of hot polymer-modified binders, and they therefore have greater potential for consistent binder cohesion strength, stone retention and therefore improved pavement performance.

  3. Confocal laser-scanning microscopy of capillaries in normal and psoriatic skin

    Science.gov (United States)

    Archid, Rami; Patzelt, Alexa; Lange-Asschenfeldt, Bernhard; Ahmad, Sufian S.; Ulrich, Martina; Stockfleth, Eggert; Philipp, Sandra; Sterry, Wolfram; Lademann, Juergen

    2012-10-01

    An important and most likely active role in the pathogenesis of psoriasis has been attributed to changes in cutaneous blood vessels. The purpose of this study was to use confocal laser-scanning microscopy (CLSM) to investigate dermal capillaries in psoriatic and normal skin. The structures of the capillary loops in 5 healthy participants were compared with those in affected skin of 13 psoriasis patients. The diameters of the capillaries and papillae were measured for each group with CLSM. All investigated psoriasis patients showed elongated, widened, and tortuous microvessels in the papillary dermis, whereas all healthy controls showed a single capillary loop in each dermal papilla. The capillaries of the papillary loop and the dermal papilla were significantly enlarged in the psoriatic skin lesions (diameters 24.39±2.34 and 146.46±28.52 μm, respectively) in comparison to healthy skin (diameters 9.53±1.8 and 69.48±17.16 μm, respectively) (P<0.001). CLSM appears to represent a promising noninvasive technique for evaluating dermal capillaries in patients with psoriasis. The diameter of the vessels could be seen as a well-quantifiable indicator for the state of psoriatic skin. CLSM could be useful for therapeutic monitoring to delay possible recurrences.

  4. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis.

    Science.gov (United States)

    Skytte, Jacob L; Ghita, Ovidiu; Whelan, Paul F; Andersen, Ulf; Møller, Flemming; Dahl, Anders B; Larsen, Rasmus

    2015-06-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented dairy products. When studying such networks, hundreds of images can be obtained, and here image analysis methods are essential for using the images in statistical analysis. Previously, methods including gray level co-occurrence matrix analysis and fractal analysis have been used with success. However, a range of other image texture characterization methods exists. These methods describe an image by a frequency distribution of predefined image features (denoted textons). Our contribution is an investigation of the choice of image analysis methods by performing a comparative study of 7 major approaches to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis, and cluster analysis. Our investigation suggests that the texton-based descriptors provide a fuller description of the images compared to gray-level co-occurrence matrix descriptors and fractal analysis, while still being as applicable and in some cases as easy to tune. © 2015 Institute of Food Technologists®

  5. Starch/carrageenan/milk proteins interactions studied using multiple staining and Confocal Laser Scanning Microscopy.

    Science.gov (United States)

    Matignon, A; Moulin, G; Barey, P; Desprairies, M; Mauduit, S; Sieffermann, J M; Michon, C

    2014-01-01

    This study focused on the effects of the interactions between modified waxy maize starch, kappa carrageenan and skim milk on the microstructure of their mixed systems using Confocal Laser Scanning Microscopy (CLSM). A multiple staining of the components was set up with a view to improving starch covalent staining. In starch/carrageenan pasted mixtures, carrageenan was found to adsorb on and penetrate slightly into the starch granules, whereas no interactions were observed between starch and milk proteins. In ternary mixtures, interactions between starch granules and carrageenan were no longer observed, even when milk proteins were added after starch swelling in the carrageenan solution, thus showing preferential interactions between carrageenan/milk proteins in comparison to carrageenan/starch granules. Modifying the blending order of the components led to microstructure differences depending on several parameters such as starch/carrageenan interactions, carrageenan/milk proteins network structure, level of starch granules disruption and amylopectin contribution to the microstructure. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Combining confocal laser scanning microscopy with serial section reconstruction in the study of adult neurogenesis.

    Directory of Open Access Journals (Sweden)

    Federico eLuzzati

    2011-05-01

    Full Text Available Current advances in imaging techniques have extended the possibility of visualizing small structures within large volumes of both fixed and live specimens without sectioning. These techniques have contributed valuable information to study neuronal plasticity in the adult brain. However, technical limits still hamper the use of these approaches to investigate neurogenic regions located far from the ventricular surface such as parenchymal neurogenic niches, or the scattered neuroblasts induced by brain lesions. Here, we present a method to combine confocal laser scanning microscopy (CLSM and serial section reconstruction in order to reconstruct large volumes of brain tissue at cellular resolution. In this method a series of thick sections are imaged with CLSM and the resulting stacks of images are registered and 3D reconstructed. This approach is based on existing freeware software and can be performed on ordinary laboratory personal computers (PC. By using this technique we have investigated the morphology and spatial organization of a group of doublecortin (DCX+ neuroblasts located in the lateral striatum of the late post-natal guinea pig. The 3D study unravelled a complex network of long and poorly ramified cell processes, often fascicled and mostly oriented along the internal capsule fibre bundles. These data support CLSM serial section reconstruction as a reliable alternative to the whole mount approaches to analyze cyto-architectural features of adult germinative niches.

  7. Use of biocytin as neuroanatomic tracer in harvested human pancreas: a confocal laser scanning microscopy analysis.

    Science.gov (United States)

    Spiga, Saturnino; Fattore, Liana; Puddu, Maria Cristina; Cappai, Antonello; Picciau, Susanna; Brotzu, Giovanni; Serra, Giuliana Paola; Petruzzo, Palmina

    2002-05-01

    To identify central neuroanatomic structure, biocytin labeling has recently been used. To date, there are no bibliographic references about the use of this molecule in investigations of the peripheral nervous system. In the present study, fresh, harvested human pancreas was used to evidence pancreatic innervations by biocytin. To investigate for the first time pancreatic innervation in harvested pancreas from human multiorgan cadaveric donors. Biocytin labeling was used as a neuroanatomic tracing method, and confocal laser scanning microscopy was used for analysis for description by means of high-resolution images. The application of biocytin-avidin staining in harvested human pancreas revealed numerous bundles of nervous fibers, intrapancreatic ganglia, few small solitary neurons, and a large number of positive supporting cells (glial-like cells). Biocytin appeared to pass through gap junctions between glial elements and neurons and among the neurons. In human pancreas, biocytin is rapidly transported in both anterograde and retrograde directions, with consequent visualization of fine details of pancreatic innervation morphology. Indeed, evidence of anterograde and retrograde transportation of biocytin has been demonstrated in the extensive labeling of pancreatic preganglionic and postganglionic fibers as well as a great number of chemical buds that wind through exocrine tissue or undetermined target cells. To our knowledge, this is the first report of the successful use of biocytin in neuronal retrograde and anterograde labeling in the human peripheral nervous system.

  8. Neutral Red as a Probe for Confocal Laser Scanning Microscopy Studies of Plant Roots

    Science.gov (United States)

    DUBROVSKY, JOSEPH G.; GUTTENBERGER, MARTIN; SARALEGUI, ANDRES; NAPSUCIALY-MENDIVIL, SELENE; VOIGT, BORIS; BALUŠKA, FRANTIŠEK; MENZEL, DIEDRIK

    2006-01-01

    • Background and Aims Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology. • Methods NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye. • Key Results The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Casparian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular–arbuscular mycorrhizas. • Conclusions Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM. PMID:16520341

  9. Confocal Raman microscopy

    CERN Document Server

    Dieing, Thomas; Hollricher, Olaf

    2018-01-01

    This second edition provides a cutting-edge overview of physical, technical and scientific aspects related to the widely used analytical method of confocal Raman microscopy. The book includes expanded background information and adds insights into how confocal Raman microscopy, especially 3D Raman imaging, can be integrated with other methods to produce a variety of correlative microscopy combinations. The benefits are then demonstrated and supported by numerous examples from the fields of materials science, 2D materials, the life sciences, pharmaceutical research and development, as well as the geosciences.

  10. CTC staining and counting of actively respiring bacteria in natural stone using confocal laser scanning microscopy.

    Science.gov (United States)

    Bartosch, S; Mansch, R; Knötzsch, K; Bock, E

    2003-01-01

    A method was established for staining and counting of actively respiring bacteria in natural stone by using the tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) in combination with confocal laser scanning microscopy (CLSM). Applying 5 mM CTC for 2 h to pure cultures of representative stone-inhabiting microorganisms showed that chemoorganotrophic bacteria and fungi-in contrast to lithoautotrophic nitrifying bacteria-were able to reduce CTC to CTF, the red fluorescing formazan crystals of CTC. Optimal staining conditions for microorganisms in stone material were found to be 15 mM CTC applied for 24 h. The cells could be visualized on transparent and nontransparent mineral materials by means of CLSM. A semi-automated method was used to count the cells within the pore system of the stone. The percentage of CTC-stained bacteria was dependent on temperature and humidity of the material. At 28 degrees C and high humidity (maximum water holding capacity) in the laboratory, about 58% of the total bacterial microflora was active. On natural stone exposed for 9 years at an urban exposure site in Germany, 52-56% of the bacterial microflora was active at the east, west, and north side of the specimen, while only 18% cells were active at the south side. This is consistent with microclimatic differences on the south side which was more exposed to sunshine thus causing UV and water stress as well as higher temperatures on a microscale level. In combination with CLSM, staining by CTC can be used as a fast method for monitoring the metabolic activity of chemoorganotrophic bacteria in monuments, buildings of historic interest or any art objects of natural stone. Due to the small size of samples required, the damage to these objects and buildings can be minimized.

  11. Confocal laser scanning microscopy of liesegang rings in odontogenic cysts: analysis of three-dimensional image reconstruction.

    Science.gov (United States)

    Scivetti, Michele; Lucchese, Alberta; Crincoli, Vito; Pilolli, Giovanni Pietro; Favia, Gianfranco

    2009-01-01

    Liesegang rings are concentric noncellular lamellar structures, occasionally found in inflammatory tissues. They have been confused with various parasites, algas, calcification, and psammoma bodies. The authors examined Liesegang rings from oral inflammatory cysts by both optical and confocal laser scanning microscopy, and perfomed a three-dimensional reconstruction. These investigations indicate that Liesegang rings are composed of multiple birefringent concentric rings, resulting from a progressive deposition of organic substances, with an unclear pathogenesis.

  12. The neuromuscular system of Pycnophyes kielensis (Kinorhyncha: Allomalorhagida investigated by confocal laser scanning microscopy

    Directory of Open Access Journals (Sweden)

    Andreas Altenburger

    2016-11-01

    Full Text Available Abstract Background Kinorhynchs are ecdysozoan animals with a phylogenetic position close to priapulids and loriciferans. To understand the nature of segmentation within Kinorhyncha and to infer a probable ancestry of segmentation within the last common ancestor of Ecdysozoa, the musculature and the nervous system of the allomalorhagid kinorhynch Pycnophyes kielensis were investigated by use of immunohistochemistry, confocal laser scanning microscopy, and 3D reconstruction software. Results The kinorhynch body plan comprises 11 trunk segments. Trunk musculature consists of paired ventral and dorsal longitudinal muscles in segments 1–10 as well as dorsoventral muscles in segments 1–11. Dorsal and ventral longitudinal muscles insert on apodemes of the cuticle inside the animal within each segment. Strands of longitudinal musculature extend over segment borders in segments 1–6. In segments 7–10, the trunk musculature is confined to the segments. Musculature of the digestive system comprises a strong pharyngeal bulb with attached mouth cone muscles as well as pharyngeal bulb protractors and retractors. The musculature of the digestive system shows no sign of segmentation. Judged by the size of the pharyngeal bulb protractors and retractors, the pharyngeal bulb, as well as the introvert, is moved passively by internal pressure caused by concerted action of the dorsoventral muscles. The nervous system comprises a neuropil ring anterior to the pharyngeal bulb. Associated with the neuropil ring are flask-shaped serotonergic somata extending anteriorly and posteriorly. A ventral nerve cord is connected to the neuropil ring and runs toward the anterior until an attachment point in segment 1, and from there toward the posterior with one ganglion in segment 6. Conclusions Segmentation within Kinorhyncha likely evolved from an unsegmented ancestor. This conclusion is supported by continuous trunk musculature in the anterior segments 1–6, continuous

  13. Confocal Raman Microscopy

    CERN Document Server

    Dieing, Thomas; Toporski, Jan

    2011-01-01

    Confocal Raman Microscopy is a relatively new technique that allows chemical imaging without specific sample preparation. By integrating a sensitive Raman spectrometer within a state-of-the-art microscope, Raman microscopy with a spatial resolution down to 200nm laterally and 500nm vertically can be achieved using visible light excitation. Recent developments in detector and computer technology as well as optimized instrument design have reduced integration times of Raman spectra by orders of magnitude, so that complete images consisting of tens of thousands of Raman spectra can be acquired in seconds or minutes rather than hours, which used to be standard just one decade ago. The purpose of this book is to provide the reader a comprehensive overview of the rapidly developing field of Confocal Raman Microscopy and its applications.

  14. An essential role for dendritic cells in vernal keratoconjunctivitis: analysis by laser scanning confocal microscopy.

    Science.gov (United States)

    Liu, M; Gao, H; Wang, T; Wang, S; Li, S; Shi, W

    2014-03-01

    CD4+ T helper type 2 cells play a central role in the pathogenesis of vernal keratoconjunctivitis (VKC), and antigen-presenting cells are required for the cell activation. In this study, we aimed to survey the density, distribution, and morphology of dendritic cells (DCs) in patients with VKC by in vivo confocal microscopy. Thirty-five patients (mean, 12.4 ± 5.3 years) affected by VKC were included. All patients were treated with 0.1% fluorometholone eye drops and 0.5% cyclosporine A eye drops. The density and morphological and distributional characteristics of DCs in each right eye were evaluated by in vivo confocal microscopy before treatment and at 1, 3, and 6 months after treatment. Thirty-five age-matched normal subjects (mean, 16.5 ± 1.8 years) were studied as controls. There was significant difference in age between the VKC group and the control group (F = 18.17, P < 0.05). Compared with normal eyes, increased numbers of DCs were found in patients with VKC, with mean cell densities of 244.09 ± 59.76 cells/mm(2) at the bulbar conjunctiva, 574.53 ± 87.34 cells/mm(2) at the limbus, and 403.32 ± 106.59 cells/mm(2) at the peripheral cornea before treatment. These DCs exhibited a typical dendritic shape. At 3 months after treatment, the DC density at the conjunctiva decreased significantly (P < 0.05), approximating that in the controls. At 3 and 6 months, the DC densities at the limbus and peripheral cornea also decreased significantly (P < 0.05), but were still statistically higher than those in the controls. These DCs, with small dendritic processes or irregular shapes, were observed to gradually locate at the epithelial basal membrane and subbasal nerve plexus. In vivo confocal microscopy appears to be a valuable tool in evaluating the dynamic change of DCs at the conjunctiva and cornea. DCs play an essential role in VKC and therefore may constitute a target for therapeutic intervention for VKC. © 2013 John Wiley & Sons Ltd.

  15. Confocal Raman Microscopy; applications in tissue engineering

    NARCIS (Netherlands)

    van Apeldoorn, Aart A.

    2005-01-01

    This dissertation describes the use of confocal Raman microscopy and spectroscopy in the field of tissue engineering. Moreover, it describes the combination of two already existing technologies, namely scanning electron microscopy and confocal Raman spectroscopy in one apparatus for the enhancement

  16. Real-time mapping of the corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy

    Science.gov (United States)

    Guthoff, Rudolf F.; Zhivov, Andrey; Stachs, Oliver

    2010-02-01

    The aim of the study was to produce two-dimensional reconstruction maps of the living corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy in real time. CLSM source data (frame rate 30Hz, 384x384 pixel) were used to create large-scale maps of the scanned area by selecting the Automatic Real Time (ART) composite mode. The mapping algorithm is based on an affine transformation. Microscopy of the sub-basal nerve plexus was performed on normal and LASIK eyes as well as on rabbit eyes. Real-time mapping of the sub-basal nerve plexus was performed in large-scale up to a size of 3.2mm x 3.2mm. The developed method enables a real-time in vivo mapping of the sub-basal nerve plexus which is stringently necessary for statistically firmed conclusions about morphometric plexus alterations.

  17. Comparison of divided and full pupil configurations for line-scanning confocal microscopy in human skin and oral mucosa

    Science.gov (United States)

    Larson, Bjorg; Abeytunge, Sanjeewa; Glazowski, Chris; Rajadhyaksha, Milind

    2012-02-01

    Confocal point-scanning microscopy has been showing promise in the detection, diagnosing and mapping of skin lesions in clinical settings. The noninvasive technique allows provides optical sectioning and cellular resolution for in vivo diagnosis of melanoma and basal cell carcinoma and pre-operative and intra-operative mapping of margins. The imaging has also enabled more accurate "guided" biopsies while minimizing the otherwise large number of "blind" biopsies. Despite these translational advances, however, point-scanning technology remains relatively complex and expensive. Line-scanning technology may offer an alternative approach to accelerate translation to the clinic. Line-scanning, using fewer optical components, inexpensive linear-array detectors and custom electronics, may enable smaller, simpler and lower-cost confocal microscopes. A line is formed using a cylindrical lens and scanned through the back focal plane of the objective with a galvanometric scanner. A linear CCD is used for detection. Two pupil configurations were compared for performance in imaging human tissue. In the full-pupil configuration, illumination and detection is made through the full objective pupil. In the divided pupil approach, half the pupil is illuminated and the other half is used for detection. The divided pupil configuration loses spatial and axial resolution due to a diminished NA, but the sectioning capability and rejection of background is improved. Imaging in skin and oral mucosa illustrate the performance of the two configurations.

  18. Prognostic significance of vascularity in cutaneous melanoma: pilot study using in vivo confocal scanning laser microscopy.

    Science.gov (United States)

    Humphrey, Shannon; Walsh, Noreen M; Delaney, Laura; Propperova, Iva; Langley, Richard G B

    2006-01-01

    Tumor vascularity may be of strong prognostic significance in cutaneous melanoma. We are the first to use a novel, noninvasive, in vivo confocal scanning laser microscope (CSLM) to evaluate vascularity in cutaneous melanoma. Our purpose was to apply a CSLM to assess vascularity in melanoma and to evaluate the prognostic significance of these findings. Patients with a suspicious pigmented lesion were prospectively recruited to undergo CSLM prior to skin biopsy, and those diagnosed with melanoma were included in this study. A blinded observer graded tumor vascularity from still digital CSLM images. The CSLM vascularity grading was correlated to tumor thickness and ulceration as a proxy for clinical prognosis. Sixty-six patients and 67 lesions underwent imaging with CSLM. Eleven patients were diagnosed with melanoma, including six in situ and five invasive melanomas. Prominent vascularity was observed in all advanced melanomas. There was an overall increase in mean tumor thickness between the absent (x = 0.315 mm) to prominent (x = 1.51 mm) categories. In this pilot study, vascularity was readily detected in cutaneous melanomas using CSLM. Prominent vascularity was observed in patients with advanced cutaneous melanomas. Our preliminary results are encouraging and indicate potential for the use of CSLM to assess vascularity in cutaneous melanoma, with potential prognostic and therapeutic implications.

  19. Cutting efficiency of apical preparation using ultrasonic tips with microprojections: confocal laser scanning microscopy study

    Directory of Open Access Journals (Sweden)

    Sang-Won Kwak

    2014-11-01

    Full Text Available Objectives The purpose of this study was to compare the cutting efficiency of a newly developed microprojection tip and a diamond-coated tip under two different engine powers. Materials and Methods The apical 3-mm of each root was resected, and root-end preparation was performed with upward and downward pressure using one of the ultrasonic tips, KIS-1D (Obtura Spartan or JT-5B (B&L Biotech Ltd.. The ultrasonic engine was set to power-1 or -4. Forty teeth were randomly divided into four groups: K1 (KIS-1D / Power-1, J1 (JT-5B / Power-1, K4 (KIS-1D / Power-4, and J4 (JT-5B / Power-4. The total time required for root-end preparation was recorded. All teeth were resected and the apical parts were evaluated for the number and length of cracks using a confocal scanning micrscope. The size of the root-end cavity and the width of the remaining dentin were recorded. The data were statistically analyzed using two-way analysis of variance and a Mann-Whitney test. Results There was no significant difference in the time required between the instrument groups, but the power-4 groups showed reduced preparation time for both instrument groups (p < 0.05. The K4 and J4 groups with a power-4 showed a significantly higher crack formation and a longer crack irrespective of the instruments. There was no significant difference in the remaining dentin thickness or any of the parameters after preparation. Conclusions Ultrasonic tips with microprojections would be an option to substitute for the conventional ultrasonic tips with a diamond coating with the same clinical efficiency.

  20. Confocal scanning Mueller polarimeter

    Science.gov (United States)

    Lompado, Arthur

    2009-08-01

    We describe the design, construction, calibration and testing of a confocal scanning Mueller polarimeter. A polarization state generator and polarization state analyzer have been inserted into the optical path of a conventional confocal scanning imager to collect the reflectance Muller matrix of samples measuring up to 6.26 mm on a side. Four sources are available for sample interrogation using diode lasers centered at 532 nm, 635 nm, 670 nm, and 785 nm. The device captures all required imagery to calculate the Mueller matrix of each image pixel in approximately 90 s. These matrices are then reduced into polarization imagery such as the diattenuation, retardance and depolarization index. Oftentimes this polarization imagery is quite different and potentially more informative than a conventional intensity image. There are a number of fields that can benefit from alternative/enhanced imagery, most notably in the biomedical, discrimination, and target recognition communities. The sensor has been designed for biomedical applications aimed at improving the technique of noninvasive detection of melanoma lesions.

  1. Noise analysis of a white-light supercontinuum light source for multiple wavelength confocal laser scanning fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    McConnell, Gail [Centre for Biophotonics, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, G4 0NR (United Kingdom)

    2005-08-07

    Intensity correlations of a Ti : sapphire, Kr/Ar and a white-light supercontinuum were performed to quantify the typical signal amplitude fluctuations and hence ascertain the comparative output stability of the white-light supercontinuum source for confocal laser scanning microscopy (CLSM). Intensity correlations across a two-pixel sample (n = 1000) of up to 98%, 95% and 94% were measured for the Ti : sapphire, Kr/Ar and white-light supercontinuum source, respectively. The white-light supercontinuum noise level is therefore acceptable for CLSM, with the added advantage of wider wavelength flexibility over traditional CLSM excitation sources. The relatively low-noise white-light supercontinuum was then used to perform multiple wavelength sequential CLSM of guinea pig detrusor to confirm the reliability of the system and to demonstrate system flexibility.

  2. Characterization by Confocal Laser Scanning Microscopy of the Phase Composition at Interfaces in Thick Films of Polymer Blends

    Directory of Open Access Journals (Sweden)

    Sandro Lattante

    2014-01-01

    Full Text Available Confocal Laser Scanning Microscopy (CLSM has been used as a fast, user-friendly, and noninvasive tool for characterizing the phase composition differences at the substrate and air interfaces in thick films of polymer blends. A clearly different phase composition at the blend/glass interface and at the blend/air interface has been detected. We show that PCBM preferentially accumulates at the glass/blend interface, while P3HT preferentially accumulates at the blend/air interface, by comparing the integrated signal intensity of the luminescence coming from both interfaces. Our results demonstrate that CLSM can be used conveniently for the fast identification of a preferential phase segregation at interfaces in polymer blends. This is useful in the research field on devices (like sensors or planar waveguides that are based on very thick layers (thickness higher than 1 μm.

  3. Fluorescence confocal microscopy for pathologists.

    Science.gov (United States)

    Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

    2014-03-01

    Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on

  4. Applications of confocal laser scanning microscopy in research into organic semiconductor thin films

    DEFF Research Database (Denmark)

    Schiek, Manuela; Balzer, Frank

    2014-01-01

    At the center of opto-electronic devices are thin layers of organic semiconductors, which need to be sandwiched between planar electrodes. With the growing demand for opto-electronic devices now and in the future, new electrode materials are needed to meet the requirements of organic semiconductors....... Control of these interfaces directly impacts on the performance and here we show with basic growth studies of model compounds on dielectric and graphitic surfaces, the formation of distinctly textured films. Silver-nanowire meshes are presented as an alternative transparent electrode material. Confocal...

  5. A video rate laser scanning confocal microscope

    Science.gov (United States)

    Ma, Hongzhou; Jiang, James; Ren, Hongwu; Cable, Alex E.

    2008-02-01

    A video-rate laser scanning microscope was developed as an imaging engine to integrate with other photonic building blocks to fulfill various microscopic imaging applications. The system is quipped with diode laser source, resonant scanner, galvo scanner, control electronic and computer loaded with data acquisition boards and imaging software. Based on an open frame design, the system can be combined with varies optics to perform the functions of fluorescence confocal microscopy, multi-photon microscopy and backscattering confocal microscopy. Mounted to the camera port, it allows a traditional microscope to obtain confocal images at video rate. In this paper, we will describe the design principle and demonstrate examples of applications.

  6. Serial Sectioning Of Cells In Three Dimensions With Confocal Scanning Laser Fluorescence Microscopy (Fl-CSLM): Microtomoscopy

    Science.gov (United States)

    Stelzer, Ernst H.; Stricker, Reiner; Pick, Reinhard; Storz, Clemens; Wijnaendts-Van-Resandt, Roelof W.

    1988-06-01

    The discrimination of out of focus contributions in fluorescence microscopy possible in a confocal setup will establish itself as a supplement to conventional fluorescence microscopy. The improvement of the contrast compared with conventional fluorescence microscopy depends mainly on the density of the fluorescing material and the thickness of the sample. The term thickness, that which microscopists refer to as the size of the specimen along the optical axis, will gain a new quality since a confocal fluorescence microscope may reveal totally different features when recording data in planes that are 0.3μm apart. Differences that have in the past been neglected suddenly become important. The following article will outline important features in the application of confocal fluorescence microscopy in the biological sciences, point out its limitatk'ns, and draw attention to expected developments.

  7. Aerogel Track Morphology: Measurement, Three Dimensional Reconstruction and Particle Location using Confocal Laser Scanning Microscopy

    Science.gov (United States)

    Kearsley, A. T.; Ball, A. D.; Wozniakiewicz, P. A.; Graham, G. A.; Burchell, M. J.; Cole, M. J.; Horz, F.; See, T. H.

    2007-01-01

    The Stardust spacecraft returned the first undoubted samples of cometary dust, with many grains embedded in the silica aerogel collector . Although many tracks contain one or more large terminal particles of a wide range of mineral compositions , there is also abundant material along the track walls. To help interpret the full particle size, structure and mass, both experimental simulation of impact by shots and numerical modeling of the impact process have been attempted. However, all approaches require accurate and precise measurement of impact track size parameters such as length, width and volume of specific portions. To make such measurements is not easy, especially if extensive aerogel fracturing and discoloration has occurred. In this paper we describe the application and limitations of laser confocal imagery for determination of aerogel track parameters, and for the location of particle remains.

  8. Data on characterization of nano- and micro-structures resulting from glycine betaine surfactant/kappa-carrageenan interactions by Laser Scanning Confocal Microscopy and Transmission Electron Microscopy.

    Science.gov (United States)

    Gaillard, Cédric; Wang, Yunhui; Covis, Rudy; Vives, Thomas; Benoit, Maud; Benvegnu, Thierry

    2016-12-01

    This article contains data on the Laser Scanning Confocal Microscopy (LSCM) and Transmission Electron Microscopy (TEM) images related to multi-scaled self-assemblies resulting from 'green' cationic glycine betaine surfactant/anionic kappa-carrageenan interactions. These data gave clear evidence of the evolution of the micron-, nano-sized structures obtained at two surfactant/polymer molar ratios (3.5 and 0.8) and after the dilution of the aqueous dispersions with factors of 5 and 10 times. This data article is related to the research article entitled, "Monitoring the architecture of anionic ĸ-carrageenan/cationic glycine betaine amide surfactant assemblies by dilution: A multiscale approach" (Gaillard et al., 2017) [1].

  9. A new approach for the spatially resolved qualitative analysis of the protein distribution in hydrogel beads based on confocal laser scanning microscopy

    NARCIS (Netherlands)

    Heinemann, Matthias; Wagner, Thomas; Doumèche, Bastien; Ansorge-Schumacher, Marion; Büchs, Jochen

    2002-01-01

    To investigate the spatial distribution of white egg albumin (WEA) in alginate beads, a new method based on confocal laser scanning microscopy (CLSM) was developed. In contrast to the existing CLSM methods, misleading conclusions are prevented with the application of the new method which does not

  10.   In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Dige, Irene; Kilian, Mogens; Nilsson, Holger

    2007-01-01

    Confocal laser scanning microscopy (CLSM) has been employed as a method for studying intact natural biofilm. When combined with fluorescence in situ hybridization (FISH) it is possible to analyze spatial relationships and changes of specific members of microbial populations over time. The aim...

  11. Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

    Science.gov (United States)

    Auty, M. A. E.; Gardiner, G. E.; McBrearty, S. J.; O'Sullivan, E. O.; Mulvihill, D. M.; Collins, J. K.; Fitzgerald, G. F.; Stanton, C.; Ross, R. P.

    2001-01-01

    The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (diluent. PMID:11133474

  12. Porosity of natural stone and use of confocal laser scanning microscopy on calcitic marble aged in laboratory

    Directory of Open Access Journals (Sweden)

    Ana Mladenovič

    2008-06-01

    Full Text Available Porosity is one of the key characteristics of natural stone, which influences ondurability as well as functionality of stone as building material. Further, deterioration processes themselves are also characterized by change of porosity. Different direct and indirect techniques can be used for porosity determination. In the following paper overview of these methods, as well as their advantages and disadvantages, is given. Confocal laser scanning microscopy (CLSM is indirect (microscopic technique. Despite its numerous advantages, among which 3D visualizationof pore structure is of major importance, this technique is less known in the area of building materials. An example how CLSM can be applied for qualitative and quantitative evaluation of porosity of calcitic polygonal granoblastic marble is given in this paper. Studied marble has been, despite of its poor durability, often used as building material, especially in the case of claddings. It is shown that thermal hydric factors of deterioration can influence porosity significantly,especially formation of intergranular cracks.This kind of deterioration can be successfully evaluated with use of CLSM method, if samples are suitable prepared and if suitable image analysis tools are developed.

  13. Diffractive elements performance in chromatic confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Garzon, J; Duque, D; Alean, A; Toledo, M [Grupo de Optica y EspectroscopIa, Centro de Ciencia Basica, Universidad Pontificia Bolivariana. Medellin (Colombia); Meneses, J [Laboratorio de Optica y Tratamiento de Senales, Instituto de Fisica, Universidad Industrial de Santander, Bucaramanga (Colombia); Gharbi, T, E-mail: jgarzonr10@une.net.co [Laboratoire d' Optique P. M. Duffieux, UMR-6603 CNR/Universite de Franche-Comte. 16 route de Gray, 25030 Besancon Cedex (France)

    2011-01-01

    The Confocal Laser Scanning Microscopy (CLSM) has been widely used in the semiconductor industry and biomedicine because of its depth discrimination capability. Subsequent to this technique has been developed in recent years Chromatic Confocal Microscopy. This method retains the same principle of confocal and offers the added advantage of removing the axial movement of the moving system. This advantage is usually accomplished with an optical element that generates a longitudinal chromatic aberration and a coding system that relates the axial position of each point of the sample with the wavelength that is focused on each. The present paper shows the performance of compact chromatic confocal microscope when some different diffractive elements are used for generation of longitudinal chromatic aberration. Diffractive elements, according to the process and manufacturing parameters, may have different diffraction efficiency and focus a specific wavelength in a specific focal position. The performance assessment is carried out with various light sources which exhibit an incoherent behaviour and a broad spectral width.

  14. Confocal microscopy imaging of the biofilm matrix

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke L

    2017-01-01

    The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens...

  15. Penetrability of AH plus and MTA fillapex after endodontic treatment and retreatment: a confocal laser scanning microscopy study.

    Science.gov (United States)

    Kok, Daniela; Rosa, Ricardo Abreu da; Barreto, Mirela Sangoi; Busanello, Fernanda Hoffmann; Santini, Manuela Favarin; Pereira, Jefferson Ricardo; Só, Marcus Vinícius Reis

    2014-06-01

    The aim of the study was to assess the penetrability of two endodontic sealers (AH Plus and MTA Fillapex) into dentinal tubules, submitted to endodontic treatment and subsequently to endodontic retreatment. Thirty ex vivo incisors were prepared using ProTaper rotary system up to F3 instrument and divided in three groups according to the endodontic sealer used for root canal filling: AH Plus (AHP), MTA Fillapex (MTAF), and control group (CG) without using EDTA previously to the root canal filling. Rhodamine B dye (red) was incorporated to the sealers in order to provide the fluorescence which will enable confocal laser scanning microscopy (CLSM) assessment. All specimens were filled with gutta-percha cones using the lateral compaction technique. The specimens were submitted to endodontic retreatment using ProTaper Retreatment system, re-prepared up to F5 instruments and filled with gutta-percha cones and the same sealer used during endodontic retreatment. Fluorescein dye (green) was incorporated to the sealer in order to distinguish from the first filling. The roots were sectioned 2 mm from the apex and assessed by CLSM. No difference was found between the two experimental groups (P > 0.05). On the other hand, in the control group the sealers were not capable to penetrate into dentinal tubules after endodontic treatment (P > 0.05). In retreatment cases, none of the sealers were able to penetrate into dentin tubules. It can be concluded that sealer penetrability is high during endodontic treatment. However, MTA Fillapex and AH Plus do not penetrate into dentinal tubules after endodontic retreatment. © 2014 Wiley Periodicals, Inc.

  16. Analysis of a marine phototrophic biofilm by confocal laser scanning microscopy using the new image quantification software PHLIP

    Directory of Open Access Journals (Sweden)

    Almeida Jonas S

    2006-01-01

    Full Text Available Abstract Background Confocal laser scanning microscopy (CLSM is the method of choice to study interfacial biofilms and acquires time-resolved three-dimensional data of the biofilm structure. CLSM can be used in a multi-channel modus where the different channels map individual biofilm components. This communication presents a novel image quantification tool, PHLIP, for the quantitative analysis of large amounts of multichannel CLSM data in an automated way. PHLIP can be freely downloaded from http://phlip.sourceforge.net. Results PHLIP is an open source public license Matlab toolbox that includes functions for CLSM imaging data handling and ten image analysis operations describing various aspects of biofilm morphology. The use of PHLIP is here demonstrated by a study of the development of a natural marine phototrophic biofilm. It is shown how the examination of the individual biofilm components using the multi-channel capability of PHLIP allowed the description of the dynamic spatial and temporal separation of diatoms, bacteria and organic and inorganic matter during the shift from a bacteria-dominated to a diatom-dominated phototrophic biofilm. Reflection images and weight measurements complementing the PHLIP analyses suggest that a large part of the biofilm mass consisted of inorganic mineral material. Conclusion The presented case study reveals new insight into the temporal development of a phototrophic biofilm where multi-channel imaging allowed to parallel monitor the dynamics of the individual biofilm components over time. This application of PHLIP presents the power of biofilm image analysis by multi-channel CLSM software and demonstrates the importance of PHLIP for the scientific community as a flexible and extendable image analysis platform for automated image processing.

  17. The simplicity of males: dwarf males of four species of Osedax (Siboglinidae; Annelida) investigated by confocal laser scanning microscopy.

    Science.gov (United States)

    Worsaae, Katrine; Rouse, Greg W

    2010-02-01

    Dwarf males of the bone-eating worms Osedax (Siboglinidae, Annelida) have been proposed to develop from larvae that settle on females rather than on bone. The apparent arrest in somatic development and resemblance of the males to trochophore larvae has been posited as an example of paedomorphosis. Here, we present the first investigation of the entire muscle and nervous system in dwarf males of Osedax frankpressi, O. roseus, O. rubiplumus, and O. "spiral" analyzed by multistaining and confocal laser scanning microscopy. Sperm shape and spermiogenesis, the sperm duct and internal and external ciliary patterns were likewise visualized. The males of all four species possess morphological traits typical of newly settled siboglinid larvae: a prostomium, a peristomium with a prototroch, one elongate segment and a second shorter segment. Each segment has a ring of eight long-handled hooked chaetae. The longitudinal muscles are distributed as evenly spaced strands forming a grid with the thin outer circular muscles. Oblique protractor and retractor muscles are associated with each of the chaetal sacs. The nervous system comprises a cerebral ganglion, a prototroch nerve ring, paired dorsolateral longitudinal nerves, five ventral longitudinal nerves with paired, posterior ganglia and a terminal commissure, as well as a net of fine peripheral transverse plexuses surrounding the first segment. Internal ciliation occurs as paired ventrolateral bands along the first segment. The bands appear to lead the free mature sperm to a ciliated duct and seminal vesicle lying just behind the prototroch region. A duct then runs from the seminal vesicle into the dorsal part of the prostomium. The similarity of Osedax males to the larvae of Osedax and other siboglinid annelids as well as similarities shown here to the neuromuscular organization seen in other annelid larvae supports the hypothesis of paedomorphosis in males of Osedax.

  18. Effects of two desensitizing dentifrices on dentinal tubule occlusion with citric acid challenge: Confocal laser scanning microscopy study

    Directory of Open Access Journals (Sweden)

    Sneha Anil Rajguru

    2017-01-01

    Full Text Available Background: Dentin hypersensitivity results when patent tubules are exposed to pain-inducing external stimuli. Aim: This study aims to compare the effects of two desensitizing dentifrices containing NovaMin and arginine on dentinal tubule occlusion with and without citric acid challenge in vitro using confocal laser scanning microscopy (CLSM. Materials and Methods: Forty dentin discs were randomly divided into Groups I and II containing twenty specimens each, treated with NovaMin and arginine-containing dentifrices, respectively. Groups I and II were divided into subgroups A and B where IA and IIA underwent CLSM analysis to determine the percentage of tubule occlusion while IB and IIB underwent 0.3% citric acid challenge and CLSM analysis. A novel grading system was devised to categorize tubule occlusion. Results: In Group II, the percentage of occluded tubules was highest for IIA (72.25% ± 10.57% and least for IIB (42.55% ± 8.65% having statistical significance (P < 0.0005. In Group I, the difference between IA (49.9% ± 12.96% and IB (43.15% ± 12.43% was statistically insignificant (P = 0.249. On the comparison between IB and IIB statistically indifferent result was obtained (P = 0.901, whereas the difference between IA and IIA was statistically significant (P < 0.001. The results of grading system were for IA 50% of samples belonged to Grade 2, for IIA 60% - Grade 3, and for IB 70% and for IIB 90% - Grade 2. Conclusion: Dentinal tubule occlusion with arginine-containing dentifrice was significantly higher than NovaMin. However, it could not resist citric acid challenge as effectively as NovaMin. The effects of NovaMin were more sustainable as compared to arginine-containing dentifrice, thus proving to be a better desensitizing agent.

  19. Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

    Directory of Open Access Journals (Sweden)

    Linney Elwood

    2007-07-01

    Full Text Available Abstract Background The zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated. Results This technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM, which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis. Conclusion These data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope.

  20. Confocal microscopy in the diagnosis of melanoma

    Directory of Open Access Journals (Sweden)

    Apostolović-Stojanović Milica

    2013-01-01

    Full Text Available Melanoma is the most deadly form of skin cancer of melanocytic origin. The tumor has a high malignant potential and early metastasis. Prognosis is directly linked to the stage of the disease. Diagnosing thin melanoma at an early stage offers patients their best chance for survival. The crucial innovation in the early recognition of melanoma was the development of in vivo examination of the skin in high-resolution, by confocal microscopy. Confocal microscopy and its modifications provides a “virtual biopsy“, owing to melanosomes and melanin, which are a source of endogenous contrast. Confocal scanning laser microscopy (CSLM provides visualization of microanatomical structures and cellular detail in real time (pigmented keratinocytes, melanocytes, melanosomes and melanophages in the epidermis, dermoepidermal junction and superficial dermis at a resolution equivalent to the resolution of conventional microscopes. [Projekat Ministarstva nauke Republike Srbije, br. 41002

  1. Distribution of biomolecules in porous nitrocellulose membrane pads using confocal laser scanning microscopy and high-speed cameras.

    Science.gov (United States)

    Mujawar, Liyakat Hamid; Maan, Abid Aslam; Khan, Muhammad Kashif Iqbal; Norde, Willem; van Amerongen, Aart

    2013-04-02

    The main focus of our research was to study the distribution of inkjet printed biomolecules in porous nitrocellulose membrane pads of different brands. We produced microarrays of fluorophore-labeled IgG and bovine serum albumin (BSA) on FAST, Unisart, and Oncyte-Avid slides and compared the spot morphology of the inkjet printed biomolecules. The distribution of these biomolecules within the spot embedded in the nitrocellulose membrane was analyzed by confocal laser scanning microscopy in the "Z" stack mode. By applying a "concentric ring" format, the distribution profile of the fluorescence intensity in each horizontal slice was measured and represented in a graphical color-coded way. Furthermore, a one-step diagnostic antibody assay was performed with a primary antibody, double-labeled amplicons, and fluorophore-labeled streptavidin in order to study the functionality and distribution of the immune complex in the nitrocellulose membrane slides. Under the conditions applied, the spot morphology and distribution of the primary labeled biomolecules was nonhomogenous and doughnut-like on the FAST and Unisart nitrocellulose slides, whereas a better spot morphology with more homogeneously distributed biomolecules was observed on the Oncyte-Avid slide. Similar morphologies and distribution patterns were observed when the diagnostic one-step nucleic acid microarray immunoassay was performed on these nitrocellulose slides. We also investigated possible reasons for the differences in the observed spot morphology by monitoring the dynamic behavior of a liquid droplet on and in these nitrocellulose slides. Using high speed cameras, we analyzed the wettability and fluid flow dynamics of a droplet on the various nitrocellulose substrates. The spreading of the liquid droplet was comparable for the FAST and Unisart slides but different, i.e., slower, for the Oncyte-Avid slide. The results of the spreading of the droplet and the penetration behavior of the liquid in the

  2. 3D digital image processing for biofilm quantification from confocal laser scanning microscopy: Multidimensional statistical analysis of biofilm modeling

    Science.gov (United States)

    Zielinski, Jerzy S.

    The dramatic increase in number and volume of digital images produced in medical diagnostics, and the escalating demand for rapid access to these relevant medical data, along with the need for interpretation and retrieval has become of paramount importance to a modern healthcare system. Therefore, there is an ever growing need for processed, interpreted and saved images of various types. Due to the high cost and unreliability of human-dependent image analysis, it is necessary to develop an automated method for feature extraction, using sophisticated mathematical algorithms and reasoning. This work is focused on digital image signal processing of biological and biomedical data in one- two- and three-dimensional space. Methods and algorithms presented in this work were used to acquire data from genomic sequences, breast cancer, and biofilm images. One-dimensional analysis was applied to DNA sequences which were presented as a non-stationary sequence and modeled by a time-dependent autoregressive moving average (TD-ARMA) model. Two-dimensional analyses used 2D-ARMA model and applied it to detect breast cancer from x-ray mammograms or ultrasound images. Three-dimensional detection and classification techniques were applied to biofilm images acquired using confocal laser scanning microscopy. Modern medical images are geometrically arranged arrays of data. The broadening scope of imaging as a way to organize our observations of the biophysical world has led to a dramatic increase in our ability to apply new processing techniques and to combine multiple channels of data into sophisticated and complex mathematical models of physiological function and dysfunction. With explosion of the amount of data produced in a field of biomedicine, it is crucial to be able to construct accurate mathematical models of the data at hand. Two main purposes of signal modeling are: data size conservation and parameter extraction. Specifically, in biomedical imaging we have four key problems

  3. Dynamic behavior of binary component ion-exchange displacement chromatography of proteins visualized by confocal laser scanning microscopy.

    Science.gov (United States)

    Shi, Qing-Hong; Shi, Zhi-Cong; Sun, Yan

    2012-09-28

    Confocal laser scanning microscopy (CLSM) was introduced to visualize particle-scale binary component protein displacement behavior in Q Sepharose HP column. To this end, displacement chromatography of two intrinsic fluorescent proteins, enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP), were developed using sodium saccharin (NaSac) as a displacer. The results indicated that RFP as well as eGFP could be effectively displaced in the single-component experiments by 50 mmol/L NaSac at 120 and 140 mmol/L NaCl whereas a fully developed displacement train with eGFP and RFP was only observed at 120 mmol/L NaCl in binary component displacement. At 140 mmol/L NaCl, there was a serious overlapping of the zones of the two proteins, indicating the importance of induced-salt effect on the formation of an isotachic displacement train. CLSM provided particle-scale evidence that induced-salt effect occurred likewise in the interior of an adsorbent and was synchronous to the introduction of the displacer. CLSM results at 140 mmol/L NaCl also demonstrated that both the proteins had the same fading rate at 50 mmol/L NaSac in the initial stage, suggesting the same displacement ability of NaSac to both the proteins. In the final stage, the fading rate of RFP in the adsorbent became slow, particularly at lower displacer concentrations. In the binary component displacement, the two proteins exhibited distinct fading rates as compared to the single component displacement and the remarkable lagging of the fading rate was observed in protein displacements. It suggested that the co-adsorbed proteins had significant influence on the formation of an isotachic train and the displacement chromatography of the proteins. Therefore, this research provided particle-scale insight into the dynamic behavior and complexity in the displacement of proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. In vivo laser scanning confocal microscopy of the cornea in patients with silicone oil tamponade after vitreoretinal surgery.

    Science.gov (United States)

    Le, Qihua; Wang, Xin; Lv, Jiahua; Sun, Xinghuai; Xu, Jianjiang

    2012-08-01

    To evaluate the morphological changes in the cornea by in vivo laser scanning confocal microscopy (LSCM) in a large case series with silicone oil endotamponade after vitreoretinal surgery and to explore the value of LSCM in the early detection of silicone keratopathy (SK). Ninety-nine patients (99 eyes) with silicone oil endotamponade after vitreoretinal surgery were included in the current study. Slit-lamp examination and measurement of intraocular pressure (IOP) were performed first. Then the central corneas of the subjects' eyes were examined by in vivo LSCM. The analysis of images of each corneal layer was performed and the endothelial cellular density (ECD), endothelial cellular area (ECA), coefficient of variation of cell size (CoV), and percentage of hexagonal cells (PHC) were measured. Moreover, the total size of stromal deposits was measured, and the correlation between the size of deposits and the parameters of endothelial cells was analyzed. Clinically recognizable abnormalities involving the cornea were identified in only 12 eyes (12.1%) under slit-lamp biomicroscopy, whereas in vivo LSCM revealed morphological abnormalities in 40 eyes (40.4%). The manifestations of endothelial lesions varied from decreased cellular density, increased polymegathism and pleomorphism to hyperreflective silicone oil membrane or droplets adhering to the endothelium. Moreover, hyperreflective deposits with various shapes could be identified in both posterior and anterior stroma, along with the infiltration of Langerhans cells beneath the epithelium. The average ECD and PHC of eyes with corneal abnormalities were significantly lower than those of normal corneas, whereas the average ECA and CoV were significantly larger (all Ps < 0.001). The patients with corneal abnormalities were significantly older than those others (P = 0.003). The rate of pseudophakic and aphakic eyes having corneal abnormalities was significantly higher than that of phakic eyes (P = 0.045). Interestingly

  5. Real-time demonstration of split skin graft inosculation and integra dermal matrix neovascularization using confocal laser scanning microscopy.

    Science.gov (United States)

    Greenwood, John; Amjadi, Mahyar; Dearman, Bronwyn; Mackie, Ian

    2009-08-20

    During the first 48 hours after placement, an autograft "drinks" nutrients and dissolved oxygen from fluid exuding from the underlying recipient bed ("plasmatic imbibition"). The theory of inosculation (that skin grafts subsequently obtain nourishment via blood vessel "anastomosis" between new vessels invading from the wound bed and existing graft vessels) was hotly debated from the late 19th to mid-20th century. This study aimed to noninvasively observe blood flow in split skin grafts and Integra dermal regeneration matrix to provide further proof of inosculation and to contrast the structure of vascularization in both materials, reflecting mechanism. Observations were made both clinically and using confocal microscopy on normal skin, split skin graft, and Integra. The VivaScope allows noninvasive, real-time, in vivo images of tissue to be obtained. Observations of blood flow and tissue architecture in autologous skin graft and Integra suggest that 2 very different processes are occurring in the establishment of circulation in each case. Inosculation provides rapid circulatory return to skin grafts whereas slower neovascularization creates an unusual initial Integra circulation. The advent of confocal laser microscopy like the VivaScope 1500, together with "virtual" journals such as ePlasty, enables us to provide exciting images and distribute them widely to a "reading" audience. The development of the early Integra vasculature by neovascularization results in a large-vessel, high-volume, rapid flow circulation contrasting markedly from the inosculatory process in skin grafts and the capillary circulation in normal skin and merits further (planned) investigation.

  6. Evaluation of transdermal delivery of nanoemulsions in ex vivo porcine skin using two-photon microscopy and confocal laser-scanning microscopy

    Science.gov (United States)

    Choi, Sanghoon; Kim, Jin Woong; Lee, Yong Joong; Delmas, Thomas; Kim, Changhwan; Park, Soyeun; Lee, Ho

    2014-10-01

    This study experimentally evaluates the self-targeting ability of asiaticoside-loaded nanoemulsions compared with nontargeted nanoemulsions in ex vivo experiments with porcine skin samples. Homebuilt two-photon and confocal laser-scanning microscopes were employed to noninvasively examine the transdermal delivery of two distinct nanoemulsions. Prior to the application of nanoemulsions, we noninvasively observed the morphology of porcine skin using two-photon microscopy. We have successfully visualized the distributions of the targeted and nontargeted nanoemulsions absorbed into the porcine skin samples. Asiaticoside-loaded nanoemulsions showed an improved ex vivo transdermal delivery through the stratum corneum compared with nonloaded nanoemulsions. As a secondary measure, nanoemulsions-applied samples were sliced in the depth direction with a surgical knife in order to obtain the complete depth-direction distribution profile of Nile red fluorescence. XZ images demonstrated that asiaticoside-loaded nanoemulsion penetrated deeper into the skin compared with nontargeted nanoemulsions. The basal layer boundary is clearly visible in the case of the asiaticoside-loaded skin sample. These results reaffirm the feasibility of using self-targeting ligands to improve permeation through the skin barrier for cosmetics and topical drug applications.

  7. Penetration of tamoxifen citrate loaded ethosomes and liposomes across human skin: a comparative study with confocal laser scanning microscopy.

    Science.gov (United States)

    Sarwa, Khomendra K; Suresh, Preeti K; Rudrapal, Mithun; Verma, Vinod K

    2014-01-01

    In the present study, ethosomal and liposomal formulations containing tamoxifen citrate were prepared and evaluated for their penetration properties in human cadaver skin using Franz diffusion cell and confocal laser scanning microscope (CLSM). The results clearly revealed that ethosomal vesicles showed a better drug permeation profile than that of liposomal vesicles. In addition, low fluorescence intensity in CLSM was recorded with liposomes as compared to ethosomes, indicating lower cumulative amount of drug permeation from liposomal vesicles. Furthermore, CLSM showed uniform fluorescence intensity across the entire depth of skin in ethosomal treatment, indicating high penetrability of ethosomal vesicles through human cadaver skin. In contrast, low penetrability of conventional liposomal vesicles was recorded as penetration was limited to the 7(th) section (i.e. upper epidermis layer) of skin as evident from visualization of intact liposomal vesicles in CLSM.

  8. Optimal pupil design for confocal microscopy

    Science.gov (United States)

    Patel, Yogesh G.; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2010-02-01

    Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current instruments are large, complex, and expensive. A simpler, confocal line-scanning microscope may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A confocal reflectance microscope may use a beamsplitter, transmitting and detecting through the pupil, or a divided pupil, or theta configuration, with half used for transmission and half for detection. The divided pupil may offer better sectioning and contrast. We present a Fourier optics model and compare the on-axis irradiance of a confocal point-scanning microscope in both pupil configurations, optimizing the profile of a Gaussian beam in a circular or semicircular aperture. We repeat both calculations with a cylindrical lens which focuses the source to a line. The variable parameter is the fillfactor, h, the ratio of the 1/e2 diameter of the Gaussian beam to the diameter of the full aperture. The optimal values of h, for point scanning are 0.90 (full) and 0.66 for the half-aperture. For line-scanning, the fill-factors are 1.02 (full) and 0.52 (half). Additional parameters to consider are the optimal location of the point-source beam in the divided-pupil configuration, the optimal line width for the line-source, and the width of the aperture in the divided-pupil configuration. Additional figures of merit are field-of-view and sectioning. Use of optimal designs is critical in comparing the experimental performance of the different configurations.

  9. In situ microspatial imaging using two-photon and confocal laser scanning microscopy of bacteria and extracellular polymeric secretions (EPS) within marine stromatolites.

    Science.gov (United States)

    Kawaguchi, Tomohiro; Decho, Alan W

    2002-03-01

    The combination of a hydrophilic embedding resin, Nanoplast, with fluorescent probes, and subsequent imaging using two-photon and confocal laser scanning microscopy (2P-LSM and CLSM) has allowed in imaging of the in situ microspatial arrangements of microbial cells and their extracellular polymeric secretion (EPS) within marine stromatolites. Optical sectioning by 2P-LSM and CLSM allowed imaging of endolithic cyanobacteria cells, Solentia sp., seen within carbonate sand grains. 2P-LSM allowed very clear imaging with a high resolution of bacteria using DAPI, which normally require UV excitation and reduced photo-bleaching of fluorescent probes.

  10. In-vivo diagnosis and non-inasive monitoring of Imiquimod 5% cream for non-melanoma skin cancer using confocal laser scanning microscopy

    Science.gov (United States)

    Dietterle, S.; Lademann, J.; Röwert-Huber, H.-J.; Stockfleth, E.; Antoniou, C.; Sterry, W.; Astner, S.

    2008-10-01

    Basal cell carcinoma (BCC) is the most common cutaneous malignancy with increasing incidence rates worldwide. A number of established treatments are available, including surgical excision. The emergence of new non-invasive treatment modalities has prompted the development of non-invasive optical devices for therapeutic monitoring and evaluating treatment efficacy. This study was aimed to evaluate the clinical applicability of a fluorescence confocal laser scanning microscope (CFLSM) for non-invasive therapeutic monitoring of basal cell carcinoma treated with Imiquimod (Aldara®) as topical immune-response modifier. Eight participants with a diagnosis of basal cell carcinoma (BCC) were enrolled in this investigation. Sequential evaluation during treatment with Imiquimod showed progressive normalization of the confocal histomorphologic parameters in correlation with normal skin. Confocal laser scanning microscopy was able to identify characteristic features of BCC and allowed the visualization of therapeutic effects over time. Thus our results indicate the clinical applicability of CFLSM imaging to evaluate treatment efficacy in vivo and non-invasively.

  11. Noninvasive redox and back-scattered light imaging of keratocyte cells in the cornea: two-photon excitation and scanning slit confocal microscopy

    Science.gov (United States)

    Masters, Barry R.

    1995-04-01

    The ability to image and monitor the metabolic activity of keratocytes is important for the investigation of wound healing and repair mechanisms in the cornea. After laser refractive surgery there is activation of the stromal keratocytes in the human cornea. Two-photon excitation laser scanning microscopy was used to monitor the NAD(P)H levels in keratocytes in the cornea. The autofluorescence was confirmed to be mostly of NAD(P)H origin by treatment with cyanide which caused an increase in the fluorescence by a factor of two. We used a real-time scanning slit confocal microscope to image the distribution of keratocytes in the full thickness of the cornea. This microscope has the ability to image the cellular processes as well as the nuclei of the stromal keratocytes. Noninvasive optical imaging may provide a useful tool to investigate keratocyte activation after laser surgery or wound healing.

  12. Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin

    NARCIS (Netherlands)

    P.J. Caspers (Peter); G.W. Lucassen (Gerald); G.J. Puppels (Gerwin)

    2003-01-01

    textabstractIn vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections

  13. Digital differential confocal microscopy based on spatial shift transformation.

    Science.gov (United States)

    Liu, J; Wang, Y; Liu, C; Wilson, T; Wang, H; Tan, J

    2014-11-01

    Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three-dimensions. We describe a simple alternative based on spatial transformation of a through-focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

  14. Imaging of intracellular behavior of polymeric nanoparticles in Staphylococcus epidermidis biofilms by slit-scanning confocal Raman microscopy and scanning electron microscopy with energy-dispersive X-ray spectroscopy.

    Science.gov (United States)

    Takahashi, Chisato; Ueno, Kusuo; Aoyama, Junichi; Adachi, Mariko; Yamamoto, Hiromitsu

    2017-07-01

    In drug delivery systems employing polymeric nanoparticles, accurate delivery of drugs to target sites such as bacterial cells, cell tissues, and organelles is essential. In particular, when designing drug delivery systems for the treatment of the biofilm infections, evaluation of the interaction between polymeric nanoparticles and biofilm or bacterial cells using a simple technique is of significant importance. Here we develop two types of novel techniques for the biological imaging of the intracellular behavior of two types of polymeric nanoparticles, biodegradable chitosan-modified poly (dl-lactide-co-glycolide) (PLGA) nanoparticles and chitosan-modified polyvinyl caprolactam - polyvinyl acetate -polyethylene glycol graft copolymer (Soluplus®, Sol) nanoparticles, within a Staphylococcus epidermidis biofilm. As the first technique, Raman imaging of unstained biological materials using slit-scanning confocal Raman microscopy (unstained Raman imaging) was performed, and as the second, field-emission scanning electron microscopy with energy-dispersive X-ray spectroscopy analysis of biological materials labeled with quantum dots (SEM-QD imaging) was demonstrated. These analyses revealed differing localization of the respective nanoparticles within the biofilm in accordance with the specific interactions of PLGA nanoparticles and Sol nanoparticles with the biofilm. These novel techniques open the door to biological imaging and analyses with high spatial resolution, which will help to understand the efficacy of drug delivery to target materials. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Digital confocal microscopy through a multimode fiber.

    Science.gov (United States)

    Loterie, Damien; Farahi, Salma; Papadopoulos, Ioannis; Goy, Alexandre; Psaltis, Demetri; Moser, Christophe

    2015-09-07

    Acquiring high-contrast optical images deep inside biological tissues is still a challenging problem. Confocal microscopy is an important tool for biomedical imaging since it improves image quality by rejecting background signals. However, it suffers from low sensitivity in deep tissues due to light scattering. Recently, multimode fibers have provided a new paradigm for minimally invasive endoscopic imaging by controlling light propagation through them. Here we introduce a combined imaging technique where confocal images are acquired through a multimode fiber. We achieve this by digitally engineering the excitation wavefront and then applying a virtual digital pinhole on the collected signal. In this way, we are able to acquire images through the fiber with significantly increased contrast. With a fiber of numerical aperture 0.22, we achieve a lateral resolution of 1.5µm, and an axial resolution of 12.7µm. The point-scanning rate is currently limited by our spatial light modulator (20Hz).

  16. Can scanning near-field optical microscopy be compared with confocal laser scanning microscopy? A preliminary study on alpha-sarcoglycan and beta1D-integrin in human skeletal muscle.

    Science.gov (United States)

    Anastasi, G; Cutroneo, G; Pisani, A; Bruschetta, D; Milardi, D; Princi, P; Gucciardi, P G; Bramanti, P; Soscia, L; Favaloro, A

    2007-12-01

    The dystrophin-glycoprotein complex and the vinculin-talin-integrin system constitute, together a protein machinery, called costameres. The dystrophin-glycoprotein complex contains, among other proteins, also dystrophin and the sarcoglycans subcomplex, proteins playing a key role in the pathogenesis of many muscular dystrophies and linking the cytoplasmic myofibrillar contractile elements to the signal transducing molecules of the extracellular matrix, also providing structural support to the sarcolemma. The vinculin-talin-integrin system connects some components of the extracellular matrix with intermediate filaments of desmin, forming transverse bridges between Z and M lines. In our previous reports we always studied these systems by confocal laser scanning microscopy (CLSM). In this paper we report on the first applications of optical near-field fluorescence microscopy to the spatial localization of alpha-sarcoglycan and beta1D-integrin in human skeletal muscle fibres in order to better compare and test the images obtained with conventional CLSM and with scanning near-field optical microscopy (SNOM). In addition, the analysis of the surface morphology, and the comparison with the fluorescence map is put forward and analyzed for the first time on human muscle fibres. In aperture-SNOM the sample is excited through the nanometre-scale aperture produced at the apex of an optical fibre after tapering and subsequent metal coating. The acquisition of the topography map, simultaneously to the optical signal, by SNOM, permits to exactly overlap the fluorescence images obtained from the two consecutive scans needed for the double localization. Besides, the differences between the topography and the optical spatial patterns permit to assess the absence of artefacts in the fluorescence maps. Although the SNOM represented a good method of analysis, this technique remains a complementary method to the CLSM and it can be accepted in order to confirm the hypothesis advanced by

  17. Investigation of the cutaneous penetration behavior of dexamethasone loaded to nano-sized lipid particles by EPR spectroscopy, and confocal Raman and laser scanning microscopy.

    Science.gov (United States)

    Lohan, Silke B; Saeidpour, Siavash; Solik, Agnieszka; Schanzer, Sabine; Richter, Heike; Dong, Pin; Darvin, Maxim E; Bodmeier, Roland; Patzelt, Alexa; Zoubari, Gaith; Unbehauen, Michael; Haag, Rainer; Lademann, Jürgen; Teutloff, Christian; Bittl, Robert; Meinke, Martina C

    2017-07-01

    An improvement of the penetration efficiency combined with the controlled release of actives in the skin can facilitate the medical treatment of skin diseases immensely. Dexamethasone (Dx), a synthetic glucocorticoid, is frequently used for the treatment of inflammatory skin diseases. To investigate the penetration of nano-sized lipid particles (NLP) loaded with Dx in comparison to a commercially available base cream, different techniques were applied. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the penetration of Dx, which was covalently labeled with the spin probe 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA). The penetration into hair follicles was studied using confocal laser scanning microscopy (CLSM) with curcumin-loaded NLP. The penetration of the vehicle was followed by confocal Raman microscopy (CRM). Penetration studies using excised porcine skin revealed a more than twofold higher penetration efficiency for DxPCA into the stratum corneum (SC) after 24h incubation compared to 4h incubation when loaded to the NLP, whereas when applied in the base cream, almost no further penetration was observed beyond 4h. The distribution of DxPCA within the SC was investigated by consecutive tape stripping. The release of DxPCA from the base cream after 24h in deeper SC layers and the viable epidermis was shown by EPR. For NLP, no release from the carrier was observed, although DxPCA was detectable in the skin after the complete SC was removed. This phenomenon can be explained by the penetration of the NLP into the hair follicles. However, penetration profiles measured by CRM indicate that NLP did not penetrate as deeply into the SC as the base cream formulation. In conclusion, NLP can improve the accumulation of Dx in the skin and provide a reservoir within the SC and in the follicular infundibula. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. A novel method to analyse in vivo the physiological state and cell viability of phototrophic microorganisms by confocal laser scanning microscopy using a dual laser.

    Science.gov (United States)

    Millach, Laia; Obiol, Aleix; Solé, Antonio; Esteve, Isabel

    2017-10-01

    Phototrophic microorganisms are very abundant in extreme environments, where are subjected to frequent and strong changes in environmental parameters. Nevertheless, little is known about the physiological effects of these changing environmental conditions on viability of these microorganisms, which are difficult to grow in solid media and have the tendency to form aggregates. For that reason, it is essential to develop methodologies that provide data in short time consuming, in vivo and with minimal manipulating the samples, in response to distinct stress conditions. In this paper, we present a novel method using Confocal Laser Scanning Microscopy and a Dual Laser (CLSM-DL) for determining the cell viability of phototrophic microorganisms without the need of either staining or additional use of image treating software. In order to differentiate viable and nonviable Scenedesmus sp. DE2009 cells, a sequential scan in two different channels was carried out from each same xyz optical section. On the one hand, photosynthetic pigments fluorescence signal (living cells) was recorded at the red channel (625- to 785-nm fluorescence emission) exciting the samples with a 561-nm laser diode, and an acousto-optic tunable filter (AOTF) of 20%. On the other hand, nonphotosynthetic autofluorescence signal (dead cells) was recorded at the green channel (500- to 585-nm fluorescence emission) using a 405-nm UV laser, an AOTF of 15%. Both types of fluorescence signatures were captured with a hybrid detector. The validation of the CLSM-DL method was performed with SYTOX green fluorochrome and electron microscopic techniques, and it was also applied for studying the response of distinct light intensities, salinity doses and exposure times on a consortium of Scenedesmus sp. DE2009. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  19. Fungal keratitis - improving diagnostics by confocal microscopy.

    Science.gov (United States)

    Nielsen, E; Heegaard, S; Prause, J U; Ivarsen, A; Mortensen, K L; Hjortdal, J

    2013-09-01

    Introducing a simple image grading system to support the interpretation of in vivo confocal microscopy (IVCM) images in filamentous fungal keratitis. Clinical and confocal studies took place at the Department of Ophthalmology, Aarhus University Hospital, Denmark. Histopathological analysis was performed at the Eye Pathology Institute, Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark. A recent series of consecutive patients with filamentous fungal keratitis is presented to demonstrate the results from in-house IVCM. Based upon our experience with IVCM and previously published images, we composed a grading system for interpreting IVCM images of filamentous fungal keratitis. A recent case series of filamentous fungal keratitis from 2011 to 2012 was examined. There were 3 male and 3 female patients. Mean age was 44.5 years (range 12-69), 6 out of 17 (35%) cultures were positive and a total of 6/7 (86%) IVCM scans were positive. Three different categories of IVCM results for the grading of diagnostic certainty were formed. IVCM is a valuable tool for diagnosing filamentous fungal keratitis. In order to improve the reliability of IVCM, we suggest implementing a simple and clinically applicable grading system for aiding the interpretation of IVCM images of filamentous fungal keratitis.

  20. Fungal Keratitis - Improving Diagnostics by Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Esben Nielsen

    2013-12-01

    Full Text Available Purpose: Introducing a simple image grading system to support the interpretation of in vivo confocal microscopy (IVCM images in filamentous fungal keratitis. Setting: Clinical and confocal studies took place at the Department of Ophthalmology, Aarhus University Hospital, Denmark. Histopathological analysis was performed at the Eye Pathology Institute, Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark. Methods: A recent series of consecutive patients with filamentous fungal keratitis is presented to demonstrate the results from in-house IVCM. Based upon our experience with IVCM and previously published images, we composed a grading system for interpreting IVCM images of filamentous fungal keratitis. Results: A recent case series of filamentous fungal keratitis from 2011 to 2012 was examined. There were 3 male and 3 female patients. Mean age was 44.5 years (range 12-69, 6 out of 17 (35% cultures were positive and a total of 6/7 (86% IVCM scans were positive. Three different categories of IVCM results for the grading of diagnostic certainty were formed. Conclusion: IVCM is a valuable tool for diagnosing filamentous fungal keratitis. In order to improve the reliability of IVCM, we suggest implementing a simple and clinically applicable grading system for aiding the interpretation of IVCM images of filamentous fungal keratitis.

  1. Pupil engineering for a confocal reflectance line-scanning microscope

    Science.gov (United States)

    Patel, Yogesh G.; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2011-03-01

    Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current confocal point-scanning systems are large, complex, and expensive. A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner. We have developed a confocal line-scanner that combines both divided-pupil and full-pupil configurations. This combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast, signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions. Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging performance. Both results will be compared to theoretical calculations using our previously reported Fourier analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of confocal reflectance scanning microscopes for clinical and surgical dermatology.

  2. Comparative pattern of growth and development of Echinostoma paraensei (Digenea: Echinostomatidae) in hamster and Wistar rat using light and confocal laser scanning microscopy.

    Science.gov (United States)

    Souza, Joyce G R; Garcia, Juberlan S; Gomes, Ana Paula N; Machado-Silva, José Roberto; Maldonado, Arnaldo

    2017-12-01

    Echinostoma paraensei (Digenea: Echinostomatidae) lives in the duodenum and bile duct of rodents and is reported as a useful model for studies on the biology of flatworms. Here, we compared the growth and development of pre and post ovigerous worms collected 3, 7, 14 and 21 days post infection from experimentally infected hamster (permissive host) and Wistar rat (less permissive hosts). Linear measurements and ratios were examined by light (morphology and morphometry) and confocal laser scanning microscopy. At day 3, either worm from hamsters or rats were small with poorly developed gonads. At seven day, worms increased in size and morphometric differences between hosts are statistically significant after this time. In addition, adult worms (14 and 21 days of age) harvested from hamster showed developed gonads and vitelline glands laterally distributed on the body, whereas worms from rat showed atrophied reproductive system characterized by underdeveloped vitelline glands and stunted ovary. The worm rate recovery in rat decreased from 29.3% (day 7) to 20.6% (day 14) and 8% (day 21), whilst it remained around 37% in hamster. In conclusion, this is the first appointment demonstrating that low permissiveness influences the reproductive system of echinostome since the immature stages of development. The phenotypic analysis evidenced that hamster provides a more favorable microenvironment for gonads development than rat, confirming golden hamster as a permissive host, whereas Wistar rat is less permissive host. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Application of in vivo laser scanning confocal microscopy for evaluation of ocular surface diseases: lessons learned from pterygium, meibomian gland disease, and chemical burns.

    Science.gov (United States)

    Wang, Yan; Le, Qihua; Zhao, Feng; Hong, Jiaxu; Xu, Jianjiang; Zheng, Tianyu; Sun, Xinghuai

    2011-10-01

    In vivo laser scanning confocal microscopy (LSCM) has been widely used to evaluate the alterations caused by ocular surface diseases at a cellular level in the living eye. In this review, we focus on its use in the diagnosis of pterygium, meibomian gland (MG) disease, and chemical burns. Histopathologic changes occurring in pterygium can be examined in situ using in vivo LSCM. Alterations at the junction of the pterygium and the cornea, which cannot be observed in excised tissue samples, can be observed. MGs play an important role in maintaining the health of the ocular surface. Meibomian gland dysfunction (MGD) is one of the most common ocular surface diseases. The use of in vivo LSCM helps in the diagnosis of MGD and provides a way to examine the microstructure of MG acinar units and measure their size. In vivo LSCM also provides a new perspective in understanding the contribution of the MG to the health of the ocular surface. Chemical burns are one of the most common ocular injuries, and in vivo LSCM can provide images of the goblet cells on the corneal surface. This is a hallmark of limbal stem cell deficiency. The application of in vivo LSCM to assessing chemical burns requires extension, allowing for evaluation of the limbus structure and ocular surface changes after reconstructive ocular surgery.

  4. Biofilm formation on the Provox ActiValve: Composition and ingrowth analyzed by Illumina paired-end RNA sequencing, fluorescence in situ hybridization, and confocal laser scanning microscopy.

    Science.gov (United States)

    Timmermans, Adriana J; Harmsen, Hermie J M; Bus-Spoor, Carien; Buijssen, Kevin J D A; van As-Brooks, Corina; de Goffau, Marcus C; Tonk, Rudi H; van den Brekel, Michiel W M; Hilgers, Frans J M; van der Laan, Bernard F A M

    2016-04-01

    The most frequent cause of voice prosthesis failure is microbial biofilm formation on the silicone valve, leading to destruction of the material and transprosthetic leakage. The Provox ActiValve valve is made of fluoroplastic, which should be insusceptible to destruction. The purpose of this study was to determine if fluoroplastic is insusceptible to destruction by Candida species. Thirty-three dysfunctional Provox ActiValves (collected 2011-2013). Biofilm analysis was performed with Illumina paired-end sequencing (IPES), assessment of biofilm-material interaction with fluorescence in situ hybridization (FISH), and confocal laser scanning microscopy (CLSM). IPES (n = 10) showed that Candida albicans and Candida tropicalis are dominant populations on fluoroplastic and silicone. Microbial diversity is significantly lower on fluoroplastic. Lactobacillus gasseri is the prevalent bacterial strain on most voice prostheses. FISH and CLSM (n = 23): in none of the cases was ingrowth of Candida species present in the fluoroplastic. Fluoroplastic material of Provox ActiValve seems insusceptible to destruction by Candida species, which could help improve durability of voice prostheses. © 2015 Wiley Periodicals, Inc. Head Neck 38: E432-E440, 2016. © 2015 Wiley Periodicals, Inc.

  5. In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization.

    Science.gov (United States)

    Dige, Irene; Nilsson, Holger; Kilian, Mogens; Nyvad, Bente

    2007-12-01

    Confocal laser scanning microscopy (CLSM) has been employed as a method for studying intact natural biofilm. When combined with fluorescence in situ hybridization (FISH) it is possible to analyze spatial relationships and changes of specific members of microbial populations over time. The aim of this study was to perform a systematic description of the pattern of initial dental biofilm formation by applying 16S rRNA-targeted oligonucleotide probes to the identification of streptococci and other bacteria, and to evaluate the usefulness of the combination of CLSM and FISH for structural studies of bacterial populations in dental biofilm. Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by 10 individuals for 6, 12, 24 or 48 h. After intra-oral exposure the biofilms were labelled with probes against either streptococci (STR405) or all bacteria (EUB338) and analysed by CLSM. The current approach of using FISH techniques enabled differentiation of streptococci from other bacteria and determination of their spatio-temporal organization. The presence of chimney-like multilayered microcolonies with different microbial compositions demonstrated by this methodology provided information supplementary to our previous knowledge obtained by classical electron microscopic methods and increased our understanding of the structure of developing biofilms.

  6. The determination of firing distance applying a microscopic quantitative method and confocal laser scanning microscopy for detection of gunshot residue particles.

    Science.gov (United States)

    Neri, Margherita; Turillazzi, Emanuela; Riezzo, Irene; Fineschi, Vittorio

    2007-07-01

    In this study, we applied a microscopic quantitative method based on the use of sodium rhodizonate to verify the presence of residues and their distribution on the cutis of gunshot wounds. A total of 250 skin samples were selected from cases in which the manner of death (accidental, suicide, and homicide) and the shooting distance could be reliably determined. The samples were examined under a light microscope, in transmitted bright field illumination and phase contrast mode, and with confocal laser scanning microscopy. In all skin specimens the area of each histological section was directly measured by an image analysis system. Both the number and the size of powder particles were measured. The distribution of gunshot residues (GSR) in the epidermal and subepidermal layers was also analyzed. The evaluation of the microscopic entrance wounds demonstrated different findings related to the range of fire. The data derived from the evaluation of both macroscopic and microscopic features demonstrated that the amount and the spatial distribution of GSR deposits in the skin surrounding entrance wounds strictly correlate with shooting distance.

  7. Morphological aspects of Schistosoma mansoni adult worms isolated from nourished and undernourished mice: a comparative analysis by confocal laser scanning microscopy

    Directory of Open Access Journals (Sweden)

    Neves Renata Heisler

    2001-01-01

    Full Text Available Malnutrition hampers the course of schistosomiasis mansoni infection just as normal growth of adult worms. A comparative morphometric study on adult specimens (male and female recovered from undernourished (fed with a low protein diet - regional basic diet and nourished (rodent commercial laboratory food, NUVILAB white mice was performed. Tomographic images and morphometric analysis of the oral and ventral suckers, reproductive system and tegument were obtained by means of confocal laser scanning microscopy. Undernourished male specimens presented smaller morphometric values (length and width of the reproductive system (first, third and last testicular lobes and thickness of the tegument than controls. Besides that, it was demonstrated that the dorsal surface of the male worms bears large tubercles unevenly distributed, but kept grouped and flat. At the subtegumental region, vacuolated areas were detected. It was concluded that the inadequate nutritional status of the vertebrate host has a negative influence mainly in the reproductive system and topographical somatic development of male adult Schistosoma mansoni, inducing some alterations on the structure of the parasite.

  8. A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy

    Directory of Open Access Journals (Sweden)

    Flaviana Bombarda de ANDRADE

    2015-01-01

    Full Text Available Objectives To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM. Material and Methods Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B. These methods were modified in an attempt to improve the model (group C. Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalis during the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD® for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn’s tests were used to evaluate the live and dead cells rates, and the scores obtained. Results The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p0.05. The volume of live cells in group C was higher than in groups A and B (p<0.05. Conclusion The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.

  9. Comparison of fungiform taste-bud distribution among age groups using confocal laser scanning microscopy in vivo in combination with gustatory function.

    Science.gov (United States)

    Saito, Takehisa; Ito, Tetsufumi; Ito, Yumi; Manabe, Yasuhiro; Sano, Kazuo

    2016-04-01

    The aim of this study was to compare the distribution of taste buds in fungiform papillae (FP) and gustatory function between young and elderly age groups. Confocal laser scanning microscopy was used because it allows many FP to be observed non-invasively in a short period of time. The age of participants (n = 211) varied from 20 to 83 yr. The tip and midlateral region of the tongue were observed. Taste buds in an average of 10 FP in each area were counted. A total of 2,350 FP at the tongue tip and 2,592 FP in the midlateral region could be observed. The average number of taste buds was similar among all age groups both at the tongue tip and in the midlateral region. The taste function, measured by electrogustometry, among participants 20-29 yr of age was significantly lower than that in the other age groups; however, there was no difference among any other age groups in taste function. These results indicate that the peripheral gustatory system is well maintained anatomically and functionally in elderly people. © 2016 Eur J Oral Sci.

  10. Reproductive system abnormalities in Schistosoma mansoni adult worms isolated from Nectomys squamipes (Muridae: Sigmodontinae: brightfield and confocal laser scanning microscopy analysis

    Directory of Open Access Journals (Sweden)

    Neves Renata Heisler

    2003-01-01

    Full Text Available Schistosoma mansoni adult worms with genital anomalies isolated from Nectomys squamipes (Muridae: Sigmodontinae were studied by confocal laser scanning microscopy under the reflected mode. One male without testicular lobes (testicular agenesia/anorchism and two females, one with an atrophied ovary and another with 17 uterine eggs, were identified. The absence of testicular lobes occurred in a worm presenting otherwise normal male adult characteristics: tegument, tubercles and a gynaecophoric canal with spines. In both female specimens the digestive tube showed a vacuolated appearance, and the specimen with supernumerary uterine eggs exhibited a developing miracidium and an egg with a formed shell. The area of the ventral sucker was similar in both specimens however the tegument thickness, ovary and vitelline glands of the specimen with the atrophied ovary were smaller than those of the one with supernumerary eggs. These reported anomalies in the reproductive system call attention to the need to improve our understanding of genetic regulation and the possible role of environmental influences upon trematode development.

  11. Novel application of confocal laser scanning microscopy and 3D volume rendering toward improving the resolution of the fossil record of charcoal.

    Directory of Open Access Journals (Sweden)

    Claire M Belcher

    Full Text Available Variations in the abundance of fossil charcoals between rocks and sediments are assumed to reflect changes in fire activity in Earth's past. These variations in fire activity are often considered to be in response to environmental, ecological or climatic changes. The role that fire plays in feedbacks to such changes is becoming increasingly important to understand and highlights the need to create robust estimates of variations in fossil charcoal abundance. The majority of charcoal based fire reconstructions quantify the abundance of charcoal particles and do not consider the changes in the morphology of the individual particles that may have occurred due to fragmentation as part of their transport history. We have developed a novel application of confocal laser scanning microscopy coupled to image processing that enables the 3-dimensional reconstruction of individual charcoal particles. This method is able to measure the volume of both microfossil and mesofossil charcoal particles and allows the abundance of charcoal in a sample to be expressed as total volume of charcoal. The method further measures particle surface area and shape allowing both relationships between different size and shape metrics to be analysed and full consideration of variations in particle size and size sorting between different samples to be studied. We believe application of this new imaging approach could allow significant improvement in our ability to estimate variations in past fire activity using fossil charcoals.

  12. Confocal light scattering and absorption spectroscopic microscopy

    Science.gov (United States)

    Qiu, Le; Vitkin, Edward; Salahuddin, Saira; Zaman, Munir M.; Andersson, Charlotte; Freedman, Steven D.; Hanlon, Eugene B.; Itzkan, Irving; Perelman, Lev T.

    2008-04-01

    We have developed a novel optical method for observing submicron intracellular structures in living cells which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light scattering spectroscopy (LSS). CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales on the order of 100 nm. In addition, it provides not only size information but also information about the biochemical and physical properties of the cell.

  13. [Current application of confocal laser scanning microscope (CLSM) in stomatology].

    Science.gov (United States)

    Zhang, Yu-sen; Li, Ning-yi

    2007-04-01

    Confocal laser scanning microscopy is one kind of modern Hi-tech on the basis of confocal imaging which is characterized by depth discrimination capability. It has been widely used in the field of stomatology due to its great advantages of non-destructive and non-invasive optical sectioning and three-dimensional reconstruction of the vital objects, in situ and dynamic real-time observation of the tissues and cells can be performed at high resolution. This paper reviews the fundamentals of confocal imaging and the application of CLSM in the fields of dental material, caries, dentin bonding interface and other basic researches in stomatology in recent years.

  14. Deep stroma investigation by confocal microscopy

    Science.gov (United States)

    Rossi, Francesca; Tatini, Francesca; Pini, Roberto; Valente, Paola; Ardia, Roberta; Buzzonetti, Luca; Canovetti, Annalisa; Malandrini, Alex; Lenzetti, Ivo; Menabuoni, Luca

    2015-03-01

    Laser assisted keratoplasty is nowadays largely used to perform minimally invasive surgery and partial thickness keratoplasty [1-3]. The use of the femtosecond laser enables to perform a customized surgery, solving the specific problem of the single patient, designing new graft profiles and partial thickness keratoplasty (PTK). The common characteristics of the PTKs and that make them eligible respect to the standard penetrating keratoplasty, are: the preservation of eyeball integrity, a reduced risk of graft rejection, a controlled postoperative astigmatism. On the other hand, the optimal surgical results after these PTKs are related to a correct comprehension of the deep stroma layers morphology, which can help in the identification of the correct cleavage plane during surgeries. In the last years some studies were published, giving new insights about the posterior stroma morphology in adult subjects [4,5]. In this work we present a study performed on two groups of tissues: one group is from 20 adult subjects aged 59 +/- 18 y.o., and the other group is from 15 young subjects, aged 12+/-5 y.o.. The samples were from tissues not suitable for transplant in patients. Confocal microscopy and Environmental Scanning Electron Microscopy (ESEM) were used for the analysis of the deep stroma. The preliminary results of this analysis show the main differences in between young and adult tissues, enabling to improve the knowledge of the morphology and of the biomechanical properties of human cornea, in order to improve the surgical results in partial thickness keratoplasty.

  15. In situ observation of the growth of biofouling layer in osmotic membrane bioreactors by multiple fluorescence labeling and confocal laser scanning microscopy.

    Science.gov (United States)

    Yuan, Bo; Wang, Xinhua; Tang, Chuyang; Li, Xiufen; Yu, Guanghui

    2015-05-15

    Since the concept of the osmotic membrane bioreactor (OMBR) was introduced in 2008, it has attracted growing interests for its potential applications in wastewater treatment and reclamation; however, the fouling mechanisms of forward osmosis (FO) membrane especially the development of biofouling layer in the OMBR are not yet clear. Here, the fouled FO membranes were obtained from the OMBRs on days 3, 8 and 25 in sequence, and then the structure and growing rule of the biofouling layer formed on the FO membrane samples were in-situ characterized by multiple fluorescence labeling and confocal laser scanning microscopy (CLSM). CLSM images indicated that the variations in abundance and distribution of polysaccharides, proteins and microorganisms in the biofouling layer during the operation of OMBRs were significantly different. Before the 8th day, their biovolume dramatically increased. Subsequently, the biovolumes of β-d-glucopyranose polysaccharides and proteins continued increasing and leveled off after 8 days, respectively, while the biovolumes of α-d-glucopyranose polysaccharides and microorganisms decreased. Extracellular polymeric substances (EPS) played a significant role in the formation and growth of biofouling layer, while the microorganisms were seldom detected on the upper fouling layer after 3 days. Based on the results obtained in this study, the growth of biofouling layer on the FO membrane surface in the OMBR could be divided into three stages. Initially, EPS was firstly deposited on the FO membrane surface, and then microorganisms associated with EPS located in the initial depositing layer to form clusters. After that, the dramatic increase of the clusters of EPS and microorganisms resulted in the quick growth of biofouling layer during the flux decline of the OMBR. However, when the water flux became stable in the OMBR, some microorganisms and EPS would be detached from the FO membrane surface. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Analysis by confocal laser scanning microscopy of the MDPB bactericidal effect on S. mutans biofilm CLSM analysis of MDPB bactericidal effect on biofilm

    Directory of Open Access Journals (Sweden)

    Fabíola Galbiatti de Carvalho

    2012-10-01

    Full Text Available Since bacteria remain in the dentin following caries removal, restorative materials with antibacterial properties are desirable to help maintaining the residual microorganisms inactive. The adhesive system Clearfil Protect Bond (PB contains the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB in its primer, which has shown antimicrobial activity. However, its bactericidal effect against biofilm on the dentin has been little investigated. Objective: The aim of this study was to analyze by confocal laser scanning microscopy (CLSM and viable bacteria counting (CFU the MDPB bactericidal effect against S. mutans biofilm on the dentin surface. Material and methods: Bovine dentin surfaces were obtained and subjected to S. mutans biofilm formation in BHI broth supplemented with 1% (w/v sucrose for 18 h. Samples were divided into three groups, according to the primer application (n=3: Clearfil Protect Bond (PB, Clearfil SE Bond, which does not contain MDPB, (SE and saline (control group. After the biofilm formation, Live/ Dead stain was applied directly to the surface of each sample. Next, 10 µL of each primer were applied on the samples during 590 s for the real-time CLSM analysis. The experiment was conducted in triplicate. The primers and saline were also applied on the other dentin samples during 20, 90, 300 and 590 s (n=9 for each group and period evaluated and the CFU were assessed by colonies counting. Results: The results of the CLSM showed that with the Se application, although non-viable bacteria were detected at 20 s, there was no increase in their count during 590 s. In contrast, after the PB application there was a gradual increase of non-viable bacteria over 590 s. Conclusions: The quantitative analysis demonstrated a significant decrease of S. mutans CFU at 90 s PB exposure and only after 300 s of Se application. Protect Bond showed an earlier antibacterial effect than Se Bond.

  17. Fluorescence confocal polarizing microscopy: Three-dimensional ...

    Indian Academy of Sciences (India)

    Three-dimensional imaging of the director. O D LAVRENTOVICH. Chemical Physics ... cholesteric LCs. Keywords. 3D imaging; confocal microscopy; liquid crystals; dislocations. PACS Nos 07.60. ... magnetic resonance, x-ray diffraction, optical phase retardation, etc., suffer from the same deficiency: they produce only an ...

  18. Fluorescence confocal polarizing microscopy: Three-dimensional ...

    Indian Academy of Sciences (India)

    A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by fluorescence confocal polarizing microscopy (FCPM). The technique employs the property of LC to orient the fluorescent dye ...

  19. Scanning ultrafast electron microscopy

    OpenAIRE

    Yang, Ding-Shyue; Mohammed, Omar F.; Zewail, Ahmed H.

    2010-01-01

    Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for whic...

  20. Time-Lapse Förster Resonance Energy Transfer Imaging by Confocal Laser Scanning Microscopy for Analyzing Dynamic Molecular Interactions in the Plasma Membrane of B Cells.

    Science.gov (United States)

    Sohn, Hae Won; Brzostowski, Joseph

    2018-01-01

    For decades, various Förster resonance energy transfer (FRET) techniques have been developed to measure the distance between interacting molecules. FRET imaging by the sensitized acceptor emission method has been widely applied to study the dynamical association between two molecules at a nanometer scale in live cells. Here, we provide a detailed protocol for FRET imaging by sensitized emission using a confocal laser scanning microscope to analyze the interaction of the B cell receptor (BCR) with the Lyn-enriched lipid microdomain on the plasma membrane of live cells upon antigen binding, one of the earliest signaling events in BCR-mediated B cell activation.

  1. Reflectance Confocal Microscopy in Lentigo Maligna.

    Science.gov (United States)

    Gamo, R; Pampín, A; Floristán, U

    2016-12-01

    Lentigo maligna is the most common type of facial melanoma. Diagnosis is complicated, however, as it shares clinical and dermoscopic characteristics with other cutaneous lesions of the face. Reflectance confocal microscopy is an imaging technique that permits the visualization of characteristic features of lentigo maligna. These include a disrupted honeycomb pattern and pagetoid cells with a tendency to show folliculotropism. These cells typically have a dendritic morphology, although they may also appear as round cells measuring over 20μm with atypical nuclei. Poorly defined dermal papillae and atypical cells may be seen at the dermal-epidermal junction and can form bridges resembling mitochondrial structures. Other characteristic findings include junctional swelling with atypical cells located around the follicles, resembling caput medusae. Reflectance confocal microscopy is a very useful tool for diagnosing lentigo maligna. Copyright © 2016 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

  2. Application of scanning cytometry and confocal-microscopy-based image analysis for investigation the role of cytoskeletal elements during equine herpesvirus type 1 (EHV-1) infection of primary murine neurons.

    Science.gov (United States)

    Słońska, A; Cymerys, J; Godlewski, M M; Bańbura, M W

    2016-11-01

    Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the β-tubulin fibres within the neurites of infected cells. Alterations in β-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Clinical applications of corneal confocal microscopy

    Directory of Open Access Journals (Sweden)

    Mitra Tavakoli

    2008-06-01

    Full Text Available Mitra Tavakoli1, Parwez Hossain2, Rayaz A Malik11Division of Cardiovascular Medicine, University of Manchester and Manchester Royal Infirmary, Manchester, UK; 2University of Southampton, Southampton Eye Unit, Southampton General Hospital, Southampton, UKAbstract: Corneal confocal microscopy is a novel clinical technique for the study of corneal cellular structure. It provides images which are comparable to in-vitro histochemical techniques delineating corneal epithelium, Bowman’s layer, stroma, Descemet’s membrane and the corneal endothelium. Because, corneal confocal microscopy is a non invasive technique for in vivo imaging of the living cornea it has huge clinical potential to investigate numerous corneal diseases. Thus far it has been used in the detection and management of pathologic and infectious conditions, corneal dystrophies and ecstasies, monitoring contact lens induced corneal changes and for pre and post surgical evaluation (PRK, LASIK and LASEK, flap evaluations and Radial Keratotomy, and penetrating keratoplasty. Most recently it has been used as a surrogate for peripheral nerve damage in a variety of peripheral neuropathies and may have potential in acting as a surrogate marker for endothelial abnormalities.Keywords: corneal confocal microscopy, cornea, infective keratitis, corneal dystrophy, neuropathy

  4. Sealing ability of three root-end filling materials prepared using an erbium: Yttrium aluminium garnet laser and endosonic tip evaluated by confocal laser scanning microscopy

    Science.gov (United States)

    Nanjappa, A Salin; Ponnappa, KC; Nanjamma, KK; Ponappa, MC; Girish, Sabari; Nitin, Anita

    2015-01-01

    Aims: (1) To compare the sealing ability of mineral trioxide aggregate (MTA), Biodentine, and Chitra-calcium phosphate cement (CPC) when used as root-end filling, evaluated under confocal laser scanning microscope using Rhodamine B dye. (2) To evaluate effect of ultrasonic retroprep tip and an erbium:yttrium aluminium garnet (Er:YAG) laser on the integrity of three different root-end filling materials. Materials and Methods: The root canals of 80 extracted teeth were instrumented and obturated with gutta-percha. The apical 3 mm of each tooth was resected and 3 mm root-end preparation was made using ultrasonic tip (n = 30) and Er:YAG laser (n = 30). MTA, Biodentine, and Chitra-CPC were used to restore 10 teeth each. The samples were coated with varnish and after drying, they were immersed in Rhodamine B dye for 24 h. The teeth were then rinsed, sectioned longitudinally, and observed under confocal laser scanning microscope. Statistical Analysis Used: Data were analyzed using one-way analysis of variance (ANOVA) and a post-hoc Tukey's test at P ultrasonics, the difference was found to be statistically significant (P ultrasonics. PMID:26180420

  5. Fungal keratitis - improving diagnostics by confocal microscopy

    DEFF Research Database (Denmark)

    Nielsen, Esben; Heegaard, S; Prause, J U

    2013-01-01

    Purpose: Introducing a simple image grading system to support the interpretation of in vivo confocal microscopy (IVCM) images in filamentous fungal keratitis. Setting: Clinical and confocal studies took place at the Department of Ophthalmology, Aarhus University Hospital, Denmark. Histopathological...... analysis was performed at the Eye Pathology Institute, Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark. Methods: A recent series of consecutive patients with filamentous fungal keratitis is presented to demonstrate the results from in-house IVCM. Based upon our experience...... with IVCM and previously published images, we composed a grading system for interpreting IVCM images of filamentous fungal keratitis. Results: A recent case series of filamentous fungal keratitis from 2011 to 2012 was examined. There were 3 male and 3 female patients. Mean age was 44.5 years (range 12...

  6. A GRISM-based probe for spectrally encoded confocal microscopy.

    Science.gov (United States)

    Pitris, C; Bouma, B; Shiskov, M; Tearney, G

    2003-01-27

    Spectrally encoded confocal microscopy (SECM) is a novel approach for obtaining high resolution, depth-sectioned images of microstructure within turbid samples. By encoding one spatial dimension in wavelength, imaging probes can be greatly simplified compared to standard scanning confocal microscopes, potentially enabling endoscopic implementation. The use of a diffraction grating for spectral encoding, however, skews the optical axis through the probe, thus complicating the design of narrow diameter instruments. In this Letter, we describe a novel use of a single-optical-axis element based on high index-of-refraction prisms and a transmission holographic grating for the design of narrow diameter SECM devices. Confocal images obtained with a 10.0 mm probe demonstrate a transverse resolution of 1.1 microm and a field of view of 650 microm.

  7. Calcium oxalate crystal growth modification; investigations with confocal Raman microscopy

    Science.gov (United States)

    McMulkin, Calum J.; Massi, Massimiliano; Jones, Franca

    2017-06-01

    Confocal Raman Microscopy (CRM) in combination with a photophysical investigation has been employed to give insight into the interaction between calcium oxalate monohydrate (COM) and a series of tetrazole containing crystal growth modifier's (CGM's), in conjunction with characterisation of morphological changes using scanning electron and optical microscopy. The tetrazole CGM's were found to interact by surface adsorption with minimal morphological changes to the COM crystals however, significant interactions via chemisorption were observed; it was discovered that the chemisorption is sufficiently strong for aggregation of the tetrazole species to occur within the crystal during crystallisation.

  8. Modular Scanning Confocal Microscope with Digital Image Processing.

    Science.gov (United States)

    Ye, Xianjun; McCluskey, Matthew D

    2016-01-01

    In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength.

  9. Scanning ultrafast electron microscopy.

    Science.gov (United States)

    Yang, Ding-Shyue; Mohammed, Omar F; Zewail, Ahmed H

    2010-08-24

    Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability.

  10. Scanning electron, 3D-confocal and stereotactic microscopies morpho-structural analysis of antibiotic-loaded cement porosity in three different formulations.

    Science.gov (United States)

    Fenga, D; Vermiglio, G; Traina, F; Favaloro, A; Rosa, M A

    2017-01-01

    Chronic osteoarticular infections such as osteomyelitis or periprosthetic joint infection (PJI) have become a growing problem over the years. The “gold standard” in local antibiotic administration is still the antibiotic-loaded acrylic bone cement (ALABC) which is used in both prophylaxis, because it has been shown it can reduce the risk of infection and used in therapy during a “two-stage surgery” in PJI or in chronic osteomyelitis. We performed morphological analysis of three different formulations of antibiotic-loaded cement (ALABC) using techniques of light microscopy, scanning electron microscopy (SEM) and 3D immunofluorescence, in order to explain how the morphological aspects of cement could influence and modulate antibiotic elution.

  11. In vivo Confocal Microscopy Report after Lasik with Sequential Accelerated Corneal Collagen Cross-Linking Treatment

    National Research Council Canada - National Science Library

    Mazzotta, Cosimo; Balestrazzi, Angelo; Traversi, Claudio; Caragiuli, Stefano; Caporossi, Aldo

    2014-01-01

    ...) treatment combined with sequential high-fluence accelerated corneal collagen cross-linking, denominated Lasik XTra, by means of HRT II laser scanning in vivo confocal microscopy after a 6-month follow-up...

  12. Parallel detection experiment of fluorescence confocal microscopy using DMD.

    Science.gov (United States)

    Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

    2016-05-01

    Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

  13. Reflectance Confocal Microscopy for Inflammatory Skin Diseases.

    Science.gov (United States)

    Agozzino, M; Gonzalez, S; Ardigò, M

    2016-10-01

    In vivo reflectance confocal microscopy (RCM) is a relatively novel non-invasive tool for microscopic evaluation of the skin used prevalently for diagnosis and management of skin tumour. Its axial resolution, its non-invasive and easy clinical application represents the goals for a large diffusion of this technique. During the last 15 years, RCM has been demonstrated to be able to increase the sensibility and sensitivity of dermoscopy in the diagnosis of skin tumours integrating in real time clinic, dermoscopic and microscopic information useful for the definition of malignancy. Despite to date, no large comparative studies on inflammatory skin diseases has been published in the literature, several papers already showed that RCM has a potential for the evaluation of the descriptive features of the most common inflammatory skin diseases as psoriasis, lupus erythematosus, contact dermatitis and others. The aim of the application of this technique in non-neoplastic skin diseases has been prevalently focused on the possibility of clinical diagnosis confirmation, as well as therapeutic management. Moreover, the use of RCM as driver for an optimised skin biopsy has been also followed in order to reduce the number of unsuccessful histopathological examination. In this review article we describe the confocal features of the major groups of inflammatory skin disorders focusing on psoriasiform dermatitis, interface dermatitis and spongiotic dermatitis. Publicado por Elsevier España, S.L.U.

  14. Generalized vector wave theory for ultrahigh resolution confocal optical microscopy.

    Science.gov (United States)

    Yang, Ken; Xie, Xiangsheng; Zhou, Jianying

    2017-01-01

    Polarization modulation of a tightly focused beam in a confocal imaging scheme is considered for incident and collected light fields. Rigorous vector wave theory of a confocal optical microscopy is developed, which provides clear physical pictures without the requirement for fragmentary calculations. Multiple spatial modulations on polarization, phase, or amplitude of the illuminating and the detected beams can be mathematically described by a uniform expression. Linear and nonlinear excitation schemes are derived with tailored excitation and detection fields within this generalized theory, whose results show that the ultimate resolution achieved with the linear excitation can reach one-fifth of the excitation wavelength (or λ/5), while the nonlinear excitation scheme gives rise to a resolution better than λ/12 for two-photon fluorescence excitation and λ/20 for three-photon fluorescence excitation. Hence the resolution of optical microscopy with a near-infrared excitation can routinely reach sub-60 nm. In addition, simulations for confocal laser scanning microscopy are carried out with the linear excitation scheme and the fluorescent one, respectively.

  15. A confocal laser scanning microscopic study on thermoresponsive ...

    Indian Academy of Sciences (India)

    CdTe QDs composites using a fluorescence confocal laser scanning microscope. These composites have potential applications both in material science and biology. Keywords. Confocal ... of binary colloidal alloys and other soft matter systems.

  16. Organic pollutant clustered in the plant cuticular membranes: visualizing the distribution of phenanthrene in leaf cuticle using two-photon confocal scanning laser microscopy.

    Science.gov (United States)

    Li, Qingqing; Chen, Baoliang

    2014-05-06

    Plants play a key role in the transport and fate of organic pollutants. Cuticles on plant surfaces represent the first resistance for the uptake of airborne toxicants. In this study, a confocal scanning microscope enhanced with a two-photon laser was applied as a direct and noninvasive probe to explore the in situ uptake of a model pollutant, phenanthrene (PHE), into the cuticular membrane of a hypostomatic plant, Photinia serrulata. On the leaf cuticle surfaces, PHE forms clusters instead of being evenly distributed. The PHE distribution was quantified by the PHE fluorescence intensity. When PHE concentrations in water varying over 5 orders of magnitude were applied to the isolated cuticle, the accumulated PHE level by the cuticle was not vastly different, whether PHE was applied to the outer or inner side of the cuticle. Notably, PHE was found to diffuse via a channel-like pathway into the middle layer of the cuticle matrix, where it was identified to be composed of polymeric lipids. The strong affinity of PHE for polymeric lipids is a major contributor of the fugacity gradient driving the diffusive uptake of PHE in the cuticular membrane. Membrane lipids constitute important domains for hydrophobic interaction with pollutants, determining significant differentials of fugacities within the membrane microsystem. These, under unsteady conditions, contribute to enhance net transport and clustering along the z dimension. Moreover, the liquid-like state of polymeric lipids may promote mobility by enhancing the diffusion rate. The proposed "diffusive uptake and storage" function of polymeric lipids within the membrane characterizes the modality of accumulation of the hydrophobic contaminant at the interface between the plant and the environment. Assessing the capacity of fugacity of these constituents in detail will bring about knowledge of contaminant fate in superior plants with a higher level of accuracy.

  17. Automated identification of epidermal keratinocytes in reflectance confocal microscopy

    Science.gov (United States)

    Gareau, Dan

    2011-03-01

    Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6+/-2.8 μm and reflectance gradient b = 3.6+/-2.1 μm at the nuclear/cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

  18. Microelectrophoresis of Silica Rods Using Confocal Microscopy

    Science.gov (United States)

    2017-01-01

    The electrophoretic mobility and the zeta potential (ζ) of fluorescently labeled colloidal silica rods, with an aspect ratio of 3.8 and 6.1, were determined with microelectrophoresis measurements using confocal microscopy. In the case where the colloidal particles all move at the same speed parallel to the direction of the electric field, we record a xyz-stack over the whole depth of the capillary. This method is faster and more robust compared to taking xyt-series at different depths inside the capillary to obtain the parabolic flow profile, as was done in previous work from our group. In some cases, rodlike particles do not move all at the same speed in the electric field, but exhibit a velocity that depends on the angle between the long axis of the rod and the electric field. We measured the orientation-dependent velocity of individual silica rods during electrophoresis as a function of κa, where κ–1 is the double layer thickness and a is the radius of the rod associated with the diameter. Thus, we determined the anisotropic electrophoretic mobility of the silica rods with different sized double layers. The size of the double layer was tuned by suspending silica rods in different solvents at different electrolyte concentrations. We compared these results with theoretical predictions. We show that even at already relatively high κa when the Smoluchowski limiting law is assumed to be valid (κa > 10), an orientation dependent velocity was measured. Furthermore, we observed that at decreasing values of κa the anisotropy in the electrophoretic mobility of the rods increases. However, in low polar solvents with κa mobility of the rods decreased. We argue that this decrease is due to end effects, which was already predicted theoretically. When end effects are not taken into account, this will lead to strong underestimation of the experimentally determined zeta potential. PMID:28045541

  19. An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.

    Science.gov (United States)

    White, J G; Amos, W B; Fordham, M

    1987-07-01

    Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.

  20. Handheld reflectance confocal microscopy for the diagnosis of conjunctival tumors.

    Science.gov (United States)

    Cinotti, Elisa; Perrot, Jean-Luc; Labeille, Bruno; Campolmi, Nelly; Espinasse, Marine; Grivet, Damien; Thuret, Gilles; Gain, Philippe; Douchet, Catherine; Andrea, Caroline; Haouas, Maher; Cambazard, Frédéric

    2015-02-01

    To evaluate whether the handheld in vivo reflectance confocal microscopy that has been recently developed for the study of skin tumors is suitable for the diagnosis of conjunctival tumors. Prospective study, observational case series. We prospectively evaluated the reflectance confocal microscopy features of 53 conjunctival lesions clinically suspicious for tumors of 46 patients referred to the University Hospital of Saint-Etienne (France) by using the handheld device. Twenty-three lesions were excised (3 nevi, 10 melanomas, 5 squamous cell carcinoma, 2 lymphomas, and 3 pinguecula/pterygium) while the other 30, presenting no reflectance confocal microscopy malignant features, were under follow-up for at least 1 year. Clinical reflectance confocal microscopy and histologic diagnosis were compared. In vivo reflectance confocal microscopy diagnosis was in agreement with the histologic diagnosis in all cases and none of the lesions that were not excised show any clinical progression under follow-up. In vivo reflectance confocal microscopy with a handheld dermatology-dedicated microscope can play a role in the noninvasive diagnosis of conjunctival lesions. Further studies should be performed to better define the diagnostic ability of this technique. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model.

    Science.gov (United States)

    Alshehri, Jawaher M; Caballero-Lima, David; Hillarby, M Chantal; Shawcross, Susan G; Brahma, Arun; Carley, Fiona; Read, Nick D; Radhakrishnan, Hema

    2016-11-01

    Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas. Sixteen healthy ex vivo human corneas were divided into four study groups: (1) untreated control, (2) cross-linked, (3) infected with fungal spores, and (4) infected with fungal spores and then cross-linked. All infected corneas were inoculated with Fusarium oxysporum spores. The PACK-CXL procedure was performed 24 hours post inoculation for group 4. For PACK-CXL treatment, the corneas were debrided of epithelium; then 1% (wt/vol) isotonic riboflavin was applied dropwise at 5-minute intervals for 30 minutes and during the course of UV-A cross-linking for another 30 minutes. The corneas were imaged using a confocal microscope at 48 hours post inoculation, and the Fusarium hyphal volume and spore concentration were calculated. The infected and then cross-linked group had a significantly lower volume of Fusarium hyphae, compared to the infected (P = 0.001) group. In the infected and then cross-linked group there was significant inhibition of Fusarium sporulation compared with the infected (P = 0.007) group. A model of human corneal infection was successfully developed for investigation of the effects of PACK-CXL on fungal keratitis. A treatment regimen of combined UV-A/riboflavin-induced corneal cross-linking appears to be a valuable approach to inhibit the growth and sporulation of Fusarium and suppress the progression of fungal keratitis.

  2. Spatially resolved analyses of uranium species using a coupled system made up of confocal laser-scanning microscopy (CLSM) and laser induced fluorescence spectroscopy (LIFS); Ortsaufgeloeste Analyse von Uranspezies mittels einem Gekoppelten System aus Konfokaler Laser-Scanning Mikroskopie (CLSM) und Laser Induzierter Fluoreszenzspektroskopie (LIFS)

    Energy Technology Data Exchange (ETDEWEB)

    Brockmann, S. [Verein fuer Kernverfahrenstechnik und Analytik Rossendorf e.V. (VKTA), Dresden (Germany); Grossmann, K.; Arnold, T. [Helmholtz-Zentrum Dresden-Rossendorf e.V. (Germany). Inst. fuer Ressourcenoekologie

    2014-01-15

    The fluorescent properties of uranium when excited by UV light are used increasingly for spectroscope analyses of uranium species within watery samples. Here, alongside the fluorescent properties of the hexavalent oxidation phases, the tetra and pentavalent oxidation phases also play an increasingly important role. The detection of fluorescent emission spectrums on solid and biological samples using (time-resolved) laser induced fluorescence spectroscopy (TRLFS or LIFS respectively) has, however, the disadvantage that no statements regarding the spatial localisation of the uranium can be made. However, particularly in complex, biological samples, such statements on the localisation of the uranium enrichment in the sample are desired, in order to e.g. be able to distinguish between intra and extra-cellular uranium bonds. The fluorescent properties of uranium (VI) compounds and minerals can also be used to detect their localisation within complex samples. So the application of fluorescent microscopic methods represents one possibility to localise and visualise uranium precipitates and enrichments in biological samples, such as biofilms or cells. The confocal laser-scanning microscopy (CLSM) is especially well suited to this purpose. Coupling confocal laser-scanning microscopy (CLSM) with laser induced fluorescence spectroscopy (LIFS) makes it possible to localise and visualise fluorescent signals spatially and three-dimensionally, while at the same time being able to detect spatially resolved, fluorescent-spectroscopic data. This technology is characterised by relatively low detection limits from up to 1.10{sup -6} M for uranium (VI) compounds within the confocal volume. (orig.)

  3. Comparison of confocal biomicroscopy and noncontact specular microscopy for evaluation of the corneal endothelium.

    Science.gov (United States)

    Hara, Makiko; Morishige, Naoyuki; Chikama, Tai-Ichiro; Nishida, Teruo

    2003-08-01

    To compare the clinical efficacy of confocal biomicroscopy with that of noncontact specular microscopy for the evaluation of the corneal endothelium. The corneal endothelium was examined in 14 normal subjects (28 eyes) and in 6 patients (11 eyes) with Fuchs corneal endothelial dystrophy using a noncontact specular microscope (SP-2000P, Topcon, Japan) and a confocal biomicroscope (ConfoScan, Tomey, Japan). The images and the calculated densities of corneal endothelial cells obtained by the 2 techniques were compared. For normal subjects, the images of corneal endothelial cells obtained by the 2 techniques were almost identical, although the density of these cells determined by confocal biomicroscopy (2916 +/- 334 cells/mm2) was slightly higher than that determined by noncontact specular microscopy (2765 +/- 323 cells/mm2). In contrast, whereas clear images of corneal endothelial cells, allowing the determination of cell density, were obtained for all 11 eyes of the patient group by confocal biomicroscopy, clear images were obtained for only 4 of these 11 eyes (36.4%) by noncontact specular microscopy. Both noncontact specular microscopy and confocal biomicroscopy revealed the shapes and number of endothelial cells in the normal cornea. However, for corneas with Fuchs dystrophy, clear images were obtained only by confocal biomicroscopy. Confocal biomicroscopy is thus an effective tool for evaluation of the diseased corneal endothelium.

  4. Vancomycin sorption on activated sludge Gram(+) bacteria rather than on EPS; 3D Confocal Laser Scanning Microscopy time-lapse imaging.

    Science.gov (United States)

    Louvet, J N; Carrion, C; Stalder, T; Alrhmoun, M; Casellas, M; Potier, O; Pons, M N; Dagot, C

    2017-11-01

    Antibiotics-bacteria interactions depend on antibiotic concentration at the scale of bacteria. This study investigates how vancomycin penetrates into activated sludge flocs and can be sorbed on the bacteria and extracellular polymeric substances (EPS). The 3D structure of flocs was imaged using EPS autofluorescence. The green fluorescent BODIPY(®) FL vancomycin was introduced in a microscopic chamber containing activated sludge and penetration of vancomycin into the flocs by diffusion was observed using time-lapse microscopy. The penetration depended on the floc structure, as long and large pores could go through the whole flocs making preferential path. The antibiotic concentration into the flocs was also found to depend on the sorption rate. BODIPY(®) FL vancomycin was found to bind preferentially into Gram(+) bacteria than on EPS. The vancomycin adsorption constant on bacteria according to the linear adsorption model, Kdbacteria was estimated to be 5 times higher (SD 2.6) than the adsorption constant on EPS KdEPS. These results suggest that antibiotic removal by sorption into wastewater treatment plants could change according to the amount of bacteria in the sludge. Moreover, antibiotic concentration at the scale of bacteria could be significantly higher than the concentration in the bulk solution and this should be taken into account when studying antibiotic activity or biodegradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. UNC Pembroke Laser Scanning Confocal Microscopy Facility

    Science.gov (United States)

    2016-04-29

    decision, unless so designated by other documentation. 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS (ES) U.S. Army Research Office P.O. Box 12211...compare to histology images and to maximize data obtained from small groups of blast and toxin-treated samples, and 3) a guillotine and freezer for

  6. Chromatic confocal microscopy using staircase diffractive surface.

    Science.gov (United States)

    Rayer, Mathieu; Mansfield, Daniel

    2014-08-10

    A chromatic confocal microscope (CCM) is a high-dynamic-range noncontact distance measurement sensor; it is based on a hyperchromatic lens. The vast majority of commercial CCMs use refractive-based chromatic dispersion to chromatically code the optical axis. This approach significantly limits the range of applications and performance of the CCM. In order to be a suitable alternative to a laser triangulation gauge and laser encoder, the performance of the CCM must be improved. In this paper, it is shown how hybrid aspheric diffractive (HAD) lenses can bring the CCM to its full potential by increasing the dynamic range by a factor of 2 and the resolution by a factor of 5 while passively athermizing and increasing the light throughput efficiency of the optical head [M. Rayer, U.S. patent 1122052.2 (2011)]. The only commercially suitable manufacturing process is single-point diamond turning. However, the optical power carried by the diffractive side of a hybrid aspheric diffractive lens is limited by the manufacturing process. A theoretical study of manufacturing losses has revealed that the HAD configuration with the highest diffraction efficiency is for a staircase diffractive surface (SDS). SDS lenses have the potential to reduce light losses associated with manufacturing limits by a factor of 5 without increasing surface roughness, allowing scalar diffraction-limited optical design with a diffractive element.

  7. A New Multichannel Spectral Imaging Laser Scanning Confocal Microscope

    Directory of Open Access Journals (Sweden)

    Yunhai Zhang

    2013-01-01

    Full Text Available We have developed a new multichannel spectral imaging laser scanning confocal microscope for effective detection of multiple fluorescent labeling in the research of biological tissues. In this paper, the design and key technologies of the system are introduced. Representative results on confocal imaging, 3-dimensional sectioning imaging, and spectral imaging are demonstrated. The results indicated that the system is applicable to multiple fluorescent labeling in biological experiments.

  8. Scanning quantum decoherence microscopy.

    Science.gov (United States)

    Cole, Jared H; Hollenberg, Lloyd C L

    2009-12-09

    The use of qubits as sensitive nanoscale magnetometers has been studied theoretically and recently demonstrated experimentally. In this paper we propose a new concept, in which a scanning two-state quantum system is used to probe a sample through the subtle effects of decoherence. Mapping both the Hamiltonian and decoherence properties of a qubit simultaneously provides a unique image of the magnetic (or electric) field properties at the nanoscale. The resulting images are sensitive to the temporal as well as spatial variation in the fields created by the sample. As examples we theoretically study two applications; one from condensed matter physics, the other biophysics. The individual components required to realize the simplest version of this device (characterization and measurement of qubits, nanoscale positioning) have already been demonstrated experimentally.

  9. Confocal reference free traction force microscopy

    OpenAIRE

    Bergert, Martin; Lendenmann, Tobias; Z?ndel, Manuel; Ehret, Alexander E.; Panozzo, Daniele; Richner, Patrizia; Kim, David K.; Kress, Stephan J. P.; Norris, David J.; Sorkine-Hornung, Olga; Mazza, Edoardo; Poulikakos, Dimos; Ferrari, Aldo

    2016-01-01

    The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Traction force microscopy (TFM) enables determination of the actuating forces. Despite progress, important limitations with intrusion effects in low resolution 2D pillar-based methods or disruptive intermediate steps of cell removal and substrate relaxation in high-resolution continuum TFM methods need to be overcome. Here ...

  10. Dynamic experimentation on the confocal laser scanning microscope : application to soft-solid, composite food materials

    NARCIS (Netherlands)

    Plucknett, K.P.; Pomfret, S.J.; Normand, V.; Ferdinando, D.; Veerman, C.; Frith, W.J.; Norton, I.T.

    2001-01-01

    Confocal laser scanning microscopy (CLSM) is used to follow the dynamic structural evolution of several phase-separated mixed biopolymer gel composites. Two protein/polysaccharide mixed gel systems were examined: gelatin/maltodextrin and gelatin/agarose. These materials exhibit 'emulsion-like'

  11. In vivo confocal microscopy for the detection of canine fungal keratitis and monitoring of therapeutic response.

    Science.gov (United States)

    Ledbetter, Eric C; Norman, Mary L; Starr, Jennifer K

    2016-05-01

    To describe in vivo corneal confocal microscopy of dogs during the clinical course of fungal keratitis and correlate findings with clinical evaluations and an ex vivo experimental canine fungal keratitis model. Seven dogs with naturally acquired fungal keratitis and ex vivo canine corneas experimentally infected with clinical fungal isolates. Dogs with naturally acquired fungal keratitis were examined by in vivo laser scanning confocal microscopy. Initial confocal microscopic examinations were performed to assist in establishing the diagnosis of fungal keratitis. Serial confocal microscopic examinations were performed to guide antifungal chemotherapy. Confocal microscopy images of canine corneal fungal isolates were obtained by examination of experimentally infected ex vivo canine corneas to corroborate in vivo findings. Fungi cultured and detected by PCR from canine corneal samples included Candida albicans, Fusarium incarnatum-equiseti, Malassezia pachydermatis, and a Rhodotorula sp. Linear, branching, interlocking, hyperreflective structures were detected by confocal microscopy in dogs with filamentous fungal keratitis and round to oval hyperreflective structures were detected in dogs with yeast fungal keratitis. Antifungal chemotherapy was associated with a progressive reduction in the distribution and density of corneal fungal elements, alterations to fungal morphology, decreased leukocyte numbers, restoration of epithelial layers, and an increased number of visible keratocyte nuclei. No dogs had a recurrence of fungal keratitis following medication discontinuation. Confocal microscopic fungal morphologies were similar between in vivo and ex vivo examinations. In vivo corneal confocal microscopy is a rapid method of diagnosing fungal keratitis in dogs and provides a noninvasive mechanism for monitoring therapeutic response. © 2015 American College of Veterinary Ophthalmologists.

  12. A novel method for enhancing the lateral resolution and image SNR in confocal microscopy

    Science.gov (United States)

    Chen, Youhua; Zhu, Dazhao; Fang, Yue; Kuang, Cuifang; Liu, Xu

    2017-12-01

    There is always a tradeoff between the resolution and the signal-to-noise ratio (SNR) in confocal microscopy. In particular, the pinhole size is very important for maintaining a balance between them. In this paper, we propose a method for improving the lateral resolution and image SNR in confocal microscopy without making any changes to the hardware. By using the fluorescence emission difference (FED) approach, we divide the images acquired by different pinhole sizes into one image acquired by the central pinhole and several images acquired by ring-shaped pinholes. Then, they are added together with the deconvolution method. Simulation and experimental results for fluorescent particles and cells show that our method can achieve a far better resolution than a large pinhole and a higher SNR than a small pinhole. Moreover, our method can improve the performance of classic confocal laser scanning microscopy (CLSM) to a certain extent, especially CLSM with a continuously variable pinhole.

  13. Adaptive optics in digital micromirror based confocal microscopy

    NARCIS (Netherlands)

    Pozzi, P.; Wilding, D.; Soloviev, O.A.; Vdovine, G.V.; Verhaegen, M.H.G.; Bifano, Thomas G.; Kubby, Joel; Gigan, Sylvain

    2016-01-01

    This proceeding reports early results in the development of a new technique for adaptive optics in confocal microscopy. The term adaptive optics refers to the branch of optics in which an active element in the optical system is used to correct inhomogeneities in the media through which light

  14. Confocal microscopy-based goniometry of barnacle cyprid permanent adhesive.

    Science.gov (United States)

    Aldred, Nick; Gohad, Neeraj V; Petrone, Luigi; Orihuela, Beatriz; Liedberg, Bo; Ederth, Thomas; Mount, Andrew; Rittschof, Dan; Clare, Anthony S

    2013-06-01

    Biological adhesives are materials of particular interest in the fields of bio-inspired technology and antifouling research. The adhesive of adult barnacles has received much attention over the years; however, the permanent adhesive of the cyprid - the colonisation stage of barnacles - is a material about which very little is presently known. We applied confocal laser-scanning microscopy to the measurement of contact angles between the permanent adhesive of barnacle cyprid larvae and self-assembled monolayers of OH- and CH3-terminated thiols. Measurement of contact angles between actual bioadhesives and surfaces has never previously been achieved and the data may provide insight into the physicochemical properties and mechanism of action of these functional materials. The adhesive is a dual-phase system post-secretion, with the behaviour of the components governed separately by the surface chemistry. The findings imply that the cyprid permanent adhesion process is more complex than previously thought, necessitating broad re-evaluation of the system. Improved understanding will have significant implications for the production of barnacle-resistant coatings as well as development of bio-inspired glues for niche applications.

  15. Conversion of biocytin labelled cells and structures for the confocal laser-scanning method.

    Science.gov (United States)

    Hilbig, H; Müller, A

    1997-05-23

    The method for converting biocytin preparations of brain sections fills a gap in the application of confocal laser-scanning microscopy. Both neuronal and non-neuronal structures are converted. The background remains free of staining. The protocol can be applied to old and already existing biocytin-(diaminobenzidine)-nickel preparations which are then made accessible to evaluation with the laser-scanning microscope by the substitution of nickel with silver-gold. Sodium thiosulphate is used to remove the unbound silver. The reflection image of the laser-scanning microscopy provides more information than the transmission image.

  16. Microscopia confocal de barrido láser y su relación con la morfología de la bula de filtración Confocal laser scanning microscopy and its association to the morphology of the filtering bleb

    Directory of Open Access Journals (Sweden)

    María Teresa Ferrer Guerra

    2012-01-01

    Full Text Available Objetivo: describir los hallazgos de la microscopia confocal de barrido láser y su relación con la morfología de la bula de filtración. Métodos: estudio observacional descriptivo de corte transversal en 100 ojos. Se les realizó microscopia confocal de barrido láser con el HRT II y el módulo corneal de Rostock. Se aplicó la prueba de significación estadística de Mc. Nemar para una p=0,05. Resultados: predominó el grupo entre 61 y 80 años de edad (55,8 % y el color de la piel blanca (46,8 %. En el grupo de descenso de la presión intraocular en más del 30 % se ubicó la mayor cantidad de bulas de todos los tamaños. Las bulas de mediano tamaño fueron las que más disminuyeron las cifras de presión intraocular, con 24 ojos (55,8 %, p=0,00, seguidas por las de pequeño tamaño (p=0,14. Las bulas de filtración aplanadas fueron las más frecuentes en 55 % de los casos (p=0,00, y 67,4 % de estas se ubicaron en el grupo de descenso de presión intraocular de más de 30 % (p=0,01. Las bulas medianas presentaron la mayor cantidad de estroma en malla porosa (60 % y de microquistes epiteliales (56 % p=0,00. Conclusiones: la configuración aplanada y el tamaño mediano de la bula de filtración se relacionaron con la presencia de variables histológicas que infieren buen funcionamiento de la bula (microquistes epiteliales y estroma en malla porosa. También se relacionaron con un mayor descenso de la presión intraocular.Objective: to describe the findings in the confocal laser scanning microscopy and its relation with the morphology of the filtering bleb. Methods: cross-sectional, observational and descriptive study of 100 eyes that underwent confocal laser scanning microscopy with the Retinal Heidelberg Tomography and the Rostock corneal module. Mc Nemar’s statistical significance test was made to obtain p=0,05. Results: the 61-80 years old age group (55,8 % and the caucasians predominated. The group of eyes with over 30 % decrease of

  17. Confocal microscopy for astrocyte in vivo imaging: Recycle and reuse in microscopy

    Science.gov (United States)

    Pérez-Alvarez, Alberto; Araque, Alfonso; Martín, Eduardo D.

    2013-01-01

    In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo. PMID:23658537

  18. Vacuum scanning capillary photoemission microscopy

    DEFF Research Database (Denmark)

    Aseyev, S.A.; Cherkun, A P; Mironov, B N

    2017-01-01

    We demonstrate the use of a conical capillary in a scanning probe microscopy for surface analysis. The probe can measure photoemission from a substrate by transmitting photoelectrons along the capillary as a function of probe position. The technique is demonstrated on a model substrate consisting...

  19. High Resolution Scanning Ion Microscopy

    NARCIS (Netherlands)

    Castaldo, V.

    2011-01-01

    The structure of the thesis is the following. The first chapter is an introduction to scanning microscopy, where the path that led to the Focused Ion Beam (FIB) is described and the main differences between electrons and ion beams are highlighted. Chapter 2 is what is normally referred to (which I

  20. Concepts in Imaging and Microscopy: Exploring Biological Structure and Function with Confocal Microscopy

    National Research Council Canada - National Science Library

    Michael Dailey; Glen Marrs; Jakob Satz; Marc Waite

    1999-01-01

    .... The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus providing sharp, high-contrast images of cells and subcellular structures within thick samples...

  1. Three-Dimensional Visualization of Interfacial Phenomena Using Confocal Microscopy

    Science.gov (United States)

    Shieh, Ian C.

    Surfactants play an integral role in numerous functions ranging from stabilizing the emulsion in a favorite salad dressing to organizing the cellular components that make life possible. We are interested in lung surfactant, which is a mixture of lipids and proteins essential for normal respiration because it modulates the surface tension of the air-liquid interface of the thin fluid lining in the lungs. Through this surface tension modulation, lung surfactant ensures effortless lung expansion and prevents lung collapse during exhalation, thereby effecting proper oxygenation of the bloodstream. The function of lung surfactant, as well as numerous interfacial lipid systems, is not solely dictated by the behavior of materials confined to the two-dimensional interface. Rather, the distributions of materials in the liquid subphase also greatly influence the performance of interfacial films of lung surfactant. Therefore, to better understand the behavior of lung surfactant and other interfacial lipid systems, we require a three-dimensional characterization technique. In this dissertation, we have developed a novel confocal microscopy methodology for investigating the interfacial phenomena of surfactants at the air-liquid interface of a Langmuir trough. Confocal microscopy provides the excellent combination of in situ, fast, three-dimensional visualization of multiple components of the lung surfactant system that other characterization techniques lack. We detail the solutions to the numerous challenges encountered when imaging a dynamic air-liquid interface with a high-resolution technique like confocal microscopy. We then use confocal microscopy to elucidate the distinct mechanisms by which a polyelectrolyte (chitosan) and nonadsorbing polymer (polyethylene glycol) restore the function of lung surfactant under inhibitory conditions mimicking the effects of lung trauma. Beyond this physiological model, we also investigate several one- and two-component interfacial films

  2. Comparison of Noncontact Specular and Confocal Microscopy for Evaluation of Corneal Endothelium.

    Science.gov (United States)

    Huang, Jianyan; Maram, Jyotsna; Tepelus, Tudor C; Sadda, Srinivas R; Chopra, Vikas; Lee, Olivia L

    2017-03-24

    To compare endothelial cell analysis obtained by noncontact specular and confocal microscopy, using the Konan NSP-9900 and Nidek ConfoScan4 systems, respectively. Three groups including 70 healthy eyes, 49 eyes with Fuchs endothelial corneal dystrophy (FECD), and 78 eyes with glaucoma were examined with both the Konan NSP-9900 specular microscope and the Nidek ConfocScan4 confocal microscope. Certified graders at the Doheny Image Reading Center compared corneal endothelial images from both instruments side by side to assess image quality. Endothelial cell density (ECD) measurements were calculated and compared using three different modalities: (1) each instrument's fully automated analysis; (2) each instrument's semiautomatic analysis with grader input; and (3) manual grading methods by certified grader. All normal eyes yielded gradable endothelial images, and most but not all glaucomatous eyes yielded images with high enough image quality to allow grading. In addition, in corneas with severe FECD, poor image quality precluded ECD grading by specular microscopy in 20 eyes (40.8%) but in only 4 (8.2%) confocal images from the same eyes. For the gradable images, the ECD values obtained using the manual grading method from either device were comparable with no statistically significant difference (P>0.05) between specular and confocal devices. Machine-generated ECD values were significantly different from manual results, measuring greater in all cases with specular microscopy. Machine-generated ECD values from confocal microscopy also differed significantly from manual determinations, but not in a consistent direction. Semiautomatic methods for both instruments obtained clinically acceptable ECD values. Automatic machine-generated ECD measurements differed significantly from manual assessments of corneal endothelium by both specular and confocal microscopy, suggesting that automated results should be used with caution. But ECD values derived manually were comparable

  3. Confocal microscopy on the beamline: novel three-dimensional imaging and sample positioning

    OpenAIRE

    Khan, I.; Gillilan, R; Kriksunov, I.; Williams, R.; Zipfel, W.R.; Englich, U.

    2012-01-01

    Possibilities of applying confocal microscopy on an X-ray beamline have been explored. Confocal microscopy images have the potential to give detailed, on-axis and three-dimensional views of protein crystals on a synchrotron beamline.

  4. Microscopia confocal en operados de queratoplastia perforante Confocal microscopy in patients operated from penetrating keratoplasty

    Directory of Open Access Journals (Sweden)

    Zulema Gómez Castillo

    2009-06-01

    Full Text Available La microscopia confocal es un examen exploratorio, práctico y poco invasivo que permite conocer las características microscópicas del tejido corneal después del trasplante, por lo que constituye una herramienta muy útil en el manejo de los pacientes operados de queratoplastia. El presente trabajo tiene como finalidad describir las características del tejido corneal en pacientes operados de este tipo de trasplante, mediante la microscopia confocal in vivo. MÉTODOS: Se realizó un estudio descriptivo, de corte transversal, en 40 ojos de 40 pacientes operados de queratoplastia perforante, en el Servicio de Córnea del Instituto Cubano de Oftalmología "Ramón Pando Ferrer", de marzo de 2006 a marzo de 2007. Se confeccionó una historia clínica oftalmológica y se les realizó a todos el examen de microscopia confocal en el injerto corneal con el microscopio confocal CONFOSCAN 4. RESULTADOS: La queratopatía bullosa pseudofáquica fue la afección más frecuente previa a la cirugía y estuvo presente en el 77,5 % de los pacientes. En el 72,5 % de los intervenidos se encontró una disminución del grosor corneal. El epitelio presentó alteraciones en el 62,5 % de los pacientes. Todos presentaron afectación de la forma y el tamaño celular endotelial. En el 82,5 % de los pacientes se observó ausencia de plexos nerviosos. CONCLUSIONES: La microscopia confocal como nueva ciencia en el campo de la oftalmología, favorece el seguimiento evolutivo de las queratoplastias perforantes y con esto no solo a prevenir la aparición de posibles complicaciones, sino además de garantizar el éxito de la cirugía y la función refractiva de la córnea.Confocal microscopy is a practical, exploratory and less invassive examination that allows finding out the microscopic characteristics of the corneal tissue after transplantation, so it is a very useful tool for the management of patients operated from keratoplasty. The present paper was aimed at describing

  5. A Video Rate Confocal Laser Beam Scanning Light Microscope Using An Image Dissector

    Science.gov (United States)

    Goldstein, Seth R.; Hubin, Thomas; Rosenthal, Scott; Washburn, Clayton

    1989-12-01

    A video rate confocal reflected light microscope with no moving parts has been developed. Return light from an acousto-optically raster scanned laser beam is imaged from the microscope stage onto the photocathode of an Image Dissector Tube (IDT). Confocal operation is achieved by appropriately raster scanning with the IDT x and y deflection coils so as to continuously "sample" that portion of the photocathode that is being instantaneously illuminated by the return image of the scanning laser spot. Optimum IDT scan parameters and geometric distortion correction parameters are determined under computer control within seconds and are then continuously applied to insure system alignment. The system is operational and reflected light images from a variety of objects have been obtained. The operating principle can be extended to fluorescence and transmission microscopy.

  6. In vivo confocal microscopy in goldenhar syndrome: a case report.

    Science.gov (United States)

    Triolo, Giacinto; Ferrari, Giulio; Doglioni, Claudio; Rama, Paolo

    2013-10-16

    Goldenhar Syndrome is characterized by malformations of multiple anatomical districts. Between these, bulbar dermoids are common and represent a significant clinical problem as they can affect both ocular function and aesthetic comfort.Histologic characterization of dermoids has been extensively performed; however, no reports exist describing in vivo confocal microscopy (IVCM) of these lesions. We aimed to (i) describe the in vivo confocal morphology of limbal dermoids in Goldenhar syndrome and (ii) compare these findings with standard light microscopy. A 15-year-old Caucasian female affected by Goldenhar Syndrome showed a left, infero-temporal, limbal neoformation, with extension to the left orbital region. Prior to surgical removal, IVCM was performed with the Heidelberg Retina Tomograph II, Cornea Module, using the "section" modality. The IVCM sections showed structures resembling corneal epithelium and vascular structures. Surgical removal of the lesion was decided as it caused poor eyelid closure. After surgical removal, sectioning and standard optical microscopy were performed. The comparison between IVCM imaging and standard microscopy sections were highly correlated in the detection of the pilar and vascular structures. This study showed that IVCM may be a useful technique to study limbal dermoids, given its ability to detect typical microscopic features and its comparability to optical microscopy, which is the current standard.

  7. A simple way to identify non-viable cells within living plant tissue using confocal microscopy

    Directory of Open Access Journals (Sweden)

    Truernit Elisabeth

    2008-06-01

    Full Text Available Abstract Background Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP fluorescence was possible. Conclusion The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy.

  8. Confocal microscopy patterns in nonmelanoma skin cancer and clinical applications.

    Science.gov (United States)

    González, S; Sánchez, V; González-Rodríguez, A; Parrado, C; Ullrich, M

    2014-06-01

    Reflectance confocal microscopy is currently the most promising noninvasive diagnostic tool for studying cutaneous structures between the stratum corneum and the superficial reticular dermis. This tool gives real-time images parallel to the skin surface; the microscopic resolution is similar to that of conventional histology. Numerous studies have identified the main confocal features of various inflammatory skin diseases and tumors, demonstrating the good correlation of these features with certain dermatoscopic patterns and histologic findings. Confocal patterns and diagnostic algorithms have been shown to have high sensitivity and specificity in melanoma and nonmelanoma skin cancer. Possible present and future applications of this noninvasive technology are wide ranging and reach beyond its use in noninvasive diagnosis. This tool can also be used, for example, to evaluate dynamic skin processes that occur after UV exposure or to assess tumor response to noninvasive treatments such as photodynamic therapy. We explain the characteristic confocal features found in the main nonmelanoma skin tumors and discuss possible applications for this novel diagnostic technique in routine dermatology practice. Copyright © 2012 Elsevier España, S.L. and AEDV. All rights reserved.

  9. CONFOCAL MICROSCOPY STUDY OF BIOLOGICAL PECULIARITIES OF SCAFFOLD MADE FROM RECOMBINANT SPIDER SILK

    Directory of Open Access Journals (Sweden)

    O. L. Pustovalova

    2009-01-01

    Full Text Available We studied the viability and dynamic of cell distribution during long-term cultivation of broblasts 3T3 in spider silk spidroin 1-based scaffold. Laser scanning confocal microscopy is shown to have advantages for visualization of cells situated on the external and internal surfaces of scaffold. Fibroblasts maintain high proliferative ability and viability during long term cultivation. Spidroin 1-based scaffold are the perspective materials for bioengineering. 

  10. In vivo confocal microscopy in dermatology: from research to clinical application

    Science.gov (United States)

    Ulrich, Martina; Lange-Asschenfeldt, Susanne

    2013-06-01

    Confocal laser scanning microscopy (CLSM) represents an emerging technique for the noninvasive histomorphological analysis of skin in vivo and has shown its applicability for dermatological research as well as its value as an adjunct tool in the clinical management of skin cancer patients. Herein, we aim to give an overview on the current clinical indications for CLSM in dermatology and also highlight the diverse applications of CLSM in dermatological research.

  11. Line-scanning confocal microendoscope for nuclear morphometry imaging

    Science.gov (United States)

    Tang, Yubo; Carns, Jennifer; Richards-Kortum, Rebecca R.

    2017-11-01

    Fiber-optic endomicroscopy is a minimally invasive method to image cellular morphology in vivo. Using a coherent fiber bundle as an image relay, it allows additional imaging optics to be placed at the distal end of the fiber outside the body. In this research, we use this approach to demonstrate a compact, low-cost line-scanning confocal fluorescence microendoscope that can be constructed for pathological conditions.

  12. Imaging inflammation in mouse colon using a rapid stage-scanning confocal fluorescence microscope.

    Science.gov (United States)

    Saldua, Meagan A; Olsovsky, Cory A; Callaway, Evelyn S; Chapkin, Robert S; Maitland, Kristen C

    2012-01-01

    Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333 lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7 mm/sec. A single 1 × 60 mm(2) field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion.

  13. Full-field interferometric confocal microscopy using a VCSEL array.

    Science.gov (United States)

    Redding, Brandon; Bromberg, Yaron; Choma, Michael A; Cao, Hui

    2014-08-01

    We present an interferometric confocal microscope using an array of 1200 vertical cavity surface emitting lasers (VCSELs) coupled to a multimode fiber. Spatial coherence gating provides ~18,000 continuous virtual pinholes, allowing an entire en face plane to be imaged in a snapshot. This approach maintains the same optical sectioning as a scanning confocal microscope without moving parts, while the high power of the VCSEL array (∼5  mW per laser) enables high-speed image acquisition with integration times as short as 100 μs. Interferometric detection also recovers the phase of the image, enabling quantitative phase measurements and improving the contrast when imaging phase objects.

  14. Scanning electron microscopy of bone.

    Science.gov (United States)

    Boyde, Alan

    2012-01-01

    This chapter described methods for Scanning Electron Microscopical imaging of bone and bone cells. Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. This chapter considers preparing and imaging samples of unembedded bone having 3D detail in a 3D surface, topography-free, polished or micromilled, resin-embedded block surfaces, and resin casts of space in bone matrix. The chapter considers methods for fixation, drying, looking at undersides of bone cells, and coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralised matrix is described in detail. Attention is given especially to methods for 3D BSE SEM imaging of bone samples and recommendations for the types of resin embedding of bone for BSE imaging are given. Correlated confocal and SEM imaging of PMMA-embedded bone requires the use of glycerol to coverslip. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualising fluorescent mineralising front labels such as calcein and tetracyclines. Making spatial casts from PMMA or other resin embedded samples is an important use of this material. Correlation with other imaging means, including microradiography and microtomography is important. Shipping wet bone samples between labs is best done in glycerol. Environmental SEM (ESEM, controlled vacuum mode) is valuable in eliminating -"charging" problems which are common with complex, cancellous bone samples.

  15. Atherosclerotic plaque detection by confocal Brillouin and Raman microscopies

    Science.gov (United States)

    Meng, Zhaokai; Basagaoglu, Berkay; Yakovlev, Vladislav V.

    2015-02-01

    Atherosclerosis, the development of intraluminal plaque, is a fundamental pathology of cardiovascular system and remains the leading cause of morbidity and mortality worldwide. Biomechanical in nature, plaque rupture occurs when the mechanical properties of the plaque, related to the morphology and viscoelastic properties, are compromised, resulting in intraluminal thrombosis and reduction of coronary blood flow. In this report, we describe the first simultaneous application of confocal Brillouin and Raman microscopies to ex-vivo aortic wall samples. Such a non-invasive, high specific approach allows revealing a direct relationship between the biochemical and mechanical properties of atherosclerotic tissue.

  16. Multifunctional scanning ion conductance microscopy.

    Science.gov (United States)

    Page, Ashley; Perry, David; Unwin, Patrick R

    2017-04-01

    Scanning ion conductance microscopy (SICM) is a nanopipette-based technique that has traditionally been used to image topography or to deliver species to an interface, particularly in a biological setting. This article highlights the recent blossoming of SICM into a technique with a much greater diversity of applications and capability that can be used either standalone, with advanced control (potential-time) functions, or in tandem with other methods. SICM can be used to elucidate functional information about interfaces, such as surface charge density or electrochemical activity (ion fluxes). Using a multi-barrel probe format, SICM-related techniques can be employed to deposit nanoscale three-dimensional structures and further functionality is realized when SICM is combined with scanning electrochemical microscopy (SECM), with simultaneous measurements from a single probe opening up considerable prospects for multifunctional imaging. SICM studies are greatly enhanced by finite-element method modelling for quantitative treatment of issues such as resolution, surface charge and (tip) geometry effects. SICM is particularly applicable to the study of living systems, notably single cells, although applications extend to materials characterization and to new methods of printing and nanofabrication. A more thorough understanding of the electrochemical principles and properties of SICM provides a foundation for significant applications of SICM in electrochemistry and interfacial science.

  17. Cellular resolution expression profiling using confocal detection of NBT/BCIP precipitate by reflection microscopy.

    Science.gov (United States)

    Jékely, Gáspár; Arendt, Detlev

    2007-06-01

    The determination of gene expression patterns in three dimensions with cellular resolution is an important goal in developmental biology. However the most sensitive, efficient, and widely used staining technique for whole-mount in situ hybridization (WMISH), nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase, could not yet be combined with the most precise, high-resolution detection technique, confocal laser-scanning microscopy (CLSM). Here we report the efficient visualization of the NBT/BCIP precipitate using confocal reflection microscopy for WMISH samples of Drosophila, zebrafish, and the marine annelid worm, Platynereis dumerilii. In our simple WMISH protocol for reflection CLSM, NBT/BCIP staining can be combined with fluorescent WMISH, immunostainings, or transgenic green fluorescent protein (GFP) marker lines, allowing double labeling of cell types or of embryological structures of interest. Whole-mount reflection CLSM will thus greatly facilitate large-scale cellular resolution expression profiling in vertebrate and invertebrate model organisms.

  18. Assessment of corneal alterations by confocal microscopy in vernal keratoconjunctivitis.

    Science.gov (United States)

    Nebbioso, Marcella; Zicari, Anna Maria; Lollobrigida, Valeria; Marenco, Marco; Duse, Marzia

    2015-01-01

    Vernal keratoconjunctivitis (VKC) is a bilateral chronic, seasonally exacerbated inflammation of the ocular surface that especially affects male children and young boys. To evaluate the corneal microscopic features of patients affected by VKC and to assess whether some corneal changes were associated with specific ocular symptoms and/or signs. 20 children aged between 4 and 14 years were enrolled. All patients underwent corneal confocal microscopy by Confoscan CS3 (Nidek). 350 images of the central cornea of each eye were obtained with a ×40 noncontact lens 3,5 micron gap in automode. Some alterations of the sub-basal and stromal corneal nerves were detected. These alterations were more evident in patients with higher severity of photophobia. On the other hand, there were scarce other signs of the anterior segment of the eye. Our preliminary findings show that there is another group of patients affected by VKC, characterized by an intense photophobia caused by corneal damage and without other significant ocular alterations. Therefore confocal microscopy may be useful for an early identification of corneal alterations before the onset of severe ocular symptoms and to set an appropriate therapeutic management.

  19. Scanning Electrochemical Microscopy in Neuroscience

    Science.gov (United States)

    Schulte, Albert; Nebel, Michaela; Schuhmann, Wolfgang

    2010-07-01

    This article reviews recent work involving the application of scanning electrochemical microscopy (SECM) to the study of individual cultured living cells, with an emphasis on topographical and functional imaging of neuronal and secretory cells of the nervous and endocrine system. The basic principles of biological SECM and associated negative amperometric-feedback and generator/collector-mode SECM imaging are discussed, and successful use of the methodology for screening soft and fragile membranous objects is outlined. The drawbacks of the constant-height mode of probe movement and the benefits of the constant-distance mode of SECM operation are described. Finally, representative examples of constant-height and constant-distance mode SECM on a variety of live cells are highlighted to demonstrate the current status of single-cell SECM in general and of SECM in neuroscience in particular.

  20. Scanning Probe Microscopy of Graphene

    Science.gov (United States)

    Tautz, Pamela

    2011-10-01

    Scanning tunneling microscopy has been used to study the unusual electronic properties of graphene. In an effort to support the graphene with minimal interaction with the substrate, we used a hexagonal boron nitride (hBN) substrate. To minimize contaminants between the CVD graphene and boron nitride, the graphene samples were cleaned with distilled water and isopropanol prior to transfer to hBN substrate. We have also examined the growth of graphene flakes by chemical vapor deposition. In particular, we examined the relationship between the orientations of the first and second layer of CVD grown graphene. We found the growth mechanism preferentially resulted in rotations of 9^o or less indicating flakes with first and second layers aligned.

  1. Automatic, high-accuracy image registration in confocal microscopy.

    Science.gov (United States)

    Liu, Jian; Li, Yong; Wang, Weibo; Wang, Yuhang; Zhang, He; Tan, Jiubin

    2017-11-10

    We proposed a high-accuracy image registration method of confocal microscopy for a large field of view and high resolution. The spatial information (edge information) and the entropy correlation coefficient have been both taken into account for higher accuracy of registration. The edge information is introduced to calculate the normalization correlation coefficient of the image. Then the normalization correlation coefficient and the entropy correlation coefficient of the original image have been used to improve the proposed similarity measures, the normalized mutual information with edge information (called EMI). Meanwhile, a parallel particle swarm optimization (pa-PSO) with the idea of conditional initialization and parallel cooperation is developed to speed up the convergence rate and further reduce the mismatch. Experiments verified that the registration accuracy can be up to 0.2 pixel and has better robustness to the noise.

  2. Reflectance confocal microscopy for cutaneous infections and infestations.

    Science.gov (United States)

    Cinotti, E; Perrot, J L; Labeille, B; Cambazard, F

    2016-05-01

    Reflectance confocal microscopy (RCM) is a high-resolution emerging imaging technique that allows non-invasive diagnosis of several cutaneous disorders. A systematic review of the literature on the use of RCM for the study of infections and infestations has been performed to evaluate the current use of this technique and its possible future applications in this field. RCM is particularly suitable for the identification of Sarcoptes scabies, Demodex folliculorum, Ixodes, Dermatophytes and Candida species in the clinical practice and for the follow-up after treatment. The cytopathic effect of herpes simplex virus, varicella zoster virus and molluscipoxvirus is also detectable by this imaging technique even in a pre-vesicular stage. In addition, thanks to its non-invasiveness, RCM allows pathophysiological studies. © 2015 European Academy of Dermatology and Venereology.

  3. Endocrine and metabolic disease: Confocal microscopy as a diagnostic aid

    Directory of Open Access Journals (Sweden)

    Jaikrit Bhutani

    2015-01-01

    Full Text Available Diabetes is a systemic disease associated with many complications. These can be prevented and managed effectively if detected promptly. Confocal microscopy (CFM is a diagnostic tool which has the potential to help in early detection of disease and timely management. CFM has the potential to serve as an excellent noninvasive modality for in vivo imaging and morphological analysis, which can aid us in assessing and monitoring various infectious and pathological diseases at the cellular level. Besides ophthalmological indications, CFM has shown good sensitivity and specificity for identifying those at risk of neuropathy and foot ulceration, monitoring evolution and therapeutic response in a wide range of neuropathies apart from diabetic neuropathy. Through this communication, we aim to sensitize the endocrinologists towards cerebral cavernous malformation as a biomarker to evaluate potential outcomes and therapies in human diabetic neuropathy.

  4. The Reliability and Reproducibility of Corneal Confocal Microscopy in Children.

    Science.gov (United States)

    Pacaud, Danièle; Romanchuk, Kenneth G; Tavakoli, Mitra; Gougeon, Claire; Virtanen, Heidi; Ferdousi, Maryam; Nettel-Aguirre, Alberto; Mah, Jean K; Malik, Rayaz A

    2015-08-01

    To assess the image and patient level interrater agreement and repeatability within 1 month for corneal nerve fiber length (CNFL) measured using in vivo corneal confocal microscopy (IVCCM) in children. Seventy-one subjects (mean [SD] age 14.3 [2.6] years, range 8-18 years; 44 with type 1 diabetes and 27 controls; 36 males and 35 females) were included. 547 images (∼6 images per subject) were analyzed manually by two independent and masked observers. One-month repeat visit images were analyzed by a single masked observer in 21 patients. Automated image analysis was then performed using a specialized computerized software (ACCMetrics). For CNFL, the ICC (95% CI) were 0.94 (0.93-0.95) for image-level, 0.86 (0.78-0.91) for patient-level, and 0.88 (0.72-0.95) for the 1-month repeat assessment, and the Bland-Altman plots showed minimal bias between observers. Although there was excellent agreement between manual and automated analysis according to an ICC 0.89 (0.82-0.93), the Bland-Altman plot showed a consistent bias with manual measurements providing higher readings. In vivo corneal confocal microscopy image analysis shows good reproducibility with excellent intraindividual and interindividual variability in pediatric subjects. Since the image-level reproducibility is stronger than the patient-level reproducibility, refinement of the method for image selection will likely further increase the robustness of this novel, rapid, and noninvasive approach to detect early neuropathy in children with diabetes. Further studies on the use of IVCCM to identify early subclinical neuropathy in children are indicated.

  5. Reflectance confocal microscopy for the evaluation of sensitive skin.

    Science.gov (United States)

    Ma, Y-F; Yuan, C; Jiang, W-C; Wang, X-L; Humbert, P

    2017-05-01

    Nowadays, the diagnosis for sensitive skin relies on subjective assessment or on the combination of subjective and objective evaluation. No quantitative evaluation is available. It could be expected that confocal microscopy imaging could be of interest to better define the condition. Total 166 healthy female subjects were recruited in this study. Firstly, all subjects completed the sensitive questionnaire. Then, the cutaneous structures were measured by the reflectance confocal microscopy (RCM) on the face and fossa cubitalia. The lactic acid sting test was conducted finally. According to the results of self-perception sensitive skin questionnaire and lactic acid stinging test to evaluate facial skin sensitivity the both positive subjects were regarded as sensitive skin group and both negative group as healthy control group. The results of RCM indicating that the proportion of 'disarranged honeycomb pattern' and 'spongiform edema' in the sensitive group and healthy control group were statistically different (P 0.05). The epidermal thickness was 38.88 ± 6.81 μm, healthy control group was 40.31 ± 9.37 μm in, respectively, sensitive skin group and healthy control group, there was no significant statistical difference between the two groups (P > 0.05). The honeycomb structure depth of sensitive group was 20.57 ± 4.86 μm. It was for 23.27 ± 6.38 μm, healthy control group the difference being statistically different between the two groups (P skin signs of RCM evaluation of sensitive skin effectively. Indeed, sensitive skin honeycomb structure depth was thinner compared with healthy control group. Such a specific pattern has good clinical and monitoring value for the further exploration. RCM could provide new data and patterns for the evaluation of sensitive skin. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery

    Directory of Open Access Journals (Sweden)

    Attila Bokros

    2013-08-01

    Full Text Available Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS methods. In the present study a combination of small molecule microarray (SMM prescreening and confocal laser scanning microscopy (CLSM was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted fluorochromes were identified. Their cytotoxicity was tested and found that between 1–10 micromolar range, they were non-toxic even during long-term incubations.

  7. Sealing ability of mineral trioxide aggregate, calcium phosphate and polymethylmethacrylate bone cements on root ends prepared using an Erbium: Yttriumaluminium garnet laser and ultrasonics evaluated by confocal laser scanning microscopy.

    Science.gov (United States)

    Girish, C Sabari; Ponnappa, Kc; Girish, Tn; Ponappa, Mc

    2013-07-01

    Surgical endodontic therapy comprises of exposure of the involved root apex, resection of the apical end of the root, preparation of a class I cavity, and insertion of a root end filling material. Mineral trioxide aggregate (MTA) is now the gold standard among all root end filling materials. MTA is however difficult to handle, expensive and has a very slow setting reaction. (1) To compare the sealing ability of MTA, polymethylmethacrylate (PMMA) bone cement and CHITRA Calcium phosphate cement (CPC) when used as root end filling material using Rhodamine B dye evaluated under a confocal laser scanning microscope. (2) To compare the seal of root ends prepared using an ultrasonic retroprep tip and an Er: YAG laser using three different root end filling materials. Statistical analysis was performed using a one-way ANOVA and a two-way ANOVA, independent samples t-test and Scheffe's post hoc test using SPSS Version 16 for Windows. All the three materials, namely MTA, PMMA BONE CEMENT and CHITRA CPC, showed microleakage. Comparison of microleakage showed maximum peak value of 0.86 mm for MTA, 0.24 mm for PMMA bone cement and 1.37 mm for CHITRA CPC. The amount of dye penetration was found to be lesser in root ends prepared using Er: YAG laser when compared with ultrasonics, but the difference was found to be not statistically significant. PMMA bone cement is a better material as root end filling material to prevent apical microleakage. MTA still continues to be a gold standard root end filling material showing minimum microleakage. Er: YAG laser is a better alternative to ultrasonics for root end preparations.

  8. 3-D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series

    NARCIS (Netherlands)

    Wouterlood, F.G.

    2014-01-01

    A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. Scanning of a specimen, with stepwise axial (Z-) movement of the stage in between each scan, produces Z-series of confocal images of a tissue volume, which then can be used

  9. Optical coherence tomography and confocal microscopy investigations of dental prostheses

    Science.gov (United States)

    Negrutiu, Meda L.; Sinescu, Cosmin; Hughes, Michael; Bradu, Adrian; Rominu, Mihai; Todea, Carmen; Dobre, George; Podoleanu, Adrian

    2008-09-01

    Dental prostheses are very complex systems, heterogenous in structure, made up from various materials, with different physical properties. An essential question mark is on the physical, chemical and mechanical compatibility between these materials. They have to satisfy high stress requirements as well as esthetic challenges. The masticatory stress may induce fractures of the prostheses, which may be triggered by initial materials defects or by alterations of the technological process. The failures of dental prostheses lead to functional, esthetic and phonetic disturbances which finally render the prosthetic treatment inefficient. The purpose of this study is to evaluate the capability of en-face optical coherence tomography as a possible non-invasive high resolution method in supplying the necessary information on the material defects of dental prostheses and microleakage at prosthetic interfaces. C-scan and B-scan OCT images as well as confocal images are acquired from a large range of samples. Gaps between the dental interfaces and material defects are clearly exposed. We conclude that OCT can successfully be used as a noninvasive analysis method.

  10. Atomic force microscopy analysis and confocal Raman microimaging of coated pellets.

    Science.gov (United States)

    Ringqvist, Ann; Taylor, Lynne S; Ekelund, Katarina; Ragnarsson, Gert; Engström, Sven; Axelsson, Anders

    2003-11-28

    Polymer-coated pellets with different coating thicknesses have been studied regarding coating morphology and drug release properties with atomic force microscopy (AFM) and confocal Raman microscopy. The results were compared with those from scanning electron microscopy (SEM) and drug release profiles, which have been measured previously for these systems and found to vary depending on coating thickness. Results from AFM studies indicated that these pellets differ in the amount of crystalline material on the surface of the coating. The amount was found to be highest on the pellet with the thinnest coating. Confocal Raman microscopy studies confirmed that the active component (remoxipride hydrochloride monohydrate) is present at or close to the surface and that the amount is higher for the thinnest coating. AFM studies in aqueous media showed that the crystalline material on the surface was almost instantaneously dissolved and released into the liquid. AFM has proven to be a powerful tool in the study of the surface of dry formulations and in the study of the controlled release mechanism of a pharmaceutical in a liquid cell. The method can be combined with Raman, giving the added possibility to identify the chemical composition in selected small areas of the coating surface.

  11. Ophthalmic applications of confocal microscopy: diagnostics, refractive surgery, and eye banking

    Science.gov (United States)

    Masters, Barry R.

    1990-11-01

    Confocal microscopy of ocular tissue provides two advantages over traditional imaging techniques: increased range and transverse resolution and increased contrast. The semitransparent cornea and ocular lens in the living eye can be optically sectioned and observed by reflected light confocal microscopy. Within the cornea we observed various cell components nerve fibers nerve cell bodies and fibrous networks. The confocal microscopic images from the in-situ ocular lens show the lens capsule the lens epithelium and the individual lens fibrils. All of the reflected light confocal microscopic images have high contrast and high resolution. Some of the applications of confocal imaging in ophthalmology include: diagnostics of the cornea and the ocular lens examination prior to and after refractive surgery examination of intraocular lenses (IOL) and examination of eye bank material. Other ophthalmic uses of confocal imaging include: studies of wound healing therapeutics and the effects of contact lenses on the cornea. The proposed features of a clinical confocal microscope are reviewed. 2.

  12. Multimodal optical workstation for simultaneous linear, nonlinear microscopy and nanomanipulation: upgrading a commercial confocal inverted microscope.

    Science.gov (United States)

    Mathew, Manoj; Santos, Susana I C O; Zalvidea, Dobryna; Loza-Alvarez, Pablo

    2009-07-01

    In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope (Nikon Confocal C1-Si) to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems (confocal and nonlinear) to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans.

  13. Effects of acids used in the microabrasion technique: Microhardness and confocal microscopy analysis.

    Science.gov (United States)

    Pini, Núbia-Inocencya-Pavesi; Lima, Débora-Alves-Nunes-Leite; Ambrosano, Gláucia-Maria-Bovi; da Silva, Wander-José; Aguiar, Flávio-Henrique-Baggio; Lovadino, José-Roberto

    2015-10-01

    This study evaluated the effects of the acids used in the microabrasion on enamel. Seventy enamel/dentine blocks (25 mm2) of bovine incisors were divided into 7 groups (n=10). Experimental groups were treated by active/passive application of 35% H3PO4 (E1/E2) or 6.6% HCl (E3/E4). Control groups were treated by microabrasion with H3PO4+pumice (C5), HCl+silica (C6), or no treatment (C7). The superficial (SMH) and cross-sectional (CSMH; depths of 10, 25, 50, and 75 µm) microhardness of enamel were analyzed. Morphology was evaluated by confocal laser-scanning microscopy (CLSM). Data were analyzed by analysis of variance (Proc Mixed), Tukey, and Dunnet tests (α=5%). Active application (E1 and E3) resulted in higher microhardness than passive application (E2 and E4), with no difference between acids. For most groups, the CSMH decreased as the depth increased. All experimental groups and negative controls (C5 and C6) showed significantly reduced CSMH values compared to the control. A significantly higher mean CSMH result was obtained with the active application of H3PO4 (E1) compared to HCl (E3). Passive application did not result in CSMH differences between acids. CLSM revealed the conditioning pattern for each group. Although the acids displayed an erosive action, use of microabrasive mixture led to less damage to the enamel layers. Enamel microabrasion, enamel microhardness, confocal laser scanning microscopy.

  14. 'En face' ex vivo reflectance confocal microscopy to help the surgery of basal cell carcinoma of the eyelid.

    Science.gov (United States)

    Espinasse, Marine; Cinotti, Elisa; Grivet, Damien; Labeille, Bruno; Prade, Virginie; Douchet, Catherine; Cambazard, Frédéric; Thuret, Gilles; Gain, Philippe; Perrot, Jean Luc

    2017-07-01

    Ex vivo confocal microscopy is a recent imaging technique for the perioperative control of skin tumour margins. Up to date, it has been used in the fluorescence mode and with vertical sections of the specimen margins. The aim of this study was to evaluate its use in the reflectance mode and with a horizontal ('en face') scanning of the surgical specimen in a series of basal cell carcinoma of the eyelid. Prospective consecutive cohort study was performed at the University Hospital of Saint-Etienne, France. Forty-one patients with 42 basal cell carcinoma of the eyelid participated in this study. Basal cell carcinomas were excised with a 2-mm-wide clinically safe margin. The surgical specimens were analysed under ex vivo confocal microscopy in the reflectance mode and with an en face scanning in order to control at a microscopic level if the margins were free from tumour invasion. Histopathogical examination was later performed in order to compare the results. Sensitivity and specificity of ex vivo confocal microscopy for the presence of tumour-free margins. Ex vivo confocal microscopy results were consistent with histopathology in all cases (tumour-free margins in 40 out of 42 samples; sensitivity and specificity of 100%). Ex vivo confocal microscopy in the reflectance mode with an 'en face' scanning can control tumour margins of eyelid basal cell carcinomas and optimize their surgical management. This procedure has the advantage on the fluorescent mode of not needing any contrast agent to examine the samples. © 2016 Royal Australian and New Zealand College of Ophthalmologists.

  15. Scanning tunneling microscopy II further applications and related scanning techniques

    CERN Document Server

    Güntherodt, Hans-Joachim

    1995-01-01

    Scanning Tunneling Microscopy II, like its predecessor, presents detailed and comprehensive accounts of the basic principles and broad range of applications of STM and related scanning probe techniques. The applications discussed in this volume come predominantly from the fields of electrochemistry and biology. In contrast to those described in STM I, these studies may be performed in air and in liquids. The extensions of the basic technique to map other interactions are described in chapters on scanning force microscopy, magnetic force microscopy, and scanning near-field optical microscopy, together with a survey of other related techniques. Also described here is the use of a scanning proximal probe for surface modification. Together, the two volumes give a comprehensive account of experimental aspects of STM. They provide essential reading and reference material for all students and researchers involved in this field. In this second edition the text has been updated and new methods are discussed.

  16. Scanning tunneling microscopy II further applications and related scanning techniques

    CERN Document Server

    Güntherodt, Hans-Joachim

    1992-01-01

    Scanning Tunneling Microscopy II, like its predecessor, presents detailed and comprehensive accounts of the basic principles and broad range of applications of STM and related scanning probe techniques. The applications discussed in this volume come predominantly from the fields of electrochemistry and biology. In contrast to those described in Vol. I, these sudies may be performed in air and in liquids. The extensions of the basic technique to map other interactions are described inchapters on scanning force microscopy, magnetic force microscopy, scanning near-field optical microscopy, together with a survey of other related techniques. Also described here is the use of a scanning proximal probe for surface modification. Togehter, the two volumes give a comprehensive account of experimental aspcets of STM. They provide essentialreading and reference material for all students and researchers involvedin this field.

  17. 4D confocal microscopy for visualisation of bone remodelling

    NARCIS (Netherlands)

    Konijn, GA; Vardaxis, NJ; Boon, ME; Kok, LP; Rietveld, DC; SCHUT, JJ

    Until recently it was very time consuming and difficult to make three-dimensional (3D) images of newly formed bone. With the advent of confocal technologies and increased computer power 3D imaging is greatly facilitated. In this paper we demonstrate that enhanced confocal visualisation of newly

  18. In vivo Confocal Microscopy Report after Lasik with Sequential Accelerated Corneal Collagen Cross-Linking Treatment

    Directory of Open Access Journals (Sweden)

    Cosimo Mazzotta

    2014-04-01

    Full Text Available We report the first pilot qualitative confocal microscopic analysis of a laser in situ keratomileusis (Lasik treatment combined with sequential high-fluence accelerated corneal collagen cross-linking, denominated Lasik XTra, by means of HRT II laser scanning in vivo confocal microscopy after a 6-month follow-up. After obtaining approval from the Siena University Hospital Institutional Review Board, a 33-year-old female patient underwent a Lasik XTra procedure in her left eye. Confocal analysis demonstrated induced slight corneal microstructural changes by the interaction between UV-A, riboflavin and corneal stromal collagen, beyond the interface to a depth of 160 µm, without adverse events at the interface and endothelial levels. This application may be considered a prophylactic biomechanical treatment, stiffening the intermediate corneal stroma to prevent corneal ectasia and stabilizing the clinical results of refractive surgery. According to our preliminary experiences, this combined approach may be useful in higher-risk Lasik patients for hyperopic treatments, high myopia and lower corneal thicknesses.

  19. Non-invasive in vivo visualization of enamel defects by reflectance confocal microscopy (RCM).

    Science.gov (United States)

    Contaldo, Maria; Di Stasio, Dario; Santoro, Rossella; Laino, Luigi; Perillo, Letizia; Petruzzi, Massimo; Lauritano, Dorina; Serpico, Rosario; Lucchese, Alberta

    2015-05-01

    The enamel defects (EDs) may present with a variety of clinical manifestations with increasing severity from the sole appearance of pale discoloration to remarkable structural alterations. EDs are responsible for higher caries receptivity. In vivo reflectance confocal microscopy (RCM) allows to image in vivo at microscopic resolution of the dental surface, thus avoiding the tooth extraction and the sample preparation because of its ability to optically scan living tissues along their depth. Aim of this study is the in vivo assessment at microscopic resolution of dental surfaces affected by EDs without resorting to invasive methods such as teeth extractions, to define histological findings occurring in chromatic and/or structural EDs. For the purpose, 15 children, referring at the Dental Clinic of the Second University of Naples, affected by several degrees of EDs, were enrolled and underwent in vivo RCM imaging to microscopically define the ED confocal features using a commercially available hand-held reflectance confocal microscope with neither injuries nor discomfort. Totally, 29 teeth were imaged. Results demonstrated images good in quality and the capability to detect EDs such as unevenness, grooves, and lack of mineralization according to their clinical degree of disarray. The present in vivo microscopic study on EDs allowed to highlight structural changes in dental enamel at microscopic resolution in real-time and in a non-invasive way, with no need for extraction or processing the samples. Further experiments could define the responsiveness to remineralizing procedures as therapeutic treatments.

  20. Nevomelanocytic atypia detection by in vivo reflectance confocal microscopy.

    Science.gov (United States)

    Vaišnorienė, Ingrida; Rotomskis, Ričardas; Kulvietis, Vytautas; Eidukevičius, Rimantas; Zalgevičienė, Violeta; Laurinavičienė, Aida; Venius, Jonas; Didžiapetrienė, Janina

    2014-01-01

    In vivo reflectance confocal microscopy (RCM) is a promising novel technology for non-invasive early diagnostics of cutaneous melanoma. However, the possibility to detect melanocytic atypia in nevi by means of in vivo RCM remains unknown. The aim of the study was to evaluate the significance of in vivo RCM features of melanocytic atypia for the diagnosis of melanocytic nevi, dysplastic nevi and cutaneous melanoma. A total of 138 melanocytic skin lesions comprising 25 melanocytic nevi, 69 dysplastic nevi and 44 melanomas were analyzed by means of dermoscopy, in vivo RCM and routine histopathology. In vivo RCM images were analyzed for the arrangement of keratinocytes in epidermis, pagetoid cells and junctional melanocytic nests and correlated refractivity aspects of nests with histopathology. Separately and all together taken the in vivo RCM features of melanocytic atypia were significant in differential diagnosis of benign and malignant melanocytic skin lesions, though none of the features was significant in discriminating nevi without cytologic atypia of dysplastic nevi. In vivo RCM feature of dense cell clusters corresponded with melanin containing nevomelanocytes on histopathology though exact correspondence of non-homogeneous and atypical sparse cell clusters remained questionable. Nevus with histopathologically confirmed nevomelanocytic atypia (dysplastic nevus) could not be distinguished from nevus without atypia using analyzed in vivo RCM features of melanocytic atypia. More accurate diagnostics by means of in vivo RCM needs further investigation on reflectance of single and nested cutaneous melanocytes in benign and malignant skin lesions. Copyright © 2014 Lithuanian University of Health Sciences. Production and hosting by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  1. Introduction to scanning tunneling microscopy

    CERN Document Server

    Chen, C Julian

    2008-01-01

    The scanning tunneling and the atomic force microscope, both capable of imaging individual atoms, were crowned with the Physics Nobel Prize in 1986, and are the cornerstones of nanotechnology today. This is a thoroughly updated version of this 'bible' in the field.

  2. Improved sampling and analysis of images in corneal confocal microscopy.

    Science.gov (United States)

    Schaldemose, E L; Fontain, F I; Karlsson, P; Nyengaard, J R

    2017-10-01

    Corneal confocal microscopy (CCM) is a noninvasive clinical method to analyse and quantify corneal nerve fibres in vivo. Although the CCM technique is in constant progress, there are methodological limitations in terms of sampling of images and objectivity of the nerve quantification. The aim of this study was to present a randomized sampling method of the CCM images and to develop an adjusted area-dependent image analysis. Furthermore, a manual nerve fibre analysis method was compared to a fully automated method. 23 idiopathic small-fibre neuropathy patients were investigated using CCM. Corneal nerve fibre length density (CNFL) and corneal nerve fibre branch density (CNBD) were determined in both a manual and automatic manner. Differences in CNFL and CNBD between (1) the randomized and the most common sampling method, (2) the adjusted and the unadjusted area and (3) the manual and automated quantification method were investigated. The CNFL values were significantly lower when using the randomized sampling method compared to the most common method (p = 0.01). There was not a statistical significant difference in the CNBD values between the randomized and the most common sampling method (p = 0.85). CNFL and CNBD values were increased when using the adjusted area compared to the standard area. Additionally, the study found a significant increase in the CNFL and CNBD values when using the manual method compared to the automatic method (p ≤ 0.001). The study demonstrated a significant difference in the CNFL values between the randomized and common sampling method indicating the importance of clear guidelines for the image sampling. The increase in CNFL and CNBD values when using the adjusted cornea area is not surprising. The observed increases in both CNFL and CNBD values when using the manual method of nerve quantification compared to the automatic method are consistent with earlier findings. This study underlines the importance of improving the analysis of the

  3. Microstructural evaluation by confocal and electron microscopy in thrombi developed in central venous catheters.

    Science.gov (United States)

    Lucas, Thabata Coaglio; Silva, Eliata Ester da; Souza, Danilo Olzon Dionysio; Santos, Amanda Rodrigues Dos; Lara, Maristela Oliveira

    2017-08-28

    Evaluating thrombi microstructure developed in central venous catheters using confocal and electron microscopy. An experimental, descriptive study carrying out a microstructural evaluation of venous thrombi developed in central venous catheters using Scanning Electron Microscopy and Confocal Laser Scanning Microscopy. A total of 78 venous catheters were collected over a period of three months. Different fibrin structures were distinguished: fibrin plates, fibrin network, and fibrin fibers. It was observed that the thrombus had thick fibrin plates adhered to the catheter wall openings in both a catheter with three days of permanence as well as in a catheter with 20 days of insertion in the patient. However, a greater amount of erythrocytes and fibrin fibers were found in the central region of the thrombus. This study contributes to improving health care and can have a positive impact on clinical practice, as easy adherence of platelets and fibrins to the catheter wall demonstrated in this study makes it possible to adopt thrombus prevention strategies such as therapy discontinuation for an extended period, blood reflux by a catheter, slow infusion rate and hypercoagulo pathyclinical conditions. Avaliar a microestrutura por microscopia confocal e eletrônica em trombos desenvolvidos em cateteres venosos centrais. Pesquisa experimental, descritiva, em que foi feita uma avaliação microestrutural de trombos venosos desenvolvidos em cateteres venosos centrais por Microscopia Eletrônica de Varredura e Microscopia Confocal de Varredura a Laser. Foram coletados 78 cateteres venosos centrais num período de três meses. Distinguiram-se diferentes estruturas de fibrina: a placa de fibrina, a rede de fibrina e as fibras de fibrina. Observou-se que tanto em um cateter com três dias de permanência quanto em um cateter com 20 dias inserido no paciente o trombo apresentou placas de fibrina espessas aderidas às paredes dos orifícios dos cateteres. Na região central do

  4. Combining confocal microscopy and optical coherence tomography for imaging in developmental biology

    Science.gov (United States)

    Bradu, A.; Ma, Lisha; Bloor, J.; Podoleanu, A.

    2008-04-01

    In-vivo Optical Coherence Tomography (OCT) imaging of the fruit fly Drosophila melanogaster larval heart allows non invasive visualizations and assesment of its cardiac functions. To image Drosophila melanogaster heart, we have developed a dedicated imaging instrument able to provide simultaneous Optical Coherence Tomography (OCT) and Laser Confocal Scanning Microscopy (LCSM) or Laser Scanning Fluorescence Microscopy (LSFM) images and can be used to produce B-scan OCT images. With this dual imaging system, the image of heart can be easily located in the specimen and the change of the heart shape in a cardiac cycle monitored. This technique therefore provides an excellent tool for large scale screen of candidate genes responsible for the contractility of the Drosophila heart. As this technique can also image the dynamic process of the heartbeat in a non-invasive fashion, it provides a new avenue to study the physiology of the heart function. En-face and B-scan OCT images of the Drosophila melanogaster heart showing its chambers have been obtained with our imaging instruments. Our results are consistent with detailed anatomical studies from the literature.

  5. Scanning Electron Microscopy in modern dentistry research

    OpenAIRE

    Paradella, Thaís Cachuté; Unesp-FOSJC; Bottino, Marco Antonio; Unesp-FOSJC

    2012-01-01

    The purpose of this article was to review the usage of Scanning Electron Microscopy (SEM) in dentistry research nowadays, through a careful and updated literature review. By using the key-words Scanning Electron Microscopy and one of the following areas of research in dentistry (Endodontics, Periodontics and Implant), in international database (PubMed), in the year of 2012 (from January to September), a total of 112 articles were found. This data was tabled and the articles were classified ac...

  6. Differential-concentration scanning ion conductance microscopy

    OpenAIRE

    Perry, David; Page, Ashley; Chen, Baoping; Frenguelli, Bruno G.; Unwin, Patrick R.

    2017-01-01

    Scanning ion conductance microscopy (SICM) is a nanopipette-based scanning probe microscopy technique that utilizes the ionic current flowing between an electrode inserted inside a nanopipette probe containing electrolyte solution and a second electrode placed in a bulk electrolyte bath, to provide information on a substrate of interest. For most applications to date, the composition and concentration of the electrolyte inside and outside the nanopipette is identical, but it is shown herein t...

  7. Fully automatic evaluation of the corneal endothelium from in vivo confocal microscopy.

    Science.gov (United States)

    Selig, Bettina; Vermeer, Koenraad A; Rieger, Bernd; Hillenaar, Toine; Luengo Hendriks, Cris L

    2015-04-26

    Manual and semi-automatic analyses of images, acquired in vivo by confocal microscopy, are often used to determine the quality of corneal endothelium in the human eye. These procedures are highly time consuming. Here, we present two fully automatic methods to analyze and quantify corneal endothelium imaged by in vivo white light slit-scanning confocal microscopy. In the first approach, endothelial cell density is estimated with the help of spatial frequency analysis. We evaluate published methods, and propose a new, parameter-free method. In the second approach, based on the stochastic watershed, cells are automatically segmented and the result is used to estimate cell density, polymegathism (cell size variability) and pleomorphism (cell shape variation). We show how to determine optimal values for the three parameters of this algorithm, and compare its results to a semi-automatic delineation by a trained observer. The frequency analysis method proposed here is more precise than any published method. The segmentation method outperforms the fully automatic method in the NAVIS software (Nidek Technologies Srl, Padova, Italy), which significantly overestimates the number of cells for cell densities below approximately 1200 mm(-2), as well as previously published methods. The methods presented here provide a significant improvement over the state of the art, and make in vivo, automated assessment of corneal endothelium more accessible. The segmentation method proposed paves the way to many possible new morphometric parameters, which can quickly and precisely be determined from the segmented image.

  8. Confocal Microscopy of Unfixed Breast Needle Core Biopsies: A Comparison to Fixed and Stained Sections

    Science.gov (United States)

    2009-01-01

    Background Needle core biopsy, often in conjunction with ultrasonic or stereotactic guided techniques, is frequently used to diagnose breast carcinoma in women. Confocal scanning laser microscopy (CSLM) is a technology that provides real-time digital images of tissues with cellular resolution. This paper reports the progress in developing techniques to rapidly screen needle core breast biopsy and surgical specimens at the point of care. CSLM requires minimal tissue processing and has the potential to reduce the time from excision to diagnosis. Following imaging, specimens can still be submitted for standard histopathological preparation. Methods Needle core breast specimens from 49 patients were imaged at the time of biopsy. These lesions had been characterized under the Breast Imaging Reporting And Data System (BI-RADS) as category 3, 4 or 5. The core biopsies were imaged with the CSLM before fixation. Samples were treated with 5% citric acid and glycerin USP to enhance nuclear visibility in the reflectance confocal images. Immediately following imaging, the specimens were fixed in buffered formalin and submitted for histological processing and pathological diagnosis. CSLM images were then compared to the standard histology. Results The pathologic diagnoses by standard histology were 7 invasive ductal carcinomas, 2 invasive lobular carcinomas, 3 ductal carcinomas in-situ (CIS), 21 fibrocystic changes/proliferative conditions, 9 fibroadenomas, and 5 other/benign; two were excluded due to imaging difficulties. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images and were comparable to standard histologic sections of the same tissue. Conclusion CSLM is a technique with the potential to screen needle core biopsy specimens in real-time. The confocal images contained sufficient information to identify stromal reactions such as fibrosis and cellular proliferations such as intra-ductal and infiltrating carcinoma, and

  9. Total Internal Reflection Fluorescence Microscopy Imaging-Guided Confocal Single-Molecule Fluorescence Spectroscopy

    OpenAIRE

    Zheng, Desheng; Kaldaras, Leonora; Lu, H. Peter

    2013-01-01

    We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscop...

  10. Towards high-speed scanning tunneling microscopy

    NARCIS (Netherlands)

    Tabak, Femke Chantal

    2013-01-01

    In this thesis, two routes towards high-speed scanning tunneling microscopy (STM) are described. The first possibility for high-speed scanning that is discussed is the use of MEMS (Micro-Electro Mechanical Systems) devices as high-speed add-ons in STM microscopes. The functionality of these devices

  11. Spiral scanning method for atomic force microscopy.

    Science.gov (United States)

    Hung, Shao-Kang

    2010-07-01

    A spiral scanning method is proposed for atomic force microscopy with thoroughgoing analysis and implementation. Comparing with the traditional line-by-line scanning method, the spiral scanning method demonstrates higher imaging speed, minor image distortion, and lower acceleration, which can damage the piezoelectric scanner. Employing the spiral scanning method to replace the line-by-line scanning method, the experiment shows that the time to complete an imaging cycle can be reduced from 800 s to 314 s without sacrificing the image resolution.

  12. An instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography.

    Science.gov (United States)

    Mochalov, Konstantin E; Chistyakov, Anton A; Solovyeva, Daria O; Mezin, Alexey V; Oleinikov, Vladimir A; Vaskan, Ivan S; Molinari, Michael; Agapov, Igor I; Nabiev, Igor; Efimov, Anton E

    2017-11-01

    In the past decade correlative microscopy, which combines the potentials of different types of high-resolution microscopies with a variety of optical microspectroscopy techniques, has been attracting increasing attention in material science and biological research. One of outstanding solutions in this area is the combination of scanning probe microscopy (SPM), which provides data on not only the topography, but also the spatial distribution of a wide range of physical properties (elasticity, conductivity, etc.), with ultramicrotomy, allowing 3D multiparametric examination of materials. The combination of SPM and ultramicrotomy (scanning probe nanotomography) is very appropriate for characterization of soft multicompound nanostructurized materials, such as polymer matrices and microstructures doped with different types of nanoparticles (magnetic nanoparticles, quantum dots, nanotubes, etc.), and biological materials. A serious problem of this technique is a lack of chemical and optical characterization tools, which may be solved by using optical microspectroscopy. Here, we report the development of an instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography in a single apparatus. This approach retains all the advantages of SPM and upright optical microspectroscopy and allows 3D multiparametric characterization using both techniques. As the first test of the system developed, we have performed correlative characterization of the morphology and the magnetic and fluorescent properties of fluorescent magnetic microspheres doped with a fluorescent dye and magnetic nanoparticles. The results of this study can be used to obtain 3D volume images of a specimen for most high-resolution near-field scanning probe microscopies: SNOM, TERS, AFM-IR, etc. This approach will result in development of unique techniques combining the advantages of SPM (nanoscale morphology and a wide range of physical parameters) and high-resolution optical

  13. Modeling of fibrin gels based on confocal microscopy and light-scattering data

    National Research Council Canada - National Science Library

    Magatti, Davide; Molteni, Matteo; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

    2013-01-01

    .... Electron and confocal microscopies show a collection of fibers that are relatively monodisperse in diameter, not uniformly distributed, and connected at nodal points with a branching order of ∼3-4...

  14. Clinical usefulness of reflectance confocal microscopy in the management of facial lentigo maligna melanoma.

    Science.gov (United States)

    Alarcón, I; Carrera, C; Puig, S; Malvehy, J

    2014-04-01

    Facial lentigo maligna melanoma can be a diagnostic challenge in daily clinical practice as it has similar clinical and morphological features to other lesions such as solar lentigines and pigmented actinic keratoses. Confocal microscopy is a noninvasive technique that provides real-time images of the epidermis and superficial dermis with cellular-level resolution. We describe 3 cases of suspected facial lentigo maligna that were assessed using dermoscopy and confocal microscopy before histopathology study. In the first case, diagnosed as lentigo maligna melanoma, presurgical mapping by confocal microscopy was performed to define the margins more accurately. In the second and third cases, with a clinical and dermoscopic suspicion of lentigo maligna melanoma, confocal microscopy was used to identify the optimal site for biopsy. Copyright © 2012 Elsevier España, S.L. and AEDV. All rights reserved.

  15. Corneal Confocal Microscopy Detects Corneal Nerve Damage in Patients Admitted With Acute Ischemic Stroke.

    Science.gov (United States)

    Khan, Adnan; Akhtar, Naveed; Kamran, Saadat; Ponirakis, Georgios; Petropoulos, Ioannis N; Tunio, Nahel A; Dargham, Soha R; Imam, Yahia; Sartaj, Faheem; Parray, Aijaz; Bourke, Paula; Khan, Rabia; Santos, Mark; Joseph, Sujatha; Shuaib, Ashfaq; Malik, Rayaz A

    2017-11-01

    Corneal confocal microscopy can identify corneal nerve damage in patients with peripheral and central neurodegeneration. However, the use of corneal confocal microscopy in patients presenting with acute ischemic stroke is unknown. One hundred thirty patients (57 without diabetes mellitus [normal glucose tolerance], 32 with impaired glucose tolerance, and 41 with type 2 diabetes mellitus) admitted with acute ischemic stroke, and 28 age-matched healthy control participants underwent corneal confocal microscopy to quantify corneal nerve fiber density, corneal nerve branch density, and corneal nerve fiber length. There was a significant reduction in corneal nerve fiber density, corneal nerve branch density, and corneal nerve fiber length in stroke patients with normal glucose tolerance ( P stroke. Corneal confocal microscopy is a rapid noninvasive ophthalmic imaging technique that identifies corneal nerve fiber loss in patients with acute ischemic stroke. © 2017 American Heart Association, Inc.

  16. Ex Vivo (Fluorescence) Confocal Microscopy in Surgical Pathology: State of the Art.

    Science.gov (United States)

    Ragazzi, Moira; Longo, Caterina; Piana, Simonetta

    2016-05-01

    First developed in 1957, confocal microscopy is a powerful imaging tool that can be used to obtain near real-time reflected light images of untreated human tissue with nearly histologic resolution. Besides its research applications, in the last decades, confocal microscopy technology has been proposed as a useful device to improve clinical diagnosis, especially in ophthalmology, dermatology, and endomicroscopy settings, thanks to advances in instrument development. Compared with the wider use of the in vivo tissue assessment, ex vivo applications of confocal microscopy are not fully explored. A comprehensive review of the current literature was performed here, focusing on the reliable applications of ex vivo confocal microscopy in surgical pathology and on some potential evolutions of this new technique from pathologists' viewpoint.

  17. Evaluation of allergic vesicular reaction to patch test using in vivo confocal microscopy.

    Science.gov (United States)

    Ardigò, Marco; Longo, Caterina; Cristaudo, Antonio; Berardesca, Enzo; Pellacani, Giovanni

    2012-02-01

    Confocal microscopy has been successfully applied both in oncologic and inflammatory diseases. In particular, it has been proved as a useful tool for the in vivo detection of microscopical changes occurring in allergic reactions. To evaluate microscopic changes occurring in positive patch test reactions. Eight patients with history of allergic dermatitis and positive patch test reaction were analysed by means of confocal microscopy. Confocal microscopy showed the presence of spongiotic vesicle preferentially localized around the adnexal ducts that appeared to be in the middle of the spongiotic phenomena. Confocal microscopy offered for the first time new insight into vesicle formation and development, showing that adnexal ducts can play a role in allergic reaction. © 2011 John Wiley & Sons A/S.

  18. WHOLE INSECT AND MAMMALIAN EMBRYO IMAGING WITH CONFOCAL MICROSCOPY: MORPHOLOGY AND APOPTOSIS

    Science.gov (United States)

    Background: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates...

  19. MEMS-BASED 3D CONFOCAL SCANNING MICROENDOSCOPE USING MEMS SCANNERS FOR BOTH LATERAL AND AXIAL SCAN.

    Science.gov (United States)

    Liu, Lin; Wang, Erkang; Zhang, Xiaoyang; Liang, Wenxuan; Li, Xingde; Xie, Huikai

    2014-08-15

    A fiber-optic 3D confocal scanning microendoscope employing MEMS scanners for both lateral and axial scan was designed and constructed. The MEMS 3D scan engine achieved a lateral scan range of over ± 26° with a 2D MEMS scanning micromirror and a depth scan of over 400 μm with a 1D MEMS tunable microlens. The lateral resolution and axial resolution of this system were experimentally measured as 1.0 μm and 7.0 μm, respectively. 2D and 3D confocal reflectance images of micro-patterns, micro-particles, onion skins and acute rat brain tissue were obtained by this MEMS-based 3D confocal scanning microendoscope.

  20. Confocal Raman microscopy for identification of bacterial species in biofilms

    Science.gov (United States)

    Beier, Brooke D.; Quivey, Robert G.; Berger, Andrew J.

    2011-03-01

    Implemented through a confocal microscope, Raman spectroscopy has been used to distinguish between biofilm samples of two common oral bacteria species, Streptococcus sanguinis and mutans, which are associated with healthy and cariogenic plaque, respectively. Biofilms of these species are studied as a model of dental plaque. A prediction model has been calibrated and validated using pure biofilms. This model has been used to identify the species of transferred and dehydrated samples (much like a plaque scraping) as well as hydrated biofilms in situ. Preliminary results of confocal Raman mapping of species in an intact two-species biofilm will be shown.

  1. Investigations in optoelectronic image processing in scanning laser microscopy

    Science.gov (United States)

    Chaliha, Hiranya Kumar

    A considerable amount of work has been done on scann-ing laser microscopy since its applications were first pointed out by Roberts and Young[1], Minsky [2] and Davidovits et al [3]. The advent of laser has made it possible to focus an intense beam of laser light in a scanning optical microscope (SOM) [4, 5] and hence explore regions of microscopy[6] uncovered by conven-tional microscopy. In the simple SOM [7, 8, 9], the upper spatial frequency in amplitude transmittance or reflectance of an object for which transfer function is nonzero is same as that in a conventional optical microscope. However, in Type II SOM [7] or confocal SOM that employs a coherent or a point detector, the spatial frequency bandwidth is twice that obtained in a conventional microscope. Besides this confocal set-up is found to be very useful in optical sectioning and consequently in 3-D image processing[10, 11, 12] specially of biological specimens. Such systems are also suitable for studies of semiconductor materials [13], super-resolution [14] and various imaginative ways of image processing[15, 16, 17] including phase imaging[18]. A brief survey of related advances in scanning optical microscopy has been covered in the chapter 1 of the thesis. The performance of SOM may be investigated by concent-rating also on signal derived by one dimensional scan of the object specimen. This simplified mode may also be adapted to give wealth of information for biological and semiconductor specimens. Hence we have investigated the design of a scanning laser system suited specifically for studies of line scan image signals of microscopic specimens when probed through a focused laser spot. An electro-mechanical method of scanning of the object specimen has been designed with this aim in mind. Chapter 2, Part A of the thesis deals with the design consider-ations of such a system. For analysis of scan signals at a later instant of time so as to facilitate further processing, an arrangement of microprocessor

  2. Confocal Raman Microscopy: new perspective on the weathering of anhydrous cement

    Science.gov (United States)

    Torres-Carrasco, M.; del Campo, A.; de la Rubia, MA; Reyes, E.; Moragues, A.; Fernández, JF

    2017-10-01

    Raman spectroscopy when is combined with Confocal microscopy is a non-destructive technique that allow us to obtain information in cementitious materials. In this study, we present non-destructive image and structural analysis of anhydrous cement with carbonation evidences by Confocal Raman Microscopy (CRM). The results obtained by CRM show a direct relationship between the presence of the weathering processes of an anhydrous cement with the presence of sulphates and surprisingly, with the existence of amorphous carbon in the medium.

  3. Magnetically Triggered Release From Giant Unilamellar Vesicles: Visualization By Means Of Confocal Microscopy

    KAUST Repository

    Nappini, Silvia

    2011-04-07

    Magnetically triggered release from magnetic giant unilamellar vesicles (GUVs) loaded with Alexa fluorescent dye was studied by means of confocal laser scanning microscopy (CLSM) under a low-frequency alternating magnetic field (LF-AMF). Core/shell cobalt ferrite nanoparticles coated with rhodamine B isothiocyanate (MP@SiO 2(RITC)) were prepared and adsorbed on the GUV membrane. The MP@SiO 2(RITC) location and distribution on giant lipid vesicles were determined by 3D-CLSM projections, and their effect on the release properties and GUV permeability under a LF-AMF was investigated by CLSM time-resolved experiments. We show that the mechanism of release of the fluorescent dye during the LF-AMF exposure is induced by magnetic nanoparticle energy and mechanical vibration, which promote the perturbation of the GUV membrane without its collapse. © 2011 American Chemical Society.

  4. Non-linear image scanning microscopy (Conference Presentation)

    Science.gov (United States)

    Gregor, Ingo; Ros, Robert; Enderlein, Jörg

    2017-02-01

    Nowadays, multiphoton microscopy can be considered as a routine method for the observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG) imaging has evolved to a powerful qualitative and label-free method for studying fibrillar structures, like collagen networks. However, examples of super-resolution non-linear microscopy are rare. So far, such approaches require complex setups and advanced synchronization of scanning elements limiting the image acquisition rates. We describe theory and realization of a super-resolution image scanning microscope [1, 2] using two-photon excited fluorescence as well as second-harmonic generation. It requires only minor modifications compared to a classical two-photon laser-scanning microscope and allows image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution. We applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further, we proofed the excellent image quality of our setup for deep tissue imaging. 1. Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101. 2. Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53-54.

  5. Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

    Science.gov (United States)

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

    2009-11-26

    Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

  6. Internal features of graphite in cast irons. Confocal microscopy: useful tool for graphite growth imaging.

    Science.gov (United States)

    Llorca-Isern, N; Tartera, J; Espanol, M; Marsal, M; Bertran, G; Castel, S

    2002-01-01

    Spherulitic crystallisation is a mode of growth of crystals from the melt. Considerable attention has been given to spheroidal graphite formation, providing detailed information about the internal microstructure of the spherulites in spheroidal (SG irons) and compacted graphite irons (CG irons) (Stefanescu, D., 1990. Cast Irons. ASM Handbook, 10th ed., vol. 1). Nevertheless, the mechanisms responsible for this mode of crystallisation are not fully understood. This study deals with the inoculation mechanisms, with particular emphasis on the study of the inclusions for the heterogeneous nucleation of graphite. It is shown that the graphite nuclei are sulfide products of the nodularizing treatment. It has been observed that when rare-earth treatment is applied, the central nucleus consists of a core and an envelope from which the graphite grows. Confocal Scanning Laser Microscopy (CSLM), in reflection mode, was used to study the internal features of the spheroidal graphite growth. Confocal reflection imaging, which has a capacity for optical sectioning of the sample, provides high-resolution images of surface and subsurface regions of interest contained within a semi-transparent sample. Furthermore, three-dimensional reconstruction of these optical sections can provide insight into the mechanism of graphite growth mechanism interpretation. With CSLM the radial growth of graphite was seen. Other techniques, such as TEM, SEM-EDS, WDS, AES and SAM were also used to corroborate the results.

  7. Customized patterned substrates for highly versatile correlative light-scanning electron microscopy

    Science.gov (United States)

    Benedetti, Lorena; Sogne, Elisa; Rodighiero, Simona; Marchesi, Davide; Milani, Paolo; Francolini, Maura

    2014-01-01

    Correlative light electron microscopy (CLEM) combines the advantages of light and electron microscopy, thus making it possible to follow dynamic events in living cells at nanometre resolution. Various CLEM approaches and devices have been developed, each of which has its own advantages and technical challenges. We here describe our customized patterned glass substrates, which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy. PMID:25391455

  8. Improved axial point spread function in a two-frequency laser scanning confocal fluorescence microscope.

    Science.gov (United States)

    Wu, Jheng-Syong; Chung, Yung-Chin; Chien, Jun-Jei; Chou, Chien

    2018-01-01

    A two-frequency laser scanning confocal fluorescence microscope (TF-LSCFM) based on intensity modulated fluorescence signal detection was proposed. The specimen-induced spherical aberration and scattering effect were suppressed intrinsically, and high image contrast was presented due to heterodyne interference. An improved axial point spread function in a TF-LSCFM compared with a conventional laser scanning confocal fluorescence microscope was demonstrated and discussed. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  9. Reproducibility of fundus autofluorescence measurements obtained using a confocal scanning laser ophthalmoscope

    OpenAIRE

    Lois, N.; Halfyard, A.; Bunce, C.; Bird, A.; Fitzke, F.

    1999-01-01

    AIM—To evaluate the reproducibility of the background fundus autofluorescence measurements obtained using a confocal scanning laser ophthalmoscope.
METHODS—10 normal volunteers and 10 patients with retinal disease were included in the study. One eye per subject was chosen randomly. Five images of the same eye of each individual were obtained, after pupillary dilatation, by two investigators using a confocal scanning laser ophthalmoscope. Background fundus autofluorescence was measured at 7 de...

  10. The Enhancement of 3D Scans Depth Resolution Obtained by Confocal Scanning of Porous Materials

    Directory of Open Access Journals (Sweden)

    Martisek Dalibor

    2017-12-01

    Full Text Available The 3D reconstruction of simple structured materials using a confocal microscope is widely used in many different areas including civil engineering. Nonetheless, scans of porous materials such as concrete or cement paste are highly problematic. The well-known problem of these scans is low depth resolution in comparison to the horizontal and vertical resolution. The degradation of the image depth resolution is caused by systematic errors and especially by different random events. Our method is focused on the elimination of such random events, mainly the additive noise. We use an averaging method based on the Lindeberg-Lévy theorem that improves the final depth resolution to a level comparable with horizontal and vertical resolution. Moreover, using the least square method, we also precisely determine the limit value of a depth resolution. Therefore, we can continuously evaluate the difference between current resolution and the optimal one. This substantially simplifies the scanning process because the operator can easily determine the required number of scans.

  11. Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate.

    Science.gov (United States)

    Lundqvist, A; Ocklind, G; Haneskog, L; Lundahl, P

    1998-01-01

    Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.

  12. Confocal laser scanning microscopic investigation of ultrasonic, sonic, and rotary sealer placement techniques

    Science.gov (United States)

    Nikhil, Vineeta; Singh, Renuka

    2013-01-01

    Background: Sealers are used to attain an impervious seal between the core material and root canal walls. Aim: To compare the depth and percentage of sealer penetration with three different placement techniques using confocal laser scanning microscopy as the evaluative tool. Materials and Methods: Root canals of 30 single-rooted teeth were prepared to a size of F3 and AH plus sealer with Rhodamine B was applied with Ultlrasonic file (Gr-1), lentulospiral (Gr-2), and Endoactivator (Gr-3). Canals were obturated with gutta-percha. The roots were sectioned at the 3 and 6-mm levels from the apical foramen and were examined on a confocal microscope. Results: A statistical significant differences among Gr-1, Gr-2, and Gr-3 were found at the 3 and 6-mm level (P < 0.05; ANOVA-Tukey tests) for the depth and percentage of sealer penetration except for Gr-1 and Gr-2 at 3-mm level. Gr-1 showed maximum mean depth of penetration (810 μm) and maximum mean percentage of sealer penetration (64.5) while Gr-3 showed minimum mean depth of penetration (112.7 μm) and minimum mean percentage of sealer penetration (26.7). Conclusion: Depth and percentage of penetration of sealer is influenced by the type of placement technique and by the root canal level with penetration decreasing apically. PMID:23956528

  13. Three-photon fluorescence imaging of melanin with a dual-wedge confocal scanning system

    Science.gov (United States)

    Mega, Yair; Kerimo, Joseph; Robinson, Joseph; Vakili, Ali; Johnson, Nicolette; DiMarzio, Charles

    2012-03-01

    Confocal microscopy can be used as a practical tool in non-invasive applications in medical diagnostics and evaluation. In particular, it is being used for the early detection of skin cancer to identify pathological cellular components and, potentially, replace conventional biopsies. The detection of melanin and its spatial location and distribution plays a crucial role in the detection and evaluation of skin cancer. Our previous work has shown that the visible emission from melanin is strong and can be easily observed with a near-infrared CW laser using low power. This is due to a unique step-wise, (SW) three-photon excitation of melanin. This paper shows that the same SW, 3-photon fluorescence can also be achieved with an inexpensive, continuous-wave laser using a dual-prism scanning system. This demonstrates that the technology could be integrated into a portable confocal microscope for clinical applications. The results presented here are in agreement with images obtained with the larger and more expensive femtosecond laser system used earlier.

  14. In vivo confocal microscopy: corneal changes of hydrogel contact lens wearers.

    Science.gov (United States)

    Yagmur, Meltem; Okay, Okan; Sizmaz, Selcuk; Unal, Ilker; Yar, Kemal

    2011-10-01

    To evaluate the corneal findings in hydrogel contact lens wearers by in vivo confocal scanning microscopy. One hundred and forty-two eyes of 71 myopic contact lens wearers (group 1) and 142 eyes of 71 non-contact lens wearers (group 2), whose age, gender and refractive error matched, were enrolled in order to detect the corneal changes by in vivo confocal microscopy through the central cornea. The average age was 25.5 ± 5.7 (16-52) and 25.6 ± 5.6 (17-49) in groups 1 and 2, respectively. The mean duration of contact lens wear was 43.9 ± 15.3 (6-240) months. Anterior keratocyte density was 667.5 ± 128.3 cells/mm(2) in group 1 and 821.4 ± 136.7 cells/mm(2) in group 2 (P = 0.001). Posterior keratocyte densities of groups 1 and 2 were 540.2 ± 87.6 cells/mm(2) and 628.2 ± 72.4 cells/mm(2), respectively (P lenses with a mean Dk/t ratio of 26.5 × 10(-9) ± 5.9 (8.9-32 × 10(-9)). Stromal microdots occurred with contact lenses with a mean Dk/t ratio of 13.2 × 10(-9) ± 17.5 × 10(-9) (8.9-20 × 10(-9)). In vivo examination of the cornea with confocal microscopy revealed a number of changes. These changes can be attributed both to the mechanical and the hypoxic effects of soft contact lenses. In soft contact lenses with a high Dk/t ratio, these changes would be less frequent.

  15. Semiconductor Surface Characterization by Scanning Probe Microscopies

    Science.gov (United States)

    2001-01-01

    potentiometry (STP)8 and ballistic electron emission microscopy (BEEM)9 which allow mapping of lateral surface potential and local subsurface Schottky...A.P.Fein. "Tunneling Spectroscopy of the Si(1 1 1)2xl Surface", Surf.Sci. 181, 295- 306, 1987. 8. P.Muralt, D.W.Pohl, "Scanning tunneling potentiometry

  16. Scanning electron microscopy study of Trichomonas gallinae.

    Science.gov (United States)

    Tasca, Tiana; De Carli, Geraldo A

    2003-12-01

    A scanning electron microscopy (SEM) study of Trichomonas gallinae (Rivolta, 1878), provided more information about the morphology of this flagellated protozoan. SEM showed the morphological features of the trophozoites; the emergence of the anterior flagella, the structure of the undulating membrane, the position and shape of the pelta, axostyle and posterior flagellum. Of special interest were the pseudocyst forms.

  17. Laser scanning laser diode photoacoustic microscopy system.

    Science.gov (United States)

    Erfanzadeh, Mohsen; Kumavor, Patrick D; Zhu, Quing

    2018-03-01

    The development of low-cost and fast photoacoustic microscopy systems enhances the clinical applicability of photoacoustic imaging systems. To this end, we present a laser scanning laser diode-based photoacoustic microscopy system. In this system, a 905 nm, 325 W maximum output peak power pulsed laser diode with 50 ns pulsewidth is utilized as the light source. A combination of aspheric and cylindrical lenses is used for collimation of the laser diode beam. Two galvanometer scanning mirrors steer the beam across a focusing aspheric lens. The lateral resolution of the system was measured to be ∼21 μm using edge spread function estimation. No averaging was performed during data acquisition. The imaging speed is ∼370 A-lines per second. Photoacoustic microscopy images of human hairs, ex vivo mouse ear, and ex vivo porcine ovary are presented to demonstrate the feasibility and potentials of the proposed system.

  18. Aberration corrected Lorentz scanning transmission electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    McVitie, S., E-mail: stephen.mcvitie@glasgow.ac.uk; McGrouther, D.; McFadzean, S.; MacLaren, D.A.; O’Shea, K.J.; Benitez, M.J.

    2015-05-15

    We present results from an aberration corrected scanning transmission electron microscope which has been customised for high resolution quantitative Lorentz microscopy with the sample located in a magnetic field free or low field environment. We discuss the innovations in microscope instrumentation and additional hardware that underpin the imaging improvements in resolution and detection with a focus on developments in differential phase contrast microscopy. Examples from materials possessing nanometre scale variations in magnetisation illustrate the potential for aberration corrected Lorentz imaging as a tool to further our understanding of magnetism on this lengthscale. - Highlights: • Demonstration of nanometre scale resolution in magnetic field free environment using aberration correction in the scanning transmission electron microscope (STEM). • Implementation of differential phase contrast mode of Lorentz microscopy in aberration corrected STEM with improved sensitivity. • Quantitative imaging of magnetic induction of nanostructures in amorphous and cross-section samples.

  19. Classifying distinct basal cell carcinoma subtype by means of dermatoscopy and reflectance confocal microscopy.

    Science.gov (United States)

    Longo, Caterina; Lallas, Aimilios; Kyrgidis, Athanassios; Rabinovitz, Harold; Moscarella, Elvira; Ciardo, Silvana; Zalaudek, Iris; Oliviero, Margaret; Losi, Amanda; Gonzalez, Salvador; Guitera, Pascale; Piana, Simonetta; Argenziano, Giuseppe; Pellacani, Giovanni

    2014-10-01

    The current guidelines for the management of basal cell carcinoma (BCC) suggest a different therapeutic approach according to histopathologic subtype. Although dermatoscopic and confocal criteria of BCC have been investigated, no specific studies were performed to evaluate the distinct reflectance confocal microscopy (RCM) aspects of BCC subtypes. To define the specific dermatoscopic and confocal criteria for delineating different BCC subtypes. Dermatoscopic and confocal images of histopathologically confirmed BCCs were retrospectively evaluated for the presence of predefined criteria. Frequencies of dermatoscopic and confocal parameters are provided. Univariate and adjusted odds ratios were calculated. Discriminant analyses were performed to define the independent confocal criteria for distinct BCC subtypes. Eighty-eight BCCs were included. Dermatoscopically, superficial BCCs (n=44) were primarily typified by the presence of fine telangiectasia, multiple erosions, leaf-like structures, and revealed cords connected to the epidermis and epidermal streaming upon RCM. Nodular BCCs (n=22) featured the classic dermatoscopic features and well outlined large basaloid islands upon RCM. Infiltrative BCCs (n=22) featured structureless, shiny red areas, fine telangiectasia, and arborizing vessels on dermatoscopy and dark silhouettes upon RCM. The retrospective design. Dermatoscopy and confocal microscopy can reliably classify different BCC subtypes. Copyright © 2014 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  20. Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

    2016-01-01

    Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

  1. Scanning Ion Conductance Microscopy of Live Keratinocytes

    Science.gov (United States)

    Hegde, V.; Mason, A.; Saliev, T.; Smith, F. J. D.; McLean, W. H. I.; Campbell, P. A.

    2012-07-01

    Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (monitoring the most delicate living structures with attendant high spatial resolutions.

  2. Analysing magnetism using scanning SQUID microscopy.

    Science.gov (United States)

    Reith, P; Renshaw Wang, X; Hilgenkamp, H

    2017-12-01

    Scanning superconducting quantum interference device microscopy (SSM) is a scanning probe technique that images local magnetic flux, which allows for mapping of magnetic fields with high field and spatial accuracy. Many studies involving SSM have been published in the last few decades, using SSM to make qualitative statements about magnetism. However, quantitative analysis using SSM has received less attention. In this work, we discuss several aspects of interpreting SSM images and methods to improve quantitative analysis. First, we analyse the spatial resolution and how it depends on several factors. Second, we discuss the analysis of SSM scans and the information obtained from the SSM data. Using simulations, we show how signals evolve as a function of changing scan height, SQUID loop size, magnetization strength, and orientation. We also investigated 2-dimensional autocorrelation analysis to extract information about the size, shape, and symmetry of magnetic features. Finally, we provide an outlook on possible future applications and improvements.

  3. Dimensional metrology of lab-on-a-chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy.

    Science.gov (United States)

    Reyes, D R; Halter, M; Hwang, J

    2015-07-01

    The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in-house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser-scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three-dimensional metrology of critical internal structures in lab-on-a-chip devices because scans can be performed rapidly and noninvasively prior to their use. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  4. Measurement of surgically induced corneal deformations using three-dimensional confocal microscopy.

    Science.gov (United States)

    Petroll, W M; Roy, P; Chuong, C J; Hall, B; Cavanagh, H D; Jester, J V

    1996-03-01

    The goal of this study was to develop and apply a new set of experimental techniques for measuring the local deformations induced by partial-thickness corneal incisions in situ. Eight adult cat eyes were enucleated and cannulated, with corneal viability maintained as close to in vivo conditions as possible and intraocular pressure (IOP) carefully controlled. Experimental measurements were made pre/post radial keratotomy (RK) surgery in situ at IOPs of 15, 30, and 45 mm Hg. Incision depth and cross-sectional profiles were measured at the midpoint of selected incisions using three-dimensional (3-D) tandem scanning confocal microscopy (TSCM); central corneal curvature was estimated using a commercial corneal topographical analysis system, and corneal thickness was assessed by both 3-D TSCM and ultrasonic pachymetry. Corneas were then processed for light microscopy and incision depth was measured histologically. Finite element models were developed for comparison with the experimental measurements. There was no significant change in central corneal thickness (-5.3 +/- 3.9%, n = 8) over the course of the experiments, demonstrating that normal endothelial cell function and normal stromal hydration was maintained. The in situ TSCM incision depth measurements were significantly correlated with the histological measurements (slope = 0.95, R = 0.854, p mechanical behavior of the cornea after refractive surgery. These data should provide the foundation for future studies into the relationships between local tissue mechanics and corneal wound healing.

  5. Clinical use of in vivo confocal microscopy through focusing in corneal refractive surgery.

    Science.gov (United States)

    Ying, Li; Xiao, Zhang; Liuxueying, Zhong; Yumei, Jin

    2006-11-01

    To illustrate the use of in vivo confocal microscopy through focusing to observe normal cornea and corneal wound healing after excimer laser refractive surgery. A total of 197 eyes, including both unoperated eyes and eyes that had undergone LASIK, photorefractive keratectomy (PRK), or laser epithelial keratomileusis (LASEK), were examined using in vivo confocal microscopy through focusing. Images of the various corneal layers resolved by confocal microscopy through focusing were recorded and analyzed. Pachymetry of the cornea, epithelium, and stroma was also recorded for all eyes. The t test was used to evaluate the differences between unoperated eyes and postoperative eyes and the change in corneal pachymetry preoperatively to postoperatively with each type of surgery. A P value <.05 was considered statistically significant. Each layer of the cornea could be resolved in unoperated eyes and eyes that had undergone refractive surgery. Wound healing could be followed over time using confocal microscopy through focusing. In eyes that underwent PRK, at 1 month postoperatively, the entire cornea and stroma were thinner than preoperatively, whereas the epithelial layer was statistically significantly thicker (P<.05). Haze after PRK is seen as reflectivity of subepithelial anterior stroma. No clinically significant haze was observed in eyes that underwent LASEK or LASIK. The features of the eyes that underwent LASIK were the same as those of unoperated eyes. Confocal microscopy through focusing was useful in documenting cellular morphology in unoperated corneas and corneas that had undergone refractive surgery. Wound-healing characteristics of eyes that had undergone refractive surgery were also documented using confocal microscopy.

  6. Confocal reader for biochip screening and fluorescence microscopy.

    Science.gov (United States)

    Ruckstuhl, Thomas; Walser, Andreas; Verdes, Dorinel; Seeger, Stefan

    2005-03-15

    We developed a fluorescence reader for the sensitive detection of surface-generated fluorescence. The system is applicable for high resolution imaging as well as for the readout of large biochips. The surface of a microscope coverslip is scanned with a laser beam focused to a waist diameter of 500 nm (FWHM) by means of a single aspheric lens. Scanning large areas with a focused beam usually evokes the need of automatic control elements to adjust the laser spot to the designated position at the surface. Due to the special design of the reader, the focus keeps at the plane of the surface even when scanning large areas, obviating the requirement of any real time control. Thus the instrument is straightforward and inexpensive. Nevertheless it features a high sensitivity and high optical resolution. The versatility of the instrument is demonstrated by imaging cells and reading out a DNA-chip. The excellent sensitivity is shown by detecting single fluorescently labeled antibodies.

  7. Scanning electron microscopy of superficial white onychomycosis*

    Science.gov (United States)

    de Almeida Jr., Hiram Larangeira; Boabaid, Roberta Oliveira; Timm, Vitor; Silva, Ricardo Marques e; de Castro, Luis Antonio Suita

    2015-01-01

    Superficial white onychomycosis is characterized by opaque, friable, whitish superficial spots on the nail plate. We examined an affected halux nail of a 20-year-old male patient with scanning electron microscopy. The mycological examination isolated Trichophyton mentagrophytes. Abundant hyphae with the formation of arthrospores were found on the nail's surface, forming small fungal colonies. These findings showed the great capacity for dissemination of this form of onychomycosis. PMID:26560225

  8. Scanning electron microscopy of molluscum contagiosum*

    OpenAIRE

    Almeida Jr,Hiram Larangeira de; Abuchaim,Martha Oliveira; Schneide, Maiko Abel; Marques, Leandra; Castro, Luis Antônio Suíta de

    2013-01-01

    Molluscum contagiosum is a disease caused by a poxvirus. It is more prevalent in children up to 5 years of age. There is a second peak of incidence in young adults. In order to examine its ultrastructure, three lesions were curetted without disruption, cut transversely with a scalpel, and routinely processed for scanning electron microscopy (SEM). The oval structure of molluscum contagiosum could be easily identified. In its core, there was a central umbilication and just below this depressio...

  9. Scanning electron microscopy of cold gases

    Science.gov (United States)

    Santra, Bodhaditya; Ott, Herwig

    2015-06-01

    Ultracold quantum gases offer unique possibilities to study interacting many-body quantum systems. Probing and manipulating such systems with ever increasing degree of control requires novel experimental techniques. Scanning electron microscopy is a high resolution technique which can be used for in situ imaging, single site addressing in optical lattices and precision density engineering. Here, we review recent advances and achievements obtained with this technique and discuss future perspectives.

  10. Measuring skin penetration by confocal Raman microscopy (CRM): correlation to results from conventional experiments

    Science.gov (United States)

    Lunter, Dominique; Daniels, Rolf

    2016-03-01

    Confocal Raman microscopy has become an advancing technique in the characterization of drug transport into the skin. In this study the skin penetration of a local anesthetic from a semisolid preparation was investigated. Furthermore, the effect of the chemical enhancers propylene glycol and POE-23-lauryl ether on its penetration was investigated. The results show that confocal Raman microscopy may provide detailed information on the penetration of APIs into the skin and may elucidate their distribution within the skin with high resolution. The results of the CRM analysis are fully in line with those of conventional permeation and penetration experiments.

  11. Confocal microscopy on the beamline: novel three-dimensional imaging and sample positioning

    Science.gov (United States)

    Khan, I.; Gillilan, R.; Kriksunov, I.; Williams, R.; Zipfel, W. R.; Englich, U.

    2012-01-01

    Confocal microscopy, a technique that has been extensively applied in cellular biological studies, may also be applied to the visualization and three-dimensional imaging of protein crystals at high resolution on synchrotron beamlines. Protein crystal samples are examined using a commercially available confocal microscope adapted for cryogenic use. A preliminary test using a custom confocal design adapted for beamline use is also presented. The confocal optics configuration is compatible with nonlinear imaging techniques such as two-photon excited fluorescence imaging and second harmonic generation. The possibilities of this method are explored using two modes: fluorescence and reflection confocal. In fluorescence mode, small amounts of dye are introduced into the crystal through soaking or growth conditions. Under such conditions, protein crystals are easily resolved from salts and amorphous precipitates, which do not generally take up dye. Reflection mode, which does not require dye, still exhibits greater resolution and sensitivity to surface detail than conventional wide-field microscopy as a result of the confocal optics configuration. The inherent three-dimensional nature of the method means that on-axis sample views (along the direction of the X-ray beam) can be reconstructed from an off-axis configuration, simplifying the beamline setup and providing uniquely detailed views of cryogenically cooled crystals. PMID:22997474

  12. Design and Performance of a Multi-Point Scan Confocal Microendoscope

    Directory of Open Access Journals (Sweden)

    Matthew D. Risi

    2014-11-01

    Full Text Available Confocal fluorescence microendoscopy provides high-resolution cellular-level imaging via a minimally invasive procedure, but requires fast scanning to achieve real-time imaging in vivo. Ideal confocal imaging performance is obtained with a point scanning system, but the scan rates required for in vivo biomedical imaging can be difficult to achieve. By scanning a line of illumination in one direction in conjunction with a stationary confocal slit aperture, very high image acquisition speeds can be achieved, but at the cost of a reduction in image quality. Here, the design, implementation, and experimental verification of a custom multi-point aperture modification to a line-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution of a line-scan system while maintaining high imaging rates. In addition, compared to the line-scanning configuration, previously reported simulations predicted that the multi-point aperture geometry greatly reduces the effects of tissue scatter on image quality. Experimental results confirming this prediction are presented.

  13. Investigation into scanning tunnelling luminescence microscopy

    CERN Document Server

    Manson-Smith, S K

    2001-01-01

    This work reports on the development of a scanning tunnelling luminescence (STL) microscope and its application to the study of Ill-nitride semiconductor materials used in the production of light emitting devices. STL microscopy is a technique which uses the high resolution topographic imaging capabilities of the scanning tunnelling microscope (STM) to generate high resolution luminescence images. The STM tunnelling current acts as a highly localised source of electrons (or holes) which generates luminescence in certain materials. Light generated at the STM tunnelling junction is collected concurrently with the height variation of the tunnelling probe as it is scanned across a sample surface, producing simultaneous topographic and luminescence images. Due to the very localised excitation source, high resolution luminescence images can be obtained. Spectroscopic resolution can be obtained by using filters. Additionally, the variation of luminescence intensity with tunnel current and with bias voltage can provi...

  14. Implementation of Accurate and Fast DNA Cytometry by Confocal Microscopy in 3D

    Directory of Open Access Journals (Sweden)

    Lennert S. Ploeger

    2005-01-01

    Full Text Available Background: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. Methods: Nuclear DNA was stained in 14 μm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0 followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of ×40 or ×100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV of the diploid peak was assessed. Results: The lowest CV (10.3% was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at ×60 or ×100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours

  15. Interferometric Synthetic Aperture Microscopy: Computed Imaging for Scanned Coherent Microscopy

    Directory of Open Access Journals (Sweden)

    Stephen A. Boppart

    2008-06-01

    Full Text Available Three-dimensional image formation in microscopy is greatly enhanced by the use of computed imaging techniques. In particular, Interferometric Synthetic Aperture Microscopy (ISAM allows the removal of out-of-focus blur in broadband, coherent microscopy. Earlier methods, such as optical coherence tomography (OCT, utilize interferometric ranging, but do not apply computed imaging methods and therefore must scan the focal depth to acquire extended volumetric images. ISAM removes the need to scan the focus by allowing volumetric image reconstruction from data collected at a single focal depth. ISAM signal processing techniques are similar to the Fourier migration methods of seismology and the Fourier reconstruction methods of Synthetic Aperture Radar (SAR. In this article ISAM is described and the close ties between ISAM and SAR are explored. ISAM and a simple strip-map SAR system are placed in a common mathematical framework and compared to OCT and radar respectively. This article is intended to serve as a review of ISAM, and will be especially useful to readers with a background in SAR.

  16. Diagnosis of thalassemia and iron deficiency anemia using confocal and atomic force microscopy

    Science.gov (United States)

    Tariq, Saira; Bilal, Muhammad; Shahzad, Shaheen; Firdous, Shamaraz; Aziz, Uzma; Ahmed, Mushtaq

    2017-11-01

    Anemia is the most prevalent blood disorder, categorized into thalassemia and iron deficiency anemia. In anemia, the morphology of erythrocytes is disturbed, thus leading to abnormal functioning of the erythrocytes. Globally, thalassemia affects 1.3% of individuals and is one of the most widespread monogenic disorders in Pakistan. All over the World, women and children are most frequently affected by a type of nutritional deficiency known as iron deficiency anemia. The morphological changes that occur in erythrocytes due to these diseases are investigated in this study at the nano-scale level. Fifty samples of blood from individuals suffering from thalassemia or iron deficiency anemia were obtained from different hospitals in Rawalpindi and Islamabad. The blood samples were scanned using atomic force microscopy (AFM) and laser scanning confocal microscopy (LSCM) to check the morphological changes in both types of anemia. According to the present study, thalassemia is most prevalent in females in the age group between 5 and 15 years old, and iron deficiency is most prevalent in females in the age groups of 16–25 and 36–45 years old. Erythrocyte morphology is the significant determinant for diagnosing and discriminating between these two types of diseases. The study reports deformed erythrocytes in anemic patients, which were different from the ones that existed in the control. Thalassemia erythrocytes showed a crenated shape, iron deficiency anemia erythrocytes showed an elliptocyte shape and healthy erythrocytes showed a biconcave disk shape when using AFM and LSCM. These techniques seem to be very promising, cheap and less time consuming in determining the structure–function relationship of erythrocytes of thalassemic and iron deficiency anemic patients. The results of LSCM and AFM are quite useful in determining the morphological changes in erythrocytes and to study the disease at the molecular level within short period of time. Hence, we encourage

  17. Scanning Probe Microscopy of Organic Solar Cells

    Science.gov (United States)

    Reid, Obadiah G.

    Nanostructured composites of organic semiconductors are a promising class of materials for the manufacture of low-cost solar cells. Understanding how the nanoscale morphology of these materials affects their efficiency as solar energy harvesters is crucial to their eventual potential for large-scale deployment for primary power generation. In this thesis we describe the use of optoelectronic scanning-probe based microscopy methods to study this efficiency-structure relationship with nanoscale resolution. In particular, our objective is to make spatially resolved measurements of each step in the power conversion process from photons to an electric current, including charge generation, transport, and recombination processes, and correlate them with local device structure. We have achieved two aims in this work: first, to develop and apply novel electrically sensitive scanning probe microscopy experiments to study the optoelectronic materials and processes discussed above; and second, to deepen our understanding of the physics underpinning our experimental techniques. In the first case, we have applied conductive-, and photoconductive atomic force (cAFM & pcAFM) microscopy to measure both local photocurrent collection and dark charge transport properties in a variety of model and novel organic solar cell composites, including polymer/fullerene blends, and polymer-nanowire/fullerene blends, finding that local heterogeneity is the rule, and that improvements in the uniformity of specific beneficial nanostructures could lead to large increases in efficiency. We have used scanning Kelvin probe microscopy (SKPM) and time resolved-electrostatic force microscopy (trEFM) to characterize all-polymer blends, quantifying their sensitivity to photochemical degradation and the subsequent formation of local charge traps. We find that while trEFM provides a sensitive measure of local quantum efficiency, SKPM is generally unsuited to measurements of efficiency, less sensitive than tr

  18. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    Directory of Open Access Journals (Sweden)

    Nohra E. Beltran

    2013-01-01

    Full Text Available The gastric mucosa ischemic tissular damage plays an important role in critical care patients’ outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine. The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10% for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (. Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia.

  19. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    Science.gov (United States)

    Beltran, Nohra E.; Garcia, Laura E.; Garcia-Lorenzana, Mario

    2013-01-01

    The gastric mucosa ischemic tissular damage plays an important role in critical care patients' outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine). The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10%) for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (P < 0.01). Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia. PMID:23841094

  20. GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method.

    Science.gov (United States)

    Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae

    2016-12-01

    We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection.

  1. [The ocular surface of severe alkali burns patients on confocal microscopy].

    Science.gov (United States)

    Zhu, Wen-qing; Xu, Jian-jiang; Sun, Xing-huai; Qiu, Ting; Hong, Jia-xu; Wang, Yan; Wang, Wen-tao

    2010-01-01

    To analyze the morphology on the ocular surface of severe alkali burns patients by in vivo laser scanning confocal microscopy. This research was a retrospective observation case series. From February to November 2008 in Eye Ear Nose and Throat Hospital of Fudan University, 39 alkali burns patients who classified as III or IV according to Roper-Hall classification were enrolled in this study. They were divided into four groups according to the course of disease: A (less than 3 months), B (3 - 6 months), C (6 - 12 months) and D (over 12 months). In vivo laser scanning confocal microscopic examinations were performed on the injured cornea, the limbus and the bulbar conjunctiva and the images were recorded. The morphology of the injured cornea, the limbus and the bulbar conjunctiva was analyzed and the densities of the inflammatory cells and dendritic cells in the limbus were calculated. One-way analysis of variance was used to compare the means of the inflammatory cells and dendritic cells. Subsequently the data between two groups were analyzed by least significant difference. The corneal epitheliums of the patients in Group A manifested large irregular features with hyperreflective cytoplasm and hyporeflective nuclei, sometimes losing cell features. There were numerous small hyperreflective inflammatory cells in groups beneath the superficial epitheliums. Shallow corneal stroma was edema, and it was hard to discriminate the morphology of the stromal cells. Deep stromal cells were in the activated state. The images of the endothelial layer were unclear. In Group B and Group C, there were the same manifestation of the superficial epitheliums as the group A and it disappeared in Group D. The inflammatory cells beneath the superficial epitheliums reduced and some residual basal epitheliums and hyperreflective conjunctiva-like epitheliums were visible in Group B and Group C. In Group D, there were small oval tight-arranged cells with punctiform hyperreflective nuclei

  2. Microscopia confocal in vivo nos depósitos corneanos por amiodarona In vivo confocal microscopy in amiodarone corneal deposits

    Directory of Open Access Journals (Sweden)

    Gustavo Victor

    2007-02-01

    Full Text Available OBJETIVO: Descrever os achados da microscopia confocal in vivo em pacientes nos diversos estágios de ceratopatia induzida por amiodarona, e correlacionar o estadiamento biomicroscópico com o estadiamento confocal. MÉTODOS: Vinte olhos de 10 pacientes (6 homens e 4 mulheres em tratamento com amiodarona, que apresentavam ceratopatia induzida pela droga, foram selecionados para o estudo, com a microscopia confocal (MC. RESULTADOS: A média de idade foi 58 ± 6,2 anos (50-66 anos e o tempo de uso da droga foi de 6 ± 3,2 anos (2-11 anos. Todos pacientes tinham acuidade visual com correção melhor ou igual a 20/40. A biomicroscopia evidenciou ceratopatia por amiodarona: dois pacientes no estágio 1, quatro no estágio 2 e quatro no estágio 3. Todas as córneas apresentaram inclusões intracelulares brilhantes e de alta refletividade na camada epitelial basal. A partir dos estágios 2 e 3, foram encontrados microdepósitos em todas camadas corneanas. Foram observados afilamento e aumento da tortuosidade dos nervos corneanos nos estágios 2 e 3 da ceratopatia. A contagem endotelial média foi de 2.524 ± 150,3 células/mm². CONCLUSÃO: O epitélio basal foi o mais acometido nos diferentes estágios da ceratopatia. Nos pacientes do estágio 1 a biomicroscopia, os microdepósitos subepiteliais são restritos ao epitélio superficial e basal, ao passo que nos pacientes dos estágios 2 e 3, os microdepósitos afetam todas camadas corneanas. À medida que a ceratopatia avança, os nervos corneanos ficam mais afilados e tortuosos.PURPOSE: To describe in vivo confocal microscopy findings in patients with different stages of amiodarone-induced keratopathy, and correlate biomicroscopy stages with confocal stages. METHODS: Twenty eyes of 10 patients (6 men and 4 women, who receive treatment with amiodarone were selected for the study with confocal microscopy (MC. RESULTS: The average age was 58 ± 6.2 years (50-66 years and time of use of the drug was 6

  3. Chemical Phenomena of Atomic Force Microscopy Scanning.

    Science.gov (United States)

    Ievlev, Anton V; Brown, Chance; Burch, Matthew J; Agar, Joshua C; Velarde, Gabriel A; Martin, Lane W; Maksymovych, Petro; Kalinin, Sergei V; Ovchinnikova, Olga S

    2018-02-12

    Atomic force microscopy is widely used for nanoscale characterization of materials by scientists worldwide. The long-held belief of ambient AFM is that the tip is generally chemically inert but can be functionalized with respect to the studied sample. This implies that basic imaging and scanning procedures do not affect surface and bulk chemistry of the studied sample. However, an in-depth study of the confined chemical processes taking place at the tip-surface junction and the associated chemical changes to the material surface have been missing as of now. Here, we used a hybrid system that combines time-of-flight secondary ion mass spectrometry with an atomic force microscopy to investigate the chemical interactions that take place at the tip-surface junction. Investigations showed that even basic contact mode AFM scanning is able to modify the surface of the studied sample. In particular, we found that the silicone oils deposited from the AFM tip into the scanned regions and spread to distances exceeding 15 μm from the tip. These oils were determined to come from standard gel boxes used for the storage of the tips. The explored phenomena are important for interpreting and understanding results of AFM mechanical and electrical studies relying on the state of the tip-surface junction.

  4. Through the looking glass : Confocal microscopy imaging of basal cell carcinoma

    NARCIS (Netherlands)

    Kadouch, D.J.

    2017-01-01

    This thesis focuses on improving diagnosis and treatment for patients suffering from BCC. Although some aspects of optical coherence tomography imaging and Raman spectroscopy will be discussed in this thesis, it focuses mainly on incorporating reflectance confocal microscopy (RCM) as noninvasive

  5. Hybrid Rayleigh, Raman and TPE fluorescence spectral confocal microscopy of living cells

    NARCIS (Netherlands)

    Pully, V.V.; Lenferink, Aufrid T.M.; Otto, Cornelis

    2010-01-01

    A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low-wavenumber-resolution Raman imaging, Rayleigh scatter imaging and two-photon fluorescence (TPE) spectral imaging, fast ‘amplitude-only’ TPE-fluorescence imaging and high-spectral-resolution Raman imaging.

  6. Fully automatic evaluation of the corneal endothelium from in vivo confocal microscopy

    NARCIS (Netherlands)

    Selig, B.; Vermeer, K.A.; Rieger, B.; Hillenaar, T.; Hendriks, C.L.L.

    2015-01-01

    Background Manual and semi-automatic analyses of images, acquired in vivo by confocal microscopy, are often used to determine the quality of corneal endothelium in the human eye. These procedures are highly time consuming. Here, we present two fully automatic methods to analyze and quantify corneal

  7. Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index

    NARCIS (Netherlands)

    Besseling, T. H.|info:eu-repo/dai/nl/35218602X; Jose, J.|info:eu-repo/dai/nl/371558158; Blaaderen, A. Van|info:eu-repo/dai/nl/092946488

    2015-01-01

    Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid,

  8. Hollow-tip scanning photoelectron microscopy

    Science.gov (United States)

    Cherkun, A. P.; Mironov, B. N.; Aseyev, S. A.; Chekalin, S. V.

    2014-07-01

    A new type of microscopy based on scanning in vacuum by a beam of charged particles transmitted through a hollow probe has been implemented. This approach provides controllable motion of spatially localized ion, electron, molecular (atomic), and soft X-ray beams and investigation of the surface in the shear force mode. In the photoelectron mode, in which electrons are transmitted through a 2-μm quartz capillary, a surface profile of gadolinium irradiated by 400-nm femtosecond laser pulses has been visualized with a subwave spatial resolution. The new method of microscopy opens an opportunity of investigations in the field of nanometer local photodesorption of molecular ions (one of the last ideas of V.S. Letokhov).

  9. Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

    Science.gov (United States)

    Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

    2016-12-01

    Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

  10. [Imaging of corneal dystrophies: Correlations between en face anterior segment OCT and in vivo confocal microscopy].

    Science.gov (United States)

    Ghouali, W; Tahiri Joutei Hassani, R; Liang, H; Dupont-Monod, S; Auclin, F; Baudouin, C; Labbé, A

    2015-05-01

    To evaluate the usefulness of en face Optical Coherence Tomography (OCT) for evaluation of corneal dystrophies and to describe correlations with in vivo confocal microscopy (IVCM). Thirty-two eyes of 16 patients with 4 types of corneal dystrophies (epithelial basement membrane dystrophy, Fuchs dystrophy, Reis-Bücklers corneal dystrophy and Crocodile Shagreen dystrophy) were enrolled in this study. Axial and reconstructed en face scans were acquired using OCT. Images were then correlated to IVCM findings. En face OCT provided new insights into the structure, size and depth of corneal tissue alterations in various corneal dystrophies. OCT en face images were well correlated with IVCM features. Despite lower resolution than IVCM, en face OCT offers the advantages of being non-invasive and allowing the analysis of larger corneal areas. En face OCT provides useful new information in corneal dystrophies. This imaging technique will probably increase in popularity in the near future for the assessment of various anterior segment diseases. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  11. Mapping of normal corneal K-structures by in vivo laser confocal microscopy.

    Science.gov (United States)

    Yokogawa, Hideaki; Kobayashi, Akira; Sugiyama, Kazuhisa

    2008-09-01

    To produce 2-dimensional reconstruction maps of normal human corneal fibrous structures beneath the Bowman layer (K-structures) by in vivo laser confocal microscopy and to show association of structures with the anterior corneal mosaic (ACM). Central corneal regions of 3 healthy volunteers were scanned. Acquired images of K-structures for each eye were arranged and mapped into a subconfluent montage. For each subject, electrical tracings of K-structures were superimposed on a slit-lamp photograph of the ACM produced by rubbing the eyelid. A mean of 677 +/- 211 images of K-structures were obtained for each eye. Mean dimensions of the mapped areas were 5.88 +/- 0.50 (horizontal) and 3.51 +/- 1.37 mm (vertical). In all subjects, K-structures formed a netlike pattern (mean area, 0.082 +/- 0.051 mm), and electrical tracings had good concordance with the ACM. This is the first study, to our knowledge, to elucidate the overall distribution of K-structures in normal human corneas. The netlike pattern of K-structures corresponded well with ACM pattern. These results support the hypothesis that the K-structures are the anterior collagen fiber bundles running at the posterior surface of the Bowman layer and thus are the structural basis for ACM formation.

  12. Grading keratinocyte atypia in actinic keratosis: a correlation of reflectance confocal microscopy and histopathology.

    Science.gov (United States)

    Pellacani, G; Ulrich, M; Casari, A; Prow, T W; Cannillo, F; Benati, E; Losi, A; Cesinaro, A M; Longo, C; Argenziano, G; Soyer, H P

    2015-11-01

    Actinic Keratosis (AK) is the clinical manifestation of cutaneous dysplasia of epidermal keratinocytes, with progressive trend towards squamous cell carcinoma. To evaluate the strength of the correlation between keratinocyte atypia, as detected by Reflectance Confocal Microscopy (RCM) and histopathology, and to develop a more objective atypia grading scale for RCM quantification, through a discrete ranking. A total of 48 AKs and two control areas (photodamaged and non-photodamaged skin) were selected for this study. All these areas were documented by RCM and biopsied for histopathology. One representative image of the epidermis was selected for RCM and for histopathology and used for side-by-side comparison with purpose written software. The assessor chose which of two images displayed more keratinocyte atypia, and an ordered list from the image showing the least to the most keratinocyte atypia was generated. Three evaluations were obtained for RCM and two for histopathology. Good interobserver correlation was obtained for RCM and histopathology grading, with high concordance between RCM and histopathology grading. Expert rater scan consistently distinguish different grades of cytological atypia. Non-invasive RCM data from in vivo imaging can be graded for keratinocyte atypia, comparable to histopathological grading. © 2015 European Academy of Dermatology and Venereology.

  13. In vivo confocal microscopy of meibomian glands and palpebral conjunctiva in vernal keratoconjunctivitis

    Directory of Open Access Journals (Sweden)

    Qiaoling Wei

    2015-01-01

    Full Text Available Purpose: To investigate the correlations between conjunctival inflammatory status and meibomian gland (MG morphology in vernal keratoconjunctivitis (VKC patients by using in vivo confocal microscopy (CM. Materials and Methods: Nineteen VKC patients (7 limbal, 7 tarsal, and 5 mixed forms and 16 normal volunteers (controls were enrolled. All subjects underwent CM scanning to obtain the images of upper palpebral conjunctiva and MGs. Inflammatory cell (IC density in palpebral conjunctival epithelial and stromal layers, Langerhans cell (LC density at lid margins and the stroma adjacent to the MG, and MG acinar unit density (MGAUD were recorded. The longest and shortest diameters of MG acinar were measured. The Kruskal-Wallis test was used to compare the parameter differences whereas the Spearman′s rank correlation analysis was applied to determine their correlations. Results: Among all groups, no significant statistical differences were found in epithelial and stromal IC densities, mean values of MG acinar unit densities, or longest and shortest diameters. Both LC parameters in the tarsal-mixed groups were significantly higher than those in the limbal and control groups. All LC densities of VKC patients showed a positive correlation with MGAUD and shortest diameter. Conclusions: In VKC patients, the conjunctival inflammatory status could be associated with the MG status. In vivo CM is a noninvasive, efficient tool in the assessment of MG status and ocular surface.

  14. In vivo confocal microscopy of meibomian glands and palpebral conjunctiva in vernal keratoconjunctivitis.

    Science.gov (United States)

    Wei, Qiaoling; Le, Qihua; Hong, Jiaxu; Xiang, Jun; Wei, Anji; Xu, Jianjiang

    2015-04-01

    To investigate the correlations between conjunctival inflammatory status and meibomian gland (MG) morphology in vernal keratoconjunctivitis (VKC) patients by using in vivo confocal microscopy (CM). Nineteen VKC patients (7 limbal, 7 tarsal, and 5 mixed forms) and 16 normal volunteers (controls) were enrolled. All subjects underwent CM scanning to obtain the images of upper palpebral conjunctiva and MGs. Inflammatory cell (IC) density in palpebral conjunctival epithelial and stromal layers, Langerhans cell (LC) density at lid margins and the stroma adjacent to the MG, and MG acinar unit density (MGAUD) were recorded. The longest and shortest diameters of MG acinar were measured. The Kruskal-Wallis test was used to compare the parameter differences whereas the Spearman's rank correlation analysis was applied to determine their correlations. Among all groups, no significant statistical differences were found in epithelial and stromal IC densities, mean values of MG acinar unit densities, or longest and shortest diameters. Both LC parameters in the tarsal-mixed groups were significantly higher than those in the limbal and control groups. All LC densities of VKC patients showed a positive correlation with MGAUD and shortest diameter. In VKC patients, the conjunctival inflammatory status could be associated with the MG status. In vivo CM is a noninvasive, efficient tool in the assessment of MG status and ocular surface.

  15. Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

    Science.gov (United States)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

  16. Soft stylus probes for scanning electrochemical microscopy.

    Science.gov (United States)

    Cortés-Salazar, Fernando; Träuble, Markus; Li, Fei; Busnel, Jean-Marc; Gassner, Anne-Laure; Hojeij, Mohamad; Wittstock, Gunther; Girault, Hubert H

    2009-08-15

    A soft stylus microelectrode probe has been developed to carry out scanning electrochemical microscopy (SECM) of rough, tilted, and large substrates in contact mode. It is fabricated by first ablating a microchannel in a polyethylene terephthalate thin film and filling it with a conductive carbon ink. After curing the carbon track and lamination with a polymer film, the V-shaped stylus was cut thereby forming a probe, with the cross section of the carbon track at the tip being exposed either by UV-photoablation machining or by blade cutting followed by polishing to produce a crescent moon-shaped carbon microelectrode. The probe properties have been assessed by cyclic voltammetry, approach curves, and line scans over electrochemically active and inactive substrates of different roughness. The influence of probe bending on contact mode imaging was then characterized using simple patterns. Boundary element method simulations were employed to rationalize the distance-dependent electrochemical response of the soft stylus probes.

  17. An interactive visualization tool for multi-channel confocal microscopy data in neurobiology research

    KAUST Repository

    Yong Wan,

    2009-11-01

    Confocal microscopy is widely used in neurobiology for studying the three-dimensional structure of the nervous system. Confocal image data are often multi-channel, with each channel resulting from a different fluorescent dye or fluorescent protein; one channel may have dense data, while another has sparse; and there are often structures at several spatial scales: subneuronal domains, neurons, and large groups of neurons (brain regions). Even qualitative analysis can therefore require visualization using techniques and parameters fine-tuned to a particular dataset. Despite the plethora of volume rendering techniques that have been available for many years, the techniques standardly used in neurobiological research are somewhat rudimentary, such as looking at image slices or maximal intensity projections. Thus there is a real demand from neurobiologists, and biologists in general, for a flexible visualization tool that allows interactive visualization of multi-channel confocal data, with rapid fine-tuning of parameters to reveal the three-dimensional relationships of structures of interest. Together with neurobiologists, we have designed such a tool, choosing visualization methods to suit the characteristics of confocal data and a typical biologist\\'s workflow. We use interactive volume rendering with intuitive settings for multidimensional transfer functions, multiple render modes and multi-views for multi-channel volume data, and embedding of polygon data into volume data for rendering and editing. As an example, we apply this tool to visualize confocal microscopy datasets of the developing zebrafish visual system.

  18. An Interactive Visualization Tool for Multi-channel Confocal Microscopy Data in Neurobiology Research

    Science.gov (United States)

    Wan, Yong; Otsuna, Hideo; Chien, Chi-Bin; Hansen, Charles

    2010-01-01

    Confocal microscopy is widely used in neurobiology for studying the three-dimensional structure of the nervous system. Confocal image data are often multi-channel, with each channel resulting from a different fluorescent dye or fluorescent protein; one channel may have dense data, while another has sparse; and there are often structures at several spatial scales: subneuronal domains, neurons, and large groups of neurons (brain regions). Even qualitative analysis can therefore require visualization using techniques and parameters fine-tuned to a particular dataset. Despite the plethora of volume rendering techniques that have been available for many years, the techniques standardly used in neurobiological research are somewhat rudimentary, such as looking at image slices or maximal intensity projections. Thus there is a real demand from neurobiologists, and biologists in general, for a flexible visualization tool that allows interactive visualization of multi-channel confocal data, with rapid fine-tuning of parameters to reveal the three-dimensional relationships of structures of interest. Together with neurobiologists, we have designed such a tool, choosing visualization methods to suit the characteristics of confocal data and a typical biologist’s workflow. We use interactive volume rendering with intuitive settings for multidimensional transfer functions, multiple render modes and multi-views for multi-channel volume data, and embedding of polygon data into volume data for rendering and editing. As an example, we apply this tool to visualize confocal microscopy datasets of the developing zebrafish visual system. PMID:19834225

  19. An FFT-based Method for Attenuation Correction in Fluorescence Confocal Microscopy

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.; Bakker, M.

    1993-01-01

    A problem in three-dimensional imaging by a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. In this paper we propose a new method to correct for these

  20. An FFT-based method for attenuation correction in fluorescence confocal microscopy

    NARCIS (Netherlands)

    J.B.T.M. Roerdink (Jos); M. Bakker (Miente)

    1993-01-01

    htmlabstractA problem in three-dimensional imaging by a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. In this paper we propose a new method to correct

  1. FFT-Based Methods for Nonlinear Image Restoration in Confocal Microscopy

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.

    1994-01-01

    Recently we developed a new method for attenuation correction in 3D imaging by a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode. The fundamental element in our approach consisted of multiplying the measured fluorescent intensity by a correction factor involving a convolution

  2. Water-Immersible MEMS scanning mirror designed for wide-field fast-scanning photoacoustic microscopy

    Science.gov (United States)

    Yao, Junjie; Huang, Chih-Hsien; Martel, Catherine; Maslov, Konstantin I.; Wang, Lidai; Yang, Joon-Mo; Gao, Liang; Randolph, Gwendalyn; Zou, Jun; Wang, Lihong V.

    2013-03-01

    By offering images with high spatial resolution and unique optical absorption contrast, optical-resolution photoacoustic microscopy (OR-PAM) has gained increasing attention in biomedical research. Recent developments in OR-PAM have improved its imaging speed, but have sacrificed either the detection sensitivity or field of view or both. We have developed a wide-field fast-scanning OR-PAM by using a water-immersible MEMS scanning mirror (MEMS-ORPAM). Made of silicon with a gold coating, the MEMS mirror plate can reflect both optical and acoustic beams. Because it uses an electromagnetic driving force, the whole MEMS scanning system can be submerged in water. In MEMS-ORPAM, the optical and acoustic beams are confocally configured and simultaneously steered, which ensures uniform detection sensitivity. A B-scan imaging speed as high as 400 Hz can be achieved over a 3 mm scanning range. A diffraction-limited lateral resolution of 2.4 μm in water and a maximum imaging depth of 1.1 mm in soft tissue have been experimentally determined. Using the system, we imaged the flow dynamics of both red blood cells and carbon particles in a mouse ear in vivo. By using Evans blue dye as the contrast agent, we also imaged the flow dynamics of lymphatic vessels in a mouse tail in vivo. The results show that MEMS-OR-PAM could be a powerful tool for studying highly dynamic and time-sensitive biological phenomena.

  3. Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions.

    Science.gov (United States)

    Zheng, Haocheng; Goldner, Lori S; Leuba, Sanford H

    2007-03-01

    Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

  4. Changing paradigms in dermatology: confocal microscopy in clinical and surgical dermatology.

    Science.gov (United States)

    González, Salvador; Swindells, Kirsty; Rajadhyaksha, Milind; Torres, Abel

    2003-01-01

    The current practice of pathology and dermatopathology depends upon the evaluation of tissue in some manner extirpated from the patient and then processed and stained. While high resolution of detail can be accomplished by this method, there are certain risks and disadvantages. Recent imaging techniques now allow for a potential of achieving noninvasive high-resolution analysis of lesions in situ in the patient. Of these, Reflectance mode confocal microscopy offers the highest resolution imaging comparable to routine histology. Being entirely non invasive, skin can be observed in its native, dynamic state. This chapter will review the fundamentals of in vivo confocal imaging and the clinical applications in general and surgical dermatology.

  5. Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin

    Science.gov (United States)

    Song, Eunjoo; Ahn, YoonJoon; Ahn, Jinhyo; Ahn, Soyeon; Kim, Changhwan; Choi, Sanghoon; Boutilier, Richard Martin; Lee, Yongjoong; Kim, Pilhan; Lee, Ho

    2015-10-01

    We examined the optical clearing assisted confocal microscopy of the transgenic mouse skin. The pinna and dorsal skin were imaged with a confocal microscope after the application of glycerol and FocusClear. In case of the glycerol-treated pinna, the clearing was minimal due to the inefficient permeability. However, the imaging depth was improved when the pinna was treated with FocusClear. In case of dorsal skin, we were able to image deeply to the subcutaneous connective tissue with both agents. Various skin structures such as the vessel, epithelium cells, cartilage, dermal cells, and hair follicles were clearly imaged.

  6. Differential-Concentration Scanning Ion Conductance Microscopy.

    Science.gov (United States)

    Perry, David; Page, Ashley; Chen, Baoping; Frenguelli, Bruno G; Unwin, Patrick R

    2017-11-21

    Scanning ion conductance microscopy (SICM) is a nanopipette-based scanning probe microscopy technique that utilizes the ionic current flowing between an electrode inserted inside a nanopipette probe containing electrolyte solution and a second electrode placed in a bulk electrolyte bath, to provide information on a substrate of interest. For most applications to date, the composition and concentration of the electrolyte inside and outside the nanopipette is identical, but it is shown herein that it can be very beneficial to lift this restriction. In particular, an ionic concentration gradient at the end of the nanopipette, generates an ionic current with a greatly reduced electric field strength, with particular benefits for live cell imaging. This differential concentration mode of SICM (ΔC-SICM) also enhances surface charge measurements and provides a new way to carry out reaction mapping measurements at surfaces using the tip for simultaneous delivery and sensing of the reaction rate. Comprehensive finite element method (FEM) modeling has been undertaken to enhance understanding of SICM as an electrochemical cell and to enable the interpretation and optimization of experiments. It is shown that electroosmotic flow (EOF) has much more influence on the nanopipette response in the ΔC-SICM configuration compared to standard SICM modes. The general model presented advances previous treatments, and it provides a framework for quantitative SICM studies.

  7. Cross-Linking Cellulosic Fibers with Photoreactive Polymers: Visualization with Confocal Raman and Fluorescence Microscopy.

    Science.gov (United States)

    Janko, Marek; Jocher, Michael; Boehm, Alexander; Babel, Laura; Bump, Steven; Biesalski, Markus; Meckel, Tobias; Stark, Robert W

    2015-07-13

    The properties of paper sheets can be tuned by adjusting the surface or bulk chemistry using functional polymers that are applied during (online) or after (offline) papermaking processes. In particular, polymers are widely used to enhance the mechanical strength of the wet state of paper sheets. However, the mechanical strength depends not only on the chemical nature of the polymeric additives but also on the distribution of the polymer on and in the lignocellulosic paper. Here, we analyze the photochemical attachment and distribution of hydrophilic polydimethylacrylamide-co-methacrylate-benzophenone P(DMAA-co-MABP) copolymers with defined amounts of photoreactive benzophenone moieties in model paper sheets. Raman microscopy was used for the unambiguous identification of P(DMAA-co-MABP) and cellulose specific bands and thus the copolymer distribution within the cellulose matrix. Two-dimensional Raman spectral maps at the intersections of overlapping cellulose fibers document that the macromolecules only partially surround the cellulose fibers, favor to attach to the fiber surface, and connect the cellulose fibers at crossings. Moreover, the copolymer appears to accumulate preferentially in holes, vacancies, and dips on the cellulose fiber surface. Correlative brightfield, Raman, and confocal laser scanning microscopy finally reveal a reticular three-dimensional distribution of the polymer and show that the polymer is predominately deposited in regions of high capillarity (i.e., in proximity to fine cellulose fibrils). These data provide deeper insights into the effects of paper functionalization with a copolymer and aid in understanding how these agents ultimately influence the local and overall properties of paper.

  8. Various confocal scan features of cysts and trophozoites in cases with Acanthamoeba keratitis.

    Science.gov (United States)

    Rezaei Kanavi, Mozhgan; Naghshgar, Nima; Javadi, Mohammad Ali; Sadat Hashemi, Marzieh

    2012-01-01

    To describe the various confocal scan features of cysts and trophozoites in patients with Acanthamoeba keratitis and to specify the associated findings. In a retrospective study of cases between June 2005 and June 2010, we reviewed all the recorded confocal scan images of patients given a high index in regards to clinical suspicion of Acanthamoeba keratitis, in order to specify the various morphometric and morphologic features of Acanthamoeba cysts and trophozoites and to characterize the associated findings in such cases. Confocal scan images of 170 eyes from 170 patients were reviewed. Bilayered, target-shaped, coffee-bean and rod-shaped appearances of the cysts were observed in 100%, 82.9%, 36.4%, and 17.5% of cases, respectively. Single file arrangement of the cysts was noticed in 22 cases. The mean size of the cysts was 18.9 µm (range 10-39.6). In all cases, trophozoites were observed as pear-shaped or irregularly wedge-shaped structures, some surrounded by a brilliant halo and some exhibiting fine pseudopodia-like extensions, with mean size of 30.2 µm (range 19.2-55.6). Keratoneuritis and the anterior stromal honeycomb pattern were seen in 28.2% and 5.9% of cases, respectively. To our knowledge, this is the largest case-series study on confocal scan features of Acanthamoeba cysts and trophozoites in cases with clinical diagnosis of Acanthamoeba keratitis specifying the morphologic and morphometric criteria of this infectious organism and the associated findings.

  9. Phase-contrast scanning transmission electron microscopy.

    Science.gov (United States)

    Minoda, Hiroki; Tamai, Takayuki; Iijima, Hirofumi; Hosokawa, Fumio; Kondo, Yukihito

    2015-06-01

    This report introduces the first results obtained using phase-contrast scanning transmission electron microscopy (P-STEM). A carbon-film phase plate (PP) with a small center hole is placed in the condenser aperture plane so that a phase shift is introduced in the incident electron waves except those passing through the center hole. A cosine-type phase-contrast transfer function emerges when the phase-shifted scattered waves interfere with the non-phase-shifted unscattered waves, which passed through the center hole before incidence onto the specimen. The phase contrast resulting in P-STEM is optically identical to that in phase-contrast transmission electron microscopy that is used to provide high contrast for weak phase objects. Therefore, the use of PPs can enhance the phase contrast of the STEM images of specimens in principle. The phase shift resulting from the PP, whose thickness corresponds to a phase shift of π, has been confirmed using interference fringes displayed in the Ronchigram of a silicon single crystal specimen. The interference fringes were found to abruptly shift at the edge of the PP hole by π. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Multimodal backside imaging of a microcontroller using confocal laser scanning and optical-beam-induced current imaging

    Science.gov (United States)

    Finkeldey, Markus; Göring, Lena; Schellenberg, Falk; Brenner, Carsten; Gerhardt, Nils C.; Hofmann, Martin

    2017-02-01

    Microscopy imaging with a single technology is usually restricted to a single contrast mechanism. Multimodal imaging is a promising technique to improve the structural information that could be obtained about a device under test (DUT). Due to the different contrast mechanisms of laser scanning microscopy (LSM), confocal laser scanning microscopy (CLSM) and optical beam induced current microscopy (OBICM), a combination could improve the detection of structures in integrated circuits (ICs) and helps to reveal their layout. While OBIC imaging is sensitive to the changes between differently doped areas and to semiconductor-metal transitions, CLSM imaging is mostly sensitive to changes in absorption and reflection. In this work we present the implementation of OBIC imaging into a CLSM. We show first results using industry standard Atmel microcontrollers (MCUs) with a feature size of about 250nm as DUTs. Analyzing these types of microcontrollers helps to improve in the field of side-channel attacks to find hardware Trojans, possible spots for laser fault attacks and for reverse engineering. For the experimental results the DUT is placed on a custom circuit board that allows us to measure the current while imaging it in our in-house built stage scanning microscope using a near infrared (NIR) laser diode as light source. The DUT is thinned and polished, allowing backside imaging through the Si-substrate. We demonstrate the possibilities using this optical setup by evaluating OBIC, LSM and CLSM images above and below the threshold of the laser source.

  11. Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope.

    Science.gov (United States)

    Kim, Dong Uk; Moon, Sucbei; Song, Hoseong; Kwon, Hyuk-Sang; Kim, Dug Young

    2011-01-01

    High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail. Copyright © 2011 Wiley Periodicals, Inc.

  12. Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

    Science.gov (United States)

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-05-05

    We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

  13. Estudio del endotelio corneal en el queratocono por microscopia confocal Study of the corneal endothelium confocal microscopy in keratoconus

    Directory of Open Access Journals (Sweden)

    María del Carmen Benítez Merino

    2011-12-01

    Full Text Available Objetivo: Describir los hallazgos morfométricos del endotelio corneal por microscopia confocal con CONFOSCAN S-4. Métodos: Estudio descriptivo transversal de 102 ojos con queratocono en el período de septiembre de 2008 a septiembre 2009. A estos pacientes se les realizó microscopia confocal con CosfoscanS-4 para el estudio del endotelio corneal atendiendo el grado de queratocono. Se analizó el comportamiento de la evolución del queratocono según edad y sexo. Las imágenes fueron analizadas y procesadas mediante un programa informático diseñado específicamente para esto. Resultados: Fueron semejantes las edades de los pacientes con queratocono grado I y II, (35,2 y 34,7 años, los grado III presentaron una edad promedio mayor (38,4 años, sin diferencias significativas (p= 0,279. El sexo femenino predominó en 80,4 % de los pacientes. El 100 % de los queratoconos grado III tuvieron endotelios patológicos. Los valores promedios de la densidad celular en los queratoconos grado III (2585,9 células/mm² resultó no significativo (p= 0,339. El polimegatismo en los queratoconos grado III para un 48,69 % fue significativo (p= 0,002. En el pleomorfismo resultó significativo las diferencias observadas entre los tres grados (p= 0,002. Conclusión: Predominó el queratocono grado II para las mujeres y el grado I para los hombres. Los hallazgos morfológicos se manifestaron en la forma y tamaño de las células endoteliales. En córneas con queratocono grado II y III confluyeron células de mediano y gran tamaño con pérdida de su hexagonalidad. La densidad celular se mantuvo dentro del rango de valores normales para cualquier grado de queratocono.Objective: To describe the morphometric findings of the corneal endothelium confocal microscopy with CONFOSCAN S-4 Methods: Descriptive cross-sectional study of 102 eyes with keratoconus performed from September 2008 to September 2009. The study patients had undergone confocal microscopy with

  14. Angular Approach Scanning Ion Conductance Microscopy.

    Science.gov (United States)

    Shevchuk, Andrew; Tokar, Sergiy; Gopal, Sahana; Sanchez-Alonso, Jose L; Tarasov, Andrei I; Vélez-Ortega, A Catalina; Chiappini, Ciro; Rorsman, Patrik; Stevens, Molly M; Gorelik, Julia; Frolenkov, Gregory I; Klenerman, David; Korchev, Yuri E

    2016-05-24

    Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. Corneal collagen cross-linking: a confocal, electron, and light microscopy study of eye bank corneas.

    Science.gov (United States)

    Dhaliwal, Jasmeet S; Kaufman, Stephen C

    2009-01-01

    The purpose of this study was to evaluate morphological changes induced by corneal collagen cross-linking in a human ex vivo cornea, using confocal, electron, and light microscopy. The central epithelium was partially removed from ex vivo human corneal buttons. Riboflavin 0.1% solution was applied before ultraviolet A light treatment and then for every 2 minutes for 30 minutes while the corneas were exposed to ultraviolet A light at a wavelength of 370 nm and intensity of 3 mW/cm(2). Each cornea was evaluated using confocal, electron, and light microscopy. Confocal microscopy demonstrated normal-appearing corneas on their initial pretreatment examination, with reduced stromal detail. After treatment, a superficial layer of highly reflective spherical structures (4-10 microm) was observed. Many of these hyperreflective structures appeared up to a depth of 300 microm. The remainder of the corneal stroma and endothelium appeared normal. Electron microscopy showed keratocyte apoptotic changes to a depth of 300 microm. No observable pathologic changes were seen on histology. Based on clinical studies, corneal cross-linking is a promising treatment that appears to be safe and to halt ectatic corneal disease progression. Initial European studies used animal models to extrapolate human protocols. In conjunction with clinical studies, we believe that human ex vivo corneal studies provide a means to evaluate the structural and morphological changes associated with this procedure, within human corneas, in a manner that cannot be accomplished in vivo.

  16. A rapid method for combined laser scanning confocal microscopic and electron microscopic visualization of biocytin or neurobiotin-labeled neurons.

    Science.gov (United States)

    Sun, X J; Tolbert, L P; Hildebrand, J G; Meinertzhagen, I A

    1998-02-01

    Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin-peroxidase conjugate, is both faster and less labor intensive than photo-oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti-serotonin as an example.

  17. In vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta

    Directory of Open Access Journals (Sweden)

    Kobayashi A

    2014-02-01

    Full Text Available Akira Kobayashi, Tomomi Higashide, Hideaki Yokogawa, Natsuko Yamazaki, Toshinori Masaki, Kazuhisa Sugiyama Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan Objective: To report the in vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta (OI with special attention to the abnormality of Bowman's layer and sub-Bowman's fibrous structures (K-structures. Patients and methods: Two patients (67-year-old male and his 26-year-old son with OI type I were included in this study. Slit lamp biomicroscopic and in vivo laser confocal microscopic examinations were performed for both patients. Central corneal thickness and central endothelial cell density were also measured. Results: Although the corneas looked clear with normal endothelial density for both eyes in both patients, they were quite thin (386 µm oculus dexter (OD (the right eye and 384 µm oculus sinister (OS (the left eye in the father and 430 µm OD and 425 µm OS in the son. In both patients, slit lamp biomicroscopic and in vivo laser confocal microscopic examination showed similar results. Anterior corneal mosaics produced by rubbing the eyelid under fluorescein were completely absent in both eyes. In vivo laser confocal microscopy revealed an absent or atrophic Bowman's layer; a trace of a presumed Bowman's layer and/or basement membrane was barely visible with high intensity. Additionally, K-structures were completely absent in both eyes. Conclusion: The absence of K-structures and fluorescein anterior corneal mosaics strongly suggested an abnormality of Bowman's layer in these OI patients. Keywords: osteogenesis imperfecta, K-structure, confocal microscopy, Bowman's layer

  18. Scanning electron microscopy of molluscum contagiosum*

    Science.gov (United States)

    de Almeida Jr, Hiram Larangeira; Abuchaim, Martha Oliveira; Schneider, Maiko Abel; Marques, Leandra; de Castro, Luis Antônio Suíta

    2013-01-01

    Molluscum contagiosum is a disease caused by a poxvirus. It is more prevalent in children up to 5 years of age. There is a second peak of incidence in young adults. In order to examine its ultrastructure, three lesions were curetted without disruption, cut transversely with a scalpel, and routinely processed for scanning electron microscopy (SEM). The oval structure of molluscum contagiosum could be easily identified. In its core, there was a central umbilication and just below this depression, there was a keratinized tunnel. Under higher magnification, a proliferation similar to the epidermis was seen. Moreover, there were areas of cells disposed like a mosaic. Under higher magnification, rounded structures measuring 0.4 micron could be observed at the end of the keratinized tunnel and on the surface of the lesion. PMID:23539009

  19. Spin-polarized scanning tunnelling microscopy

    CERN Document Server

    Bode, M

    2003-01-01

    The recent experimental progress in spin-polarized scanning tunnelling microscopy (SP-STM) - a magnetically sensitive imaging technique with ultra-high resolution - is reviewed. The basics of spin-polarized electron tunnelling are introduced as they have been investigated in planar tunnel junctions for different electrode materials, i.e. superconductors, optically excited GaAs, and ferromagnets. It is shown that ferromagnets and antiferromagnets are suitable tip materials for the realization of SP-STM. Possible tip designs and modes of operations are discussed for both classes of materials. The results of recent spatially resolved measurements as performed with different magnetic probe tips and using different modes of operation are reviewed and discussed in terms of applicability to surfaces, thin films, and nanoparticles. The limits of spatial resolution, and the impact of an external magnetic field on the imaging process.

  20. In-vivo immunofluorescence confocal microscopy of herpes simplex virus type 1 keratitis

    Science.gov (United States)

    Kaufman, Stephen C.; Laird, Jeffery A.; Beuerman, Roger W.

    1996-05-01

    The white-light confocal microscope offers an in vivo, cellular-level resolution view of the cornea. This instrument has proven to be a valuable research and diagnostic tool for the study of infectious keratitis. In this study, we investigate the direct visualization of herpes simplex virus type 1 (HSV-1)-infected corneal epithelium, with in vivo confocal microscopy, using HSV-1 immunofluorescent antibodies. New Zealand white rabbits were infected with McKrae strain of HSV-1 in one eye; the other eye of each rabbit was used as an uninfected control. Four days later, the rabbits were anesthetized and a cellulose sponge was applied to each cornea, and a drop of direct HSV fluorescein-tagged antibody was placed on each sponge every 3 to 5 minutes for 1 hour. Fluorescence confocal microscopy was then performed. The HSV-infected corneas showed broad regions of hyperfluorescent epithelial cells. The uninfected corneas revealed no background fluorescence. Thus, using the confocal microscope with a fluorescent cube, we were able to visualize HSV-infected corneal epithelial cells tagged with a direct fluorescent antibody. This process may prove to be a useful clinical tool for the in vivo diagnosis of HSV keratitis.

  1. Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning.

    Science.gov (United States)

    Wang, Thomas D; Contag, Christopher H; Mandella, Michael J; Chan, Ning Y; Kino, Gordon S

    2004-01-01

    We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution < or =4.4 microm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging.

  2. Image scanning fluorescence emission difference microscopy based on a detector array.

    Science.gov (United States)

    Li, Y; Liu, S; Liu, D; Sun, S; Kuang, C; Ding, Z; Liu, X

    2017-06-01

    We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  3. A dark mode in scanning thermal microscopy

    Science.gov (United States)

    Ramiandrisoa, Liana; Allard, Alexandre; Joumani, Youssef; Hay, Bruno; Gomés, Séverine

    2017-12-01

    The need for high lateral spatial resolution in thermal science using Scanning Thermal Microscopy (SThM) has pushed researchers to look for more and more tiny probes. SThM probes have consequently become more and more sensitive to the size effects that occur within the probe, the sample, and their interaction. Reducing the tip furthermore induces very small heat flux exchanged between the probe and the sample. The measurement of this flux, which is exploited to characterize the sample thermal properties, requires then an accurate thermal management of the probe-sample system and to reduce any phenomenon parasitic to this system. Classical experimental methodologies must then be constantly questioned to hope for relevant and interpretable results. In this paper, we demonstrate and estimate the influence of the laser of the optical force detection system used in the common SThM setup that is based on atomic-force microscopy equipment on SThM measurements. We highlight the bias induced by the overheating due to the laser illumination on the measurements performed by thermoresistive probes (palladium probe from Kelvin Nanotechnology). To face this issue, we propose a new experimental procedure based on a metrological approach of the measurement: a SThM "dark mode." The comparison with the classical procedure using the laser shows that errors between 14% and 37% can be reached on the experimental data exploited to determine the heat flux transferred from the hot probe to the sample.

  4. Dental pulp stem cells (DPSCs) differentiation study by confocal Raman microscopy

    Science.gov (United States)

    Salehi, H.; Collart-Dutilleul, P.-Y.; Gergely, C.; Cuisinier, F. J. G.

    2014-03-01

    Regenerative medicine brings a huge application for Mesenchymal stem cells such as Dental Pulp Stem Cells (DPSCs). Confocal Raman microscopy, a non-invasive, label free , real time and high spatial resolution imaging technique is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800-3000 cm-1 region (C-H stretching) and 960 cm-1 peak (phosphate PO4 3-) were collected. In Dental Pulp Stem Cells 21st day differentiated in buffer solution, phosphate peaks ν1 PO4 3- (first vibrational mode) at 960cm-1 and ν2 PO4 3- at 430cm-1 and ν4 PO4 3- at 585cm-1 are obviously present. Confocal Raman microscopy enables the detection of cell differentiation and it can be used to investigate clinical stem cell research.

  5. Confocal Raman microscopy to monitor extracellular matrix during dental pulp stem cells differentiation

    Science.gov (United States)

    Salehi, Hamideh; Collart-Dutilleul, Pierre-Yves; Gergely, Csilla; Cuisinier, Frédéric J. G.

    2015-07-01

    Regenerative medicine brings promising applications for mesenchymal stem cells, such as dental pulp stem cells (DPSCs). Confocal Raman microscopy, a noninvasive technique, is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800 to 3000 cm-1 region (C-H stretching) and the 960 cm-1 peak (ν1 PO43-) were collected (to image cells and phosphate, respectively), and the ratio of two peaks 1660 over 1690 cm-1 (amide I bands) to measure the collagen cross-linking has been calculated. Raman spectra of DPSCs after 21 days differentiation reveal several phosphate peaks: ν1 (first stretching mode) at 960 cm-1, ν2 at 430 cm-1, and ν4 at 585 cm-1 and collagen cross-linking can also be calculated. Confocal Raman microscopy enables monitoring osteogenic differentiation in vitro and can be a credible tool for clinical stem cell based research.

  6. Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques

    Directory of Open Access Journals (Sweden)

    Biliana Todorova

    2017-01-01

    Full Text Available Purpose. We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Procedures. Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM. Results. The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. Conclusions. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

  7. [Confocal microscopy as an early relapse marker after keratoplasty due to Fusarium solani keratitis].

    Science.gov (United States)

    Daas, L; Bischoff-Jung, M; Viestenz, A; Seitz, B; Viestenz, A

    2017-01-01

    In the case of therapy-resistant keratitis an infection with Fusarium solani should be taken into consideration as a rare but very severe eye disease. In the majority of cases Fusarium solani keratitis will result in a protracted clinical course despite aggressive medicinal and surgical interventions. We describe the case of a referred patient after intensive topical, intracameral and systemic antibacterial and antimycotic therapy as well as surgical treatment with emergency keratoplasty à chaud because of Fusarium solani keratitis. The patient presented to our department with persistent discomfort for further therapeutic interventions. Using confocal microscopy we were able to demonstrate the presence of fungal hyphae in the host cornea and the graft, which was important for making further surgical decisions. Furthermore, this emphasizes the role of confocal microscopy as an early relapse marker during the clinical monitoring.

  8. The use of reflectance confocal microscopy for monitoring response to therapy of skin malignancies

    OpenAIRE

    Ulrich, Martina; Lange-Asschenfeldt, Susanne; Gonzalez, Salvador

    2012-01-01

    Summary Reflectance confocal microscopy (RCM) is a new non-invasive imaging technique that enables visualizing cells and structures in living skin in real-time with resolution close to that of histological analysis. RCM has been successfully implemented in the assessment of benign and malignant lesions. Most importantly, it also enables monitoring dynamic changes in the skin over time and in response to different therapies, e.g., imiquimod, photodynamic therapy, and others. Given the often tr...

  9. AN AUTOMATIC FEATURE BASED MODEL FOR CELL SEGMENTATION FROM CONFOCAL MICROSCOPY VOLUMES

    OpenAIRE

    Delibaltov, Diana; Ghosh, Pratim; Veeman, Michael; Smith, William; Manjunath, B.S.

    2011-01-01

    We present a model for the automated segmentation of cells from confocal microscopy volumes of biological samples. The segmentation task for these images is exceptionally challenging due to weak boundaries and varying intensity during the imaging process. To tackle this, a two step pruning process based on the Fast Marching Method is first applied to obtain an over-segmented image. This is followed by a merging step based on an effective feature representation. The algorithm is applied on two...

  10. Microscopia confocal en córneas de cien ojos sanos Confocal microscopy results of one hundred healthy eye corneas

    Directory of Open Access Journals (Sweden)

    Zulema Gómez Castillo

    2012-06-01

    Full Text Available Objetivo: Analizar las estructuras celulares por microscopia confocal, Confoscan 4, en córneas sanas en nuestro medio. Métodos: Se realizó un estudio prospectivo longitudinal a 100 ojos sanos de médicos que trabajan en nuestra institución, y pacientes que asistieron al servicio de córnea. Esta investigación fue desde mayo de 2007 a mayo 2008, en el Instituto Cubano de Oftalmología "Ramón Pando Ferrer", La Habana. En los médicos se examinaron ambos ojos y en los pacientes el ojo no afectado. Se recopilaron un total de 50 casos sin afección corneal. Resultados: De los 100 ojos estudiados, 64 tenían paquimetrías por encima del valor medio. Estuvieron presentes los tres tipos de células epiteliales en casi la totalidad de los pacientes; así como los queratocitos en las diferentes profundidades del estroma corneal. La mayoría de los ojos tenían un conteo celular endotelial por encima de 2 500, cifra comprendida dentro de los valores normales. Se encontraron fibras nerviosas en cada una de sus capas. Conclusiones: La microscopia confocal se presenta como una nueva herramienta que permite observar en vivo la histología corneal y complementar las observaciones de la biomicroscopia convencional. Esto constituye un reto para el mejor entendimiento de la histopatología corneal. De esta manera podemos actuar de forma profiláctica y terapéutica, en el seguimiento y evolución de patologías corneales.Objective: This paper is aimed at analyzing the corneal cellular structures through Confoscan S4-aided confocal microscopy in apparently healthy corneas. Methods: A prospective longitudinal study of 100 healthy eyes from practicing doctors, and from patients who had attended the corneal service at “Ramón Pando Ferrer” Cuban Institute of Ophthalmology in Havana since May 2007 was conducted. Both eyes of participating doctors were examined whereas the non-affected eye was examined in the patients. A total of 50 cases with no corneal

  11. In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy

    Directory of Open Access Journals (Sweden)

    Saubaméa Bruno

    2010-03-01

    Full Text Available Abstract Background Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs. CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Methods Protein expression in whole spheroids (150 μm in diameter from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. Results We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively. Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to

  12. In vivo Confocal Microscopy in Differentiating Ipilimumab-Induced Anterior Uveitis from Metastatic Uveal Melanoma

    Directory of Open Access Journals (Sweden)

    Hayyam Kiratli

    2016-09-01

    Full Text Available This report aims to describe the facilitating role of in vivo confocal microscopy in differentiating inflammatory cells from a metastatic process in a patient with uveal melanoma and multiple systemic metastases who developed anterior uveitis while under ipilimumab treatment. A 43-year-old woman developed systemic metastases 11 months after treatment of amelanotic choroidal melanoma in her right eye with 30 Gy fractionated stereotactic radiotherapy. She first received temozolomide and then 4 cycles of ipilimumab 3 mg/kg/day. After the third cycle, severe anterior uveitis with coarse pigment clumps on the lens was seen in the left eye. Her left visual acuity declined from 20/20 to 20/80. Confocal microscopy revealed globular keratic precipitates with hyperreflective inclusions and endothelial blebs all suggestive of granulomatous uveitis. The uveitic reaction subsided after a 3-week course of topical corticosteroids, and her visual acuity was 20/20 again. Although uveal melanoma metastatic to the intraocular structures of the fellow eye is exceedingly rare and metastasis masquerading uveitis without any identifiable uveal lesion is even more unusual, it was still mandatory to rule out this distant possibility in our particular patient who already had widespread systemic metastases. Confocal microscopy was a useful complementary tool by identifying the inflammatory features of the keratic precipitates.

  13. Automated motion estimation of root responses to sucrose in two Arabidopsis thaliana genotypes using confocal microscopy.

    Science.gov (United States)

    Wuyts, Nathalie; Bengough, A Glyn; Roberts, Timothy J; Du, Chengjin; Bransby, M Fraser; McKenna, Stephen J; Valentine, Tracy A

    2011-10-01

    Root growth is a highly dynamic process influenced by genetic background and environment. This paper reports the development of R scripts that enable root growth kinematic analysis that complements a new motion analysis tool: PlantVis. Root growth of Arabidopsis thaliana expressing a plasma membrane targeted GFP (C24 and Columbia 35S:LTI6b-EGFP) was imaged using time-lapse confocal laser scanning microscopy. Displacement of individual pixels in the time-lapse sequences was estimated automatically by PlantVis, producing dense motion vector fields. R scripts were developed to extract kinematic growth parameters and report displacement to ± 0.1 pixel. In contrast to other currently available tools, Plantvis-R delivered root velocity profiles without interpolation or averaging across the root surface and also estimated the uncertainty associated with tracking each pixel. The PlantVis-R analysis tool has a range of potential applications in root physiology and gene expression studies, including linking motion to specific cell boundaries and analysis of curvature. The potential for quantifying genotype × environment interactions was examined by applying PlantVis-R in a kinematic analysis of root growth of C24 and Columbia, under contrasting carbon supply. Large genotype-dependent effects of sucrose were recorded. C24 exhibited negligible differences in elongation zone length and elongation rate but doubled the density of lateral roots in the presence of sucrose. Columbia, in contrast, increased its elongation zone length and doubled its elongation rate and the density of lateral roots.

  14. In vivo confocal microscopy for the oral cavity: Current state of the field and future potential.

    Science.gov (United States)

    Maher, N G; Collgros, H; Uribe, P; Ch'ng, S; Rajadhyaksha, M; Guitera, P

    2016-03-01

    Confocal microscopy (CM) has been shown to correlate with oral mucosal histopathology in vivo. The purposes of this review are to summarize what we know so far about in vivo CM applications for oral mucosal pathologies, to highlight some current developments with CM devices relevant for oral applications, and to formulate where in vivo CM could hold further application for oral mucosal diagnosis and management. Ovid Medline® and/or Google® searches were performed using the terms 'microscopy, confocal', 'mouth neoplasms', 'mouth mucosa', 'leukoplakia, oral', 'oral lichen planus', 'gingiva', 'cheilitis', 'taste', 'inflammatory oral confocal', 'mucosal confocal' and 'confocal squamous cell oral'. In summary, inclusion criteria were in vivo use of any type of CM for the human oral mucosa and studies on normal or pathological oral mucosa. Experimental studies attempting to identify proteins of interest and microorganisms were excluded. In total 25 relevant articles were found, covering 8 main topics, including normal oral mucosal features (n=15), oral dysplasia or neoplasia (n=7), inflamed oral mucosa (n=3), taste impairment (n=3), oral autoimmune conditions (n=2), pigmented oral pathology/melanoma (n=1), delayed type hypersensitivity (n=1), and cheilitis glandularis (n=1). The evidence for using in vivo CM in these conditions is poor, as it is limited to mainly small descriptive studies. Current device developments for oral CM include improved probe design. The authors propose that future applications for in vivo oral CM may include burning mouth syndrome, intra-operative mapping for cancer surgery, and monitoring and targeted biopsies within field cancerization. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Effect of calcium on Staphylococcus aureus biofilm architecture: a confocal laser scanning microscopic study.

    Science.gov (United States)

    Shukla, Sudhir K; Rao, T Subba

    2013-03-01

    Bacterial adhesion is a threshold event in the formation of biofilms. Several studies on molecular and biochemical aspects have highlighted that the protein matrix of the biofilm is of interest in developing strategies to combat biofouling. The prevalent role of biofilm associated protein (Bap) of Staphylococcus aureus in early adhesion and the putative presence of Ca(2+) binding EF hand motif in Bap was the motivation for this study. Biofilm assays (S. aureus strains V329 and M556) were done in micro-titer plates and confocal laser scanning microscopy (CLSM) was used to study the biofilm architecture. The results showed that Ca(2+) did not influence planktonic growth of the cultures; however, it modulated the biofilm architecture of S. aureus V329 in a dose dependent manner. Strain M556 was found to be a weak biofilm former and showed no significant change in the presence of Ca(2+). When tested with increasing NaCl concentration, there was no reversal of the Bap-dependent Ca(2+) inhibition of S. aureus V329 biofilm. This indicates that the interaction of Bap and Ca(2+) is not mere electrostatic. CLSM images of V329 biofilm showed reduction in biofilm thickness as well as altered biofilm topography with varying Ca(2+) concentrations. The inhibition effect of Ca(2+) on strain V329 biofilm disappeared in the presence of chelating agent EDTA at a non-inhibiting concentration (0.15 mM). The paper elaborates the role of Ca(2+) in biofilm architecture of S. aureus. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. In-situ imaging of interlayer nanodeformation with improved differential confocal microscopy

    Science.gov (United States)

    Li, Z.; Gao, S.; Herrmann, K.

    2008-04-01

    Nanoindentation testing has proved to be an effective tool to determine the mechanical properties of small volumes of materials applied in various micro-systems, including hardness, indentation modulus, creep and so on. Nowadays, with the help of advanced numerical methods, especially the finite element analysis (FEA) technique, further mechanical properties of the material under test (e.g. tensile strength, etc.) can be interpreted from the typical indentation curve. However, the reliability and accuracy of these analytical models have to be well tested. Recently, the deformed topography of the interlayer surface within the tip-film-substrate system has been proposed to be the reference for the evaluation of FEA and other mathematic models for indentation testing. Here an in-situ interlayer deformation imaging system based on differential confocal microscopy is therefore developed, which has the capability to measure in-situ the real-time topography deformation within a layered specimen during nanoindentation testing. By means of linear regression and interpolation of the linear region of the standard confocal microscopy, differential confocal microscopy (DCM) can achieve a very high resolution for topography measurements. However, the actual capability and measurement uncertainty of DCM would be subject to those common-mode error sources like surface heterogeneity, intensity fluctuation of the light source, etc. In this paper an improved DCM is proposed, which introduces an additional point detector to the conventional DCM, creating dual confocal signals with slight relative axial shifting. The real topography of the surface under test can then be easily deconvoluted from the dual differential signals, whilst the common-mode errors within the measurement are eliminated. A prototype was developed and applied for measuring a step-height composed of two different materials and for in-situ inspection of the interlayer deformation during nanoindentation testing

  17. Ribbon scanning confocal for high-speed high-resolution volume imaging of brain.

    Directory of Open Access Journals (Sweden)

    Alan M Watson

    Full Text Available Whole-brain imaging is becoming a fundamental means of experimental insight; however, achieving subcellular resolution imagery in a reasonable time window has not been possible. We describe the first application of multicolor ribbon scanning confocal methods to collect high-resolution volume images of chemically cleared brains. We demonstrate that ribbon scanning collects images over ten times faster than conventional high speed confocal systems but with equivalent spectral and spatial resolution. Further, using this technology, we reconstruct large volumes of mouse brain infected with encephalitic alphaviruses and demonstrate that regions of the brain with abundant viral replication were inaccessible to vascular perfusion. This reveals that the destruction or collapse of large regions of brain micro vasculature may contribute to the severe disease caused by Venezuelan equine encephalitis virus. Visualization of this fundamental impact of infection would not be possible without sampling at subcellular resolution within large brain volumes.

  18. Confocal scanning electroluminescence spectro-microscope for multidimensional light-emitting property analysis

    Science.gov (United States)

    Hong, S.; Onushkin, G.; Park, J. S.; Kim, B. K.; Lee, D.-Y.; Fomin, A.; Ko, K.; Kim, J. W.

    2007-02-01

    We report new type of micro-EL instrument and its applications for light emitting devices. Our new micro-EL, so-called confocal scanning electroluminescence sprctro-microscope (CSESM) has not only fast image acquisition time but also high image resolution. The newly developed CSESM is combined with confocal laser scanning photoluminescence micsoscope, i.e. micro-PL. Therefore, micro-EL distribution can be directly matched with micro-PL and mechanical chip structure of LED. It is fruitful for providing a fast and non-destructive method to analyze the homogeneity of LEDs in its completely proceeded form. Using this apparatus, we study local intensity and wavelength distribution of electroluminescence for InGaN/GaN blue LED chip. Our results represent that local fluctuations of electroluminescence intensity and wavelength position are closely connected with the fluctuation of local current density, i.e. current spreading features on LED chips.

  19. In Vivo Confocal Microscopy and Anterior Segment Optic Coherence Tomography Findings in Ocular Ochronosis

    Directory of Open Access Journals (Sweden)

    Elif Demirkilinc Biler

    2015-01-01

    Full Text Available Purpose. To report clinical and in vivo confocal microscopy (IVCM findings of two patients with ocular ochronosis secondary due to alkaptonuria. Materials and Methods. Complete ophthalmologic examinations, including IVCM (HRT II/Rostock Cornea Module, Heidelberg, Germany, anterior segment optical coherence tomography (AS-OCT (Topcon 3D spectral-domain OCT 2000, Topcon Medical Systems, Paramus, NJ, USA, corneal topography (Pentacam, OCULUS Optikgeräte GmbH, Wetzlar, Germany, and anterior segment photography, were performed. Results. Biomicroscopic examination showed bilateral darkly pigmented lesions of the nasal and temporal conjunctiva and episclera in both patients. In vivo confocal microscopy of the lesions revealed prominent degenerative changes, including vacuoles and fragmentation of collagen fibers in the affected conjunctival lamina propria and episclera. Hyperreflective pigment granules in different shapes were demonstrated in the substantia propria beneath the basement membrane. AS-OCT of Case 1 demonstrated hyporeflective areas. Fundus examination was within normal limits in both patients, except tilted optic discs with peripapillary atrophy in one of the patients. Corneal topography, thickness, and macular OCT were normal bilaterally in both cases. Conclusion. The degenerative and anatomic changes due to ochronotic pigment deposition in alkaptonuria can be demonstrated in detail with IVCM and AS-OCT. Confocal microscopic analysis in ocular ochronosis may serve as a useful adjunct in diagnosis and monitoring of the disease progression.

  20. Multispectral confocal microscopy images and artificial neural nets to monitor the photosensitizer uptake and degradation in Candida albicans cells

    Science.gov (United States)

    Romano, Renan A.; Pratavieira, Sebastião.; da Silva, Ana P.; Kurachi, Cristina; Guimarães, Francisco E. G.

    2017-07-01

    This study clearly demonstrates that multispectral confocal microscopy images analyzed by artificial neural networks provides a powerful tool to real-time monitoring photosensitizer uptake, as well as photochemical transformations occurred.

  1. Scanned probe microscopy for thin film superconductor development

    Energy Technology Data Exchange (ETDEWEB)

    Moreland, J. [National Institute of Standards and Technology, Boulder, CO (United States)

    1996-12-31

    Scanned probe microscopy is a general term encompassing the science of imaging based on piezoelectric driven probes for measuring local changes in nanoscale properties of materials and devices. Techniques like scanning tunneling microscopy, atomic force microscopy, and scanning potentiometry are becoming common tools in the production and development labs in the semiconductor industry. The author presents several examples of applications specific to the development of high temperature superconducting thin films and thin-film devices.

  2. Probing intracellular mass density fluctuation through confocal microscopy: application in cancer diagnostics as a case study

    CERN Document Server

    Sahay, Peeyush; Ghimire, Hemendra M; Almabadi, Huda; Yallappu, Murali M; Skalli, Omar; Jaggi, Meena; Chauhan, Subhash C; Pradhan, Prabhakar

    2015-01-01

    Intracellular structural alterations are hallmark of several disease conditions and treatment modalities. However, robust methods to quantify these changes are scarce. In view of this, we introduce a new method to quantify structural alterations in biological cells through the widely used confocal microscopy. This novel method employs optical eigenfunctions localization properties of cells and quantifies the degree of structural alterations, in terms of nano- to micron scale intracellular mass density fluctuations, in one single parameter. Such approach allows a powerful way to compare changing structures in heterogeneous cellular media irrespective of the origin of the cause. As a case study, we demonstrate its applicability in cancer detection with breast and prostate cancer cases of different tumorigenicity levels. Adding new dimensions to the confocal based studies, this technique has potentially significant applications in areas ranging from disease diagnostics to therapeutic studies, such as patient pro...

  3. Confocal Raman microscopy for in depth analysis in the field of cultural heritage

    Science.gov (United States)

    Lorenzetti, G.; Striova, J.; Zoppi, A.; Castellucci, E. M.

    2011-05-01

    In the field of cultural heritage, the main concern when a sample is analyzed is its safeguard, and this means that non-destructive techniques are required. In this work, we show how confocal Raman microscopy (CRM) may be successfully applied in the study of works of art as a valuable alternative to other well established techniques. CRM with a metallurgical objective was tested for the in depth study of thin samples that are of interest in the field of cultural heritage. The sensitivity of the instrumentation was first evaluated by analyzing single layers of pure polyethylene terephthalate (PET) films having a thickness of 12, 25, and 50 μm, respectively, and a multilayer sample of polypropylene (PP) and polyethylene (PE). Subsequently, the technique was applied to the analysis of historical dyed cotton yarns in order to check whether it was possible to achieve a better discrimination of the fibres' signals for an easier identification. A substantial improvement of the signal to noise ratio was found in the confocal arrangement with respect to the non-confocal one, suggesting the use of this technique for this kind of analysis in the field of cultural heritage. Furthermore, Raman spectroscopy in confocal configuration was exploited in the evaluation of cleaning performed on the mural painting specimens, treated with acrylic resin (Paraloid B72). Confocal Raman experiments were performed before and after laser cleaning (at different conditions) in order to monitor the presence and to approximate the polymer thickness: the method proved to be a valid comparative tool in assessment of cleaning efficiencies.

  4. Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

    Science.gov (United States)

    Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

    2015-10-01

    Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

  5. Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology

    Science.gov (United States)

    Yin, C.; Glaser, A.K.; Leigh, S. Y.; Chen, Y.; Wei, L.; Pillai, P. C. S.; Rosenberg, M. C.; Abeytunge, S.; Peterson, G.; Glazowski, C.; Sanai, N.; Mandella, M. J.; Rajadhyaksha, M.; Liu, J. T. C.

    2016-01-01

    There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo clinical use, enables our device to achieve an optical-sectioning thickness and lateral resolution of 2.0 and 1.1 microns respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate the clinical potential of this high-speed optical-sectioning microscopy device. PMID:26977337

  6. Coin-shaped epithelial lesions following an acute attack of erythema multiforme minor with confocal microscopy findings

    OpenAIRE

    Babu Kalpana; Murthy Vinay; Akki Veeresh; Prabhakaran Venkatesh; Murthy K

    2010-01-01

    We report an interesting ocular finding of bilateral multiple coin-shaped epithelial lesions along with the confocal microscopy findings in a patient following an acute attack of erythema multiforme (EM) minor. A 30-year-old male presented with a history of watering and irritation in both eyes of three days duration. He was diagnosed to have EM minor and was on oral acyclovir. Slit-lamp examination revealed multiple coin-shaped epithelial lesions. Confocal microscopy showed a corresponding co...

  7. [In vivo reflectance-mode confocal laser microscopy: basic principles and clinical and research employments in dermatology].

    Science.gov (United States)

    Levi, Assi; Ingber, Arieh; Enk, David Claes

    2012-10-01

    Reflectance-mode confocal scanning laser microscopy is a novel, non-invasive imaging technique which permits real time visualization of cellular components in the skin at a resolution close to that of conventional histology. It has been widely used in the diagnosis of both benign and malignant tumors of the skin. In recent years it was also employed in the investigation of a variety of inflammatory and infectious skin conditions. The non-invasive nature of the procedure allows examination of multiple lesions and/ or repetitive sampling of one lesion over time, making it an excellent tool for followup and for monitoring treatment outcome in medical and cosmetic dermatology. This review summarizes the main indications for the use of this novel technique in clinical and experimental dermatology.

  8. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    Science.gov (United States)

    Estrela-Lopis, I.; Romero, G.; Rojas, E.; Moya, S. E.; Donath, E.

    2011-07-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  9. Interfacial shape and contact-angle measurement of transparent samples with confocal interference microscopy.

    Science.gov (United States)

    Fischer, D G; Ovryn, B

    2000-04-01

    A model has been developed that predicts the effective optical path through a thick, refractive specimen on a reflective substrate, as measured with a scanning confocal interference microscope equipped with a high-numerical-aperture objective. Assuming that the effective pinhole of the confocal microscope has an infinitesimal diameter, only one ray in the illumination bundle (the magic ray) contributes to the differential optical path length (OPL). A pinhole with finite diameter, however, allows rays within a small angular cone centered on the magic ray to contribute to the OPL. The model was incorporated into an iterative algorithm that allows the measured phase to be corrected for refractive errors by use of an a priori estimate of the sample profile. The algorithm was validated with a reflected-light microscope equipped with a phase-shifting laser-feedback interferometer to measure the interface shape and the 68 degrees contact angle of a silicone-oil drop on a coated silicon wafer.

  10. Optical microscope illumination analysis using through-focus scanning optical microscopy.

    Science.gov (United States)

    Attota, Ravi Kiran; Park, Haesung

    2017-06-15

    Misalignment of the aperture diaphragm present in optical microscopes results in angular illumination asymmetry (ANILAS) at the sample plane. Here we show that through-focus propagation of ANILAS results in a lateral image shift with a focus position. This could lead to substantial errors in quantitative results for optical methods that use through-focus images such as three-dimensional nanoparticle tracking, confocal microscopy, and through-focus scanning optical microscopy (TSOM). A correlation exists between ANILAS and the slant in TSOM images. Hence, the slant in the TSOM image can be used to detect, analyze, and rectify the presence of ANILAS.

  11. Extracting the ridge set as a graph for actin filament length estimation from confocal laser scanning microscopic images

    Science.gov (United States)

    Birkholz, Harald

    2012-04-01

    The progress in image acquisition techniques provides life sciences with an abundance of data. Image analysis facilitates the assessment. The actin cytoskeleton plays a crucial role in understanding the behavior of osteoblastic cells on biomaterials. In the flat basal part of the cells, it can be visualized by confocal laser scanning microscopy. In the microscopic images, the stained cytoskeleton appears as a dense network of bright ridges which is so far only qualitatively assessed. For its quantification, there is a need for ridge detection techniques that provide a geometrical description of this graph feature. The state of the art methods do not cope with the systematical degradation by noise, unspecific luminance, and uneven dye uptake. This work presents the key part of a ridge-tracking technique, which makes more efficient use of context information, and evaluate it by its length measurement accuracy. Two random models illustrate the performance against ground truth. Representative microscopic images confirm the applicability.

  12. Confocal scanning laser evaluation of repeated Q-switched laser exposure and possible retinal NFL damage

    Science.gov (United States)

    Zwick, Harry; Gagliano, Donald A.; Zuclich, Joseph A.; Stuck, Bruce E.; Lund, David J.; Glickman, Randolph D.

    1995-05-01

    Repeated extended source Q-switched exposure centered on the macula has been shown to produce a Bullseye maculopathy. This paper provides a confocal scanning laser ophthalmoscopic evaluation with regard to the retinal nerve fiber layer (NFL) and deeper choroidal vascular network. Confocal imaging revealed that the punctate annular appearance of this lesion in the deeper retinal layers is associated with retinal nerve fiber bundle disruptions and small gaps in the retinal NFL. No choroidal dysfunction was noticed with Indocyanine green angiography. It is hypothesized that retinal NFL damage occurs either through disruption of retinal pigment epithelial cell layer support to the NFL or through direct exposure to high spatial peak powers within the extended source beam profile, causing direct microthermal injury to the NFL. The apparent sparring of the fovea reflects central retinal morphology rather than a lack of retinal damage to the fovea.

  13. Single Fluorescent Molecule Confocal Microscopy: A New Tool for Molecular Biology Research and Biosensor Development

    Energy Technology Data Exchange (ETDEWEB)

    Darrow, C.; Huser, T.; Campos, C.; Yan, M.; Lane, S.; Balhorn, R.

    2000-03-09

    Our original proposal was presented to the LDRD committee on February 18, 1999. The revised proposal that followed incorporated changes that addressed the issues, concerns, and suggestions put forth by the committee members both during the presentation and in subsequent discussions we've had with individual committee members. The goal of the proposal was to establish an SMD confocal microscopy capability and technology base at LLNL. Here we report on our progress during the 6-month period for which funding was available.

  14. Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

    Science.gov (United States)

    Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

    2014-05-01

    The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

  15. Amiodarone-induced multiorgan toxicity with ocular findings on confocal microscopy.

    Science.gov (United States)

    Turk, Ugur; Turk, Bengu Gerceker; Yılmaz, Suzan Guven; Tuncer, Esref; Alioğlu, Emin; Dereli, Tugrul

    2015-01-01

    Amiodarone is an antiarrhythmic medication that can adversely effect various organs including lungs, thyroid gland, liver, eyes, skin, and nerves. The risk of adverse effects increases with high doses and prolonged use. We report a 54-year-old female who presented with multiorgan toxicity after 8 months of low dose (200 mg/day) amiodarone treatment. The findings of confocal microscopy due to amiodarone-induced keratopathy are described. Amiodarone may cause multiorgan toxicity even at lower doses and for shorter treatment periods.

  16. Confocal microscopy: A new tool for erosion measurements on large scale plasma facing components in tokamaks

    Energy Technology Data Exchange (ETDEWEB)

    Gauthier, E., E-mail: eric.gauthier@cea.fr [CEA/DSM/IRFM, CEA Cadarache, Saint-Paul-lez-Durance (France); Brosset, C.; Roche, H.; Tsitrone, E.; Pégourié, B.; Martinez, A. [CEA/DSM/IRFM, CEA Cadarache, Saint-Paul-lez-Durance (France); Languille, P. [PIIM, CNRS-Université de Provence, Centre de St Jérôme, 13397 Marseille, Cedex 20 (France); Courtois, X.; Lallier, Y. [CEA/DSM/IRFM, CEA Cadarache, Saint-Paul-lez-Durance (France); Salami, M. [AVANTIS CONCEPT, 75 Rue Marcelin Berthelot, 13858 Aix en Provence (France)

    2013-07-15

    A diagnostic based on confocal microscopy was developed at CEA Cadarache in order to measure erosion on large plasma facing components during shutdown in situ in Tore Supra. This paper describes the diagnostic and presents results obtained on Beryllium and Carbon Fibre Composite (CFC) materials. Erosion in the range of 800 μm was found on one sector of the Toroidal Pumped Limiter (TPL) which provides, by integration to the full limiter a net carbon erosion of about 900 g over the period 2002–2007.

  17. Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope.

    Directory of Open Access Journals (Sweden)

    André Klauss

    Full Text Available By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as "easy-STED", achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.

  18. EDITORIAL: Scanning probe microscopy: a visionary development Scanning probe microscopy: a visionary development

    Science.gov (United States)

    Demming, Anna

    2013-07-01

    The development of scanning probe microscopy repositioned modern physics. When Rohrer and Binnig first used electronic tunnelling effects to image atoms and quantum states they did more than pin down theoretical hypotheses to real-world observables; the scanning tunnelling microscope fed imaginations, prompting researchers to consider new directions and possibilities [1]. As Rohrer once commented, 'We could show that you can easily manipulate or position something small in space with an accuracy of 10 pm.... When you can do that, you simply have ideas of what you can do' [2]. The development heralded a cavalry of scanning probe techniques—such as atomic force microscopy (AFM) [3-5], scanning near-field optical microscopy (SNOM) [6-8] and Kelvin probe force microscopy (KPFM) [9, 10]—that still continue to bring nanomaterials and nanoscale phenomena into fresh focus. Not long after the development of scanning tunnelling microscopy, Binnig, Quate and Gerber collaborating in California in the US published work on a new type of microscope also capable of atomic level resolution [3]. The original concept behind scanning tunnelling microscopy uses electrical conductance, which places substantial limitations on the systems that it can image. Binnig, Quate and Gerber developed the AFM to 'feel' the topology of surfaces like the needle of an old fashioned vinyl player. In this way insulators could be imaged as well. The development of a force modulation mode AFM extended the tool's reach to soft materials making images of biological samples accessible with the technique [4]. There have now been a number of demonstrations of image capture at rates that allow dynamics at the nanoscale to be tracked in real time, opening further possibilities in applications of the AFM as described in a recent review by Toshio Ando at Kanazawa University [5]. Researchers also found a way to retrieve optical information at 'super-resolution' [6, 7]. Optical microscopy provides spectral

  19. In vivo confocal microscopy in the normal corneas of cats, dogs and birds.

    Science.gov (United States)

    Kafarnik, Christiane; Fritsche, Jens; Reese, Sven

    2007-01-01

    To evaluate the applicability of in vivo confocal microscopy (IVCM) in veterinary ophthalmology and analyze the morphology of living, healthy cornea. ANIMALS EXAMINED: Thirty-seven dogs, 34 cats and five birds. Various corneal sublayers were visualized in the central region using an in vivo confocal corneal microscope (HRTII/RCM). An investigation method was developed and adapted for use on animals with varying skull forms and eye positions. Real-time images of the epithelial cells, the corneal stroma and the endothelial layer were obtained. The corneal stromal nerve trunks and the subepithelial and basal epithelial nerve plexus were visualized. In dogs, full corneal thickness (FCT) was 585 +/- 79 microm (mean +/- SD) and endothelial cell density (ECD) 3175 +/- 776 cells/mm(2) (mean +/- SD). In cats, FCT was 592 +/- 80 microm and ECD 2846 +/- 403 cells/mm(2). There were no significant differences between canine and feline FCT and ECD and no morphologic differences could be seen between dogs and cats. The bird images revealed a number of structural differences. Noninvasive IVCM allows accurate detection of corneal sublayers, corneal pachymetry, endothelial cell density and corneal innervation in various animal species. For clinical usage, patients must be under general anesthesia. The confocal images provided anatomic reference images of various healthy corneal structures in dogs, cats and birds.

  20. Scanning transmission electron microscopy imaging and analysis

    CERN Document Server

    Pennycook, Stephen J

    2011-01-01

    Provides the first comprehensive treatment of the physics and applications of this mainstream technique for imaging and analysis at the atomic level Presents applications of STEM in condensed matter physics, materials science, catalysis, and nanoscience Suitable for graduate students learning microscopy, researchers wishing to utilize STEM, as well as for specialists in other areas of microscopy Edited and written by leading researchers and practitioners

  1. Method and apparatus for a high-resolution three dimensional confocal scanning transmission electron microscope

    Science.gov (United States)

    de Jonge, Niels [Oak Ridge, TN

    2010-08-17

    A confocal scanning transmission electron microscope which includes an electron illumination device providing an incident electron beam propagating in a direction defining a propagation axis, and a precision specimen scanning stage positioned along the propagation axis and movable in at least one direction transverse to the propagation axis. The precision specimen scanning stage is configured for positioning a specimen relative to the incident electron beam. A projector lens receives a transmitted electron beam transmitted through at least part of the specimen and focuses this transmitted beam onto an image plane, where the transmitted beam results from the specimen being illuminated by the incident electron beam. A detection system is placed approximately in the image plane.

  2. Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara.

    Science.gov (United States)

    Borges, J N; Costa, V S; Mantovani, C; Barros, E; Santos, E G N; Mafra, C L; Santos, C P

    2017-02-01

    Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish-eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P. macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox-1) are presented. Phylogeny reconstructions linked P. macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin-fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P. macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High-resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes. © 2016 John Wiley & Sons Ltd.

  3. Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

    Science.gov (United States)

    Wu, Yong; Wu, Xundong; Lu, Rong; Zhang, Jin; Toro, Ligia; Stefani, Enrico

    2015-10-01

    Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

  4. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy.

    Directory of Open Access Journals (Sweden)

    Chao Huang

    Full Text Available Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000. We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81 and nodal tissue (n = 81. In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively. Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease.

  5. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy.

    Science.gov (United States)

    Huang, Chao; Sachse, Frank B; Hitchcock, Robert W; Kaza, Aditya K

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease.

  6. Adaptive and Background-Aware GAL4 Expression Enhancement of Co-registered Confocal Microscopy Images.

    Science.gov (United States)

    Trapp, Martin; Schulze, Florian; Novikov, Alexey A; Tirian, Laszlo; J Dickson, Barry; Bühler, Katja

    2016-04-01

    GAL4 gene expression imaging using confocal microscopy is a common and powerful technique used to study the nervous system of a model organism such as Drosophila melanogaster. Recent research projects focused on high throughput screenings of thousands of different driver lines, resulting in large image databases. The amount of data generated makes manual assessment tedious or even impossible. The first and most important step in any automatic image processing and data extraction pipeline is to enhance areas with relevant signal. However, data acquired via high throughput imaging tends to be less then ideal for this task, often showing high amounts of background signal. Furthermore, neuronal structures and in particular thin and elongated projections with a weak staining signal are easily lost. In this paper we present a method for enhancing the relevant signal by utilizing a Hessian-based filter to augment thin and weak tube-like structures in the image. To get optimal results, we present a novel adaptive background-aware enhancement filter parametrized with the local background intensity, which is estimated based on a common background model. We also integrate recent research on adaptive image enhancement into our approach, allowing us to propose an effective solution for known problems present in confocal microscopy images. We provide an evaluation based on annotated image data and compare our results against current state-of-the-art algorithms. The results show that our algorithm clearly outperforms the existing solutions.

  7. Diagnostic utility of corneal confocal microscopy and intra-epidermal nerve fibre density in diabetic neuropathy.

    Science.gov (United States)

    Alam, Uazman; Jeziorska, Maria; Petropoulos, Ioannis N; Asghar, Omar; Fadavi, Hassan; Ponirakis, Georgios; Marshall, Andrew; Tavakoli, Mitra; Boulton, Andrew J M; Efron, Nathan; Malik, Rayaz A

    2017-01-01

    Corneal confocal microscopy (CCM) is a rapid, non-invasive, reproducible technique that quantifies small nerve fibres. We have compared the diagnostic capability of CCM against a range of established measures of nerve damage in patients with diabetic neuropathy. In this cross sectional study, thirty subjects with Type 1 diabetes without neuropathy (T1DM), thirty one T1DM subjects with neuropathy (DSPN) and twenty seven non-diabetic healthy control subjects underwent detailed assessment of neuropathic symptoms and neurologic deficits, quantitative sensory testing (QST), electrophysiology, skin biopsy and corneal confocal microscopy (CCM). Subjects with DSPN were older (C vs T1DM vs DSPN: 41.0±14.9 vs 38.8±12.5 vs 53.3±11.9, P = 0.0002), had a longer duration of diabetes (Pdiabetic neuropathy with clinical signs and symptoms of neuropathy and greater neuropathy deficits quantified by QST, electrophysiology, intra-epidermal nerve fibre density and CCM. Corneal nerve fibre density (CNFD) (Spearman's Rho = 0.60 Pdiabetic neuropathy the sensitivity for CNFD was 0.77 and specificity was 0.79 with an area under the ROC curve of 0.81. IENFD had a diagnostic sensitivity of 0.61, specificity of 0.80 and area under the ROC curve of 0.73. CCM is a valid accurate non-invasive method to identify small nerve fibre pathology and is able to diagnose DPN.

  8. Biomimetic Coating on Porous Alumina for Tissue Engineering: Characterisation by Cell Culture and Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Elizabeth Kolos

    2015-06-01

    Full Text Available In this study porous alumina samples were prepared and then coated using the biomimetic coating technique using a five times Simulated Body Fluid (5.0SBF as the growth solution. A coating was achieved after pre-treatment with concentrated acid. From elemental analysis, the coating contained calcium and phosphorous, but also sodium and chlorine. Halite was identified by XRD, a sodium chloride phase. Sintering was done to remove the halite phase. Once halite was burnt off, the calcium phosphate crystals were not covered with halite and, therefore, the apatite phases can be clearly observed. Cell culturing showed sufficient cell attachment to the less porous alumina, Sample B, that has more calcium phosphate growth, while the porous alumina, Sample A, with minimal calcium phosphate growth attained very little cell attachment. This is likely due to the contribution that calcium phosphate plays in the attachment of bone-like cells to a bioinert ceramic such as alumina. These results were repeated on both SEM and confocal microscopy analysis. Confocal microscopy was a novel characterisation approach which gave useful information and was a visual aid.

  9. Quantification of whey in fluid milk using confocal Raman microscopy and artificial neural network.

    Science.gov (United States)

    Alves da Rocha, Roney; Paiva, Igor Moura; Anjos, Virgílio; Furtado, Marco Antônio Moreira; Bell, Maria José Valenzuela

    2015-06-01

    In this work, we assessed the use of confocal Raman microscopy and artificial neural network as a practical method to assess and quantify adulteration of fluid milk by addition of whey. Milk samples with added whey (from 0 to 100%) were prepared, simulating different levels of fraudulent adulteration. All analyses were carried out by direct inspection at the light microscope after depositing drops from each sample on a microscope slide and drying them at room temperature. No pre- or posttreatment (e.g., sample preparation or spectral correction) was required in the analyses. Quantitative determination of adulteration was performed through a feed-forward artificial neural network (ANN). Different ANN configurations were evaluated based on their coefficient of determination (R2) and root mean square error values, which were criteria for selecting the best predictor model. In the selected model, we observed that data from both training and validation subsets presented R2>99.99%, indicating that the combination of confocal Raman microscopy and ANN is a rapid, simple, and efficient method to quantify milk adulteration by whey. Because sample preparation and postprocessing of spectra were not required, the method has potential applications in health surveillance and food quality monitoring. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. In vivo ocular imaging of the cornea of the normal female laboratory beagle using confocal microscopy.

    Science.gov (United States)

    Strom, Ann R; Cortés, Dennis E; Thomasy, Sara M; Kass, Philip H; Mannis, Mark J; Murphy, Christopher J

    2016-01-01

    To obtain normative data for the normal laboratory beagle cornea using high-resolution in vivo confocal microscopy (IVCM). Sixteen eyes of eight healthy young female intact beagles. The central cornea was imaged using IVCM. Mixed effects linear regression was used for statistical analysis. in vivo confocal microscopy allowed detailed visualization and quantification of epithelial cells (superficial epithelial cell diameter: 43.25 ± 6.64 μm, basal cell diameter: 4.43 ± 0.67 μm), and nerve fibers (subepithelial nerve fiber diameter: 2.38 ± 0.69 μm, anterior stromal nerve fiber diameter: 16.93 ± 4.55 μm). Keratocyte density (anterior stroma 993.38 ± 134.24 cells/mm(2) , posterior stroma 789.38 ± 87.13 cells/mm(2) ) and endothelial cell density (2815.18 ± 212.59 cells/mm(2) ) were also measured. High-resolution IVCM provides detailed noninvasive evaluation of the cornea in the normal laboratory beagle. © 2015 American College of Veterinary Ophthalmologists.

  11. Assessing strain mapping by electron backscatter diffraction and confocal Raman microscopy using wedge-indented Si

    Energy Technology Data Exchange (ETDEWEB)

    Friedman, Lawrence H.; Vaudin, Mark D.; Stranick, Stephan J.; Stan, Gheorghe; Gerbig, Yvonne B.; Osborn, William; Cook, Robert F., E-mail: robert.cook@nist.gov

    2016-04-15

    The accuracy of electron backscatter diffraction (EBSD) and confocal Raman microscopy (CRM) for small-scale strain mapping are assessed using the multi-axial strain field surrounding a wedge indentation in Si as a test vehicle. The strain field is modeled using finite element analysis (FEA) that is adapted to the near-indentation surface profile measured by atomic force microscopy (AFM). The assessment consists of (1) direct experimental comparisons of strain and deformation and (2) comparisons in which the modeled strain field is used as an intermediate step. Direct experimental methods (1) consist of comparisons of surface elevation and gradient measured by AFM and EBSD and of Raman shifts measured and predicted by CRM and EBSD, respectively. Comparisons that utilize the combined FEA–AFM model (2) consist of predictions of distortion, strain, and rotation for comparison with EBSD measurements and predictions of Raman shift for comparison with CRM measurements. For both EBSD and CRM, convolution of measurements in depth-varying strain fields is considered. The interconnected comparisons suggest that EBSD was able to provide an accurate assessment of the wedge indentation deformation field to within the precision of the measurements, approximately 2×10{sup −4} in strain. CRM was similarly precise, but was limited in accuracy to several times this value. - Highlights: • We map strain by electron backscatter diffraction and confocal Raman microscopy. • The test vehicle is the multi-axial strain field of wedge-indented silicon. • Strain accuracy is assessed by direct experimental intercomparison. • Accuracy is also assessed by atomic force microscopy and finite element analyses. • Electron diffraction measurements are accurate; Raman measurements need refinement.

  12. Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy.

    Science.gov (United States)

    Terasaki, M

    1995-06-01

    A Ca2+ wave at fertilization triggers cortical granule exocytosis in sea urchin eggs. New methods for visualizing exocytosis of individual cortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cortical granule exocytosis results in the formation of long-lived depressions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal microscopy as disks. FM 1-43, a water-soluble fluorescent dye which labels membranes in contact with the sea water, seemed to label the membrane of these depressions and appeared as rings. In double-labeling experiments, the disk and ring labeling by the two types of fluorescent dyes were coincident to within 0.5 second. The fluorescent labeling is coincident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical granule exocytosis. Fluorescent labeling was simultaneous with an expansion of the space occupied by the cortical granule, and labeling by the fluorescent dextran was found to take 0.1-0.2 second. These results are consistent with, and reinforce the previous electron microscopic evidence for, long-lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exocytosis in living eggs. The fluorescence labeling methods were used with the Ca2+ indicator Ca Green-dextran to test if Ca2+ and cortical granule exocytosis are closely related spatially and temporally. In any given region of the cortex, Ca2+ increased relatively slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Optimal lens design and use in laser-scanning microscopy.

    NARCIS (Netherlands)

    Negrean, A.; Mansvelder, H.D.

    2014-01-01

    In laser-scanning microscopy often an off-the-shelf achromatic doublet is used as a scan lens which can reduce the available diffraction-limited field-of-view (FOV) by a factor of 3 and introduce chromatic aberrations that are scan angle dependent. Here we present several simple lens designs of

  14. Probing cytotoxicity of nanoparticles and organic compounds using scanning proton microscopy, scanning electron microscopy and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tong Yongpeng [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China)], E-mail: yongpengt@yahoo.com.cn; Li Changming [School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457 (Singapore); Liang Feng [Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen Jianmin [Shenzhen Municipal Hospital for Chronic Disease Control and Prevention, Guangdong 518020 (China); Zhang Hong; Liu Guoqing; Sun Huibin [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China); Luong, John H.T. [Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, H4P 2R2 (Canada)

    2008-12-15

    Scanning proton microscopy, scanning electron microscopy (SEM) and fluorescence microscopy have been used to probe the cytotoxicity effect of benzo[a]pyrene (BaP), ethidium bromide (EB) and nanoparticles (ZnO, Al{sub 2}O{sub 3} and TiO{sub 2}) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl{sub 2}) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al{sub 2}O{sub 3} and TiO{sub 2} nanoparticles, however, showed a protective effect against these two organic compounds. Such inorganic nanoparticles complexed with BaP or EB which became less toxic to the cell. Fe{sub 2}O{sub 3} nanoparticles as an insoluble particle model scavenged by macrophage were investigated in rats. They were scavenged out of the lung tissue about 48 h after infection. This result suggest that some insoluble inorganic nanoparticles of PM (particulate matters) showed protective effects on organic toxins induced acute toxic effects as they can be scavenged by macrophage cells. Whereas, some inorganic ions such as calcium ion in PM may help environmental organic toxins to penetrate cell membrane and induce higher toxic effect.

  15. Combined frequency modulated atomic force microscopy and scanning tunneling microscopy detection for multi-tip scanning probe microscopy applications

    Science.gov (United States)

    Morawski, Ireneusz; Spiegelberg, Richard; Korte, Stefan; Voigtländer, Bert

    2015-12-01

    A method which allows scanning tunneling microscopy (STM) tip biasing independent of the sample bias during frequency modulated atomic force microscopy (AFM) operation is presented. The AFM sensor is supplied by an electronic circuit combining both a frequency shift signal and a tunneling current signal by means of an inductive coupling. This solution enables a control of the tip potential independent of the sample potential. Individual tip biasing is specifically important in order to implement multi-tip STM/AFM applications. An extensional quartz sensor (needle sensor) with a conductive tip is applied to record simultaneously topography and conductivity of the sample. The high resonance frequency of the needle sensor (1 MHz) allows scanning of a large area of the surface being investigated in a reasonably short time. A recipe for the amplitude calibration which is based only on the frequency shift signal and does not require the tip being in contact is presented. Additionally, we show spectral measurements of the mechanical vibration noise of the scanning system used in the investigations.

  16. De novo melanoma and melanoma arising from pre-existing nevus: in vivo morphologic differences as evaluated by confocal microscopy.

    Science.gov (United States)

    Longo, Caterina; Rito, Cintia; Beretti, Francesca; Cesinaro, Anna Maria; Piñeiro-Maceira, Juan; Seidenari, Stefania; Pellacani, Giovanni

    2011-09-01

    Although in the majority of melanomas there is no evidence of pre-existing melanocytic nevus, it is believed that malignant transformation may sometimes occur within a benign precursor. We sought to describe the morphologic features of de novo melanoma and melanoma arising from nevi by means of in vivo confocal microscopy, and to correlate them with their corresponding histopathologic features. A total of 113 consecutive, histopathologically proven melanoma cases, 33 arising from a nevus and 80 occurring de novo, were imaged by confocal microscopy and retrospectively evaluated. Cyto-architectural features preferentially expressed in melanomas arising from nevi and in de novo melanomas were defined. By confocal microscopy, abrupt transition, localized distribution of junctional atypical cells, and the presence of dense dermal nests were the most helpful criteria for categorizing a melanoma as arising from a nevus. Melanomas arising from common and congenital nevi were predominantly composed of roundish, monomorphous cells, whereas melanomas arising either de novo or from dysplastic nevi were characterized by markedly pleomorphic cells. The study is retrospective. Confocal microscopy is effective in identifying melanoma even when a nevus is simultaneously present, confirming the clinical usefulness of this methodology. Moreover, distinctive features were observed in de novo melanomas and melanomas arising from nevi, permitting accurate distinction between the two groups. Finally, differences in cell morphology, easily detectable by confocal microscopy, seemed to characterize different melanoma types. Copyright © 2010 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

  17. The potential role of in vivo reflectance confocal microscopy for evaluating oral cavity lesions: a systematic review.

    Science.gov (United States)

    Lucchese, Alberta; Gentile, Enrica; Romano, Antonio; Maio, Claudio; Laino, Luigi; Serpico, Rosario

    2016-11-01

    Since the early 2000s, several studies have examined the application of reflectance confocal microscopy (RCM) to the oral cavity. This review gives an overview of the literature on reflectance confocal microscopy analysis of the oral cavity in vivo and identifies flaws in the studies, providing guidance to improve reflectance confocal microscopy applications and inform the design of future studies. The PubMed, ISI, Scopus, and Cochrane Library databases were searched for publications on RCM using the terms 'reflectance confocal microscopy' in combination with 'mouth' and other terms related to the topic of interest. The search gave 617 results. Seventeen studies were included in our final analysis. We decided to organize the selected articles according to four topics: healthy mucosa, autoimmune diseases, cancer and precancerous lesions, and hard dental tissues. Although reflectance confocal microscopy is promising for diagnosing and monitoring oral pathology, it has shortcomings and there are still too few publications on this topic. Further studies are needed to increase the quantity and quality of the results, to translate research into clinical practice. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Local Delivery of Fluorescent Dye For Fiber-Optics Confocal Microscopy of the Living Heart

    Directory of Open Access Journals (Sweden)

    Chao eHuang

    2014-09-01

    Full Text Available Fiber-optics confocal microscopy (FCM is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release versus foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  19. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

    Science.gov (United States)

    Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  20. Confocal line scanning of a Bessel beam for fast 3D imaging.

    Science.gov (United States)

    Zhang, P; Phipps, M E; Goodwin, P M; Werner, J H

    2014-06-15

    We have developed a light-sheet illumination microscope that can perform fast 3D imaging of transparent biological samples with inexpensive visible lasers and a single galvo mirror (GM). The light-sheet is created by raster scanning a Bessel beam with a GM, with this same GM also being used to rescan the fluorescence across a chip of a camera to construct an image in real time. A slit is used to reject out-of-focus fluorescence such that the image formed in real time has minimal contribution from the sidelobes of the Bessel beam. Compared with two-photon Bessel beam excitation or other confocal line-scanning approaches, our method is of lower cost, is simpler, and does not require calibration and synchronization of multiple GMs. We demonstrated the optical sectioning and out-of-focus background rejection capabilities of this microscope by imaging fluorescently labeled actin filaments in fixed 3T3 cells.

  1. Reflectance confocal microscope for imaging oral tissues in vivo, potentially with line scanning as a low-cost approach for clinical use

    Science.gov (United States)

    Peterson, Gary; Abeytunge, Sanjeewa; Eastman, Zachary; Rajadhyaksha, Milind

    2012-02-01

    Reflectance confocal microscopy with a line scanning approach potentially offers a smaller, simpler and less expensive approach than traditional methods of point scanning for imaging in living tissues. With one moving mechanical element (galvanometric scanner), a linear array detector and off-the-shelf optics, we designed a compact (102x102x76mm) line scanning confocal reflectance microscope (LSCRM) for imaging human tissues in vivo in a clinical setting. Custom-designed electronics, based on field programmable gate array (FPGA) logic has been developed. With 405 nm illumination and a custom objective lens of numerical aperture 0.5, lateral resolution was measured to be 0.8 um (calculated 0.64 um). The calculated optical sectioning is 3.2 um. Preliminary imaging shows nuclear and cellular detail in human skin and oral epithelium in vivo. Blood flow is also visualized in the deeper connective tissue (lamina propria) in oral mucosa. Since a line is confocal only in one dimension (parallel) but not in the other, the detection is more sensitive to multiply scattered out of focus background noise than in the traditional point scanning configuration. Based on the results of our translational studies thus far, a simpler, smaller and lower-cost approach based on a LSCRM appears to be promising for clinical imaging.

  2. QUANTIFICATION OF BIOFILMS IN MULTI-SPECTRAL DIGITAL1 VOLUMES FROM CONFOCAL LASER-SCANNING MICROSCOPES

    Directory of Open Access Journals (Sweden)

    Karsten Rodenacker

    2011-05-01

    Full Text Available Populations of bacteria in sludge flocs and biofilm marked by fluorescence marked with fluorescent probes are digitised with a confocal laser scanning microscope. These data are used to analyse the microbial community structure, to obtain information on the localisation of specific bacterial groups and to examine gene expression. This information is urgently required for an in-depth understanding of the function and, more generally, the microbial ecology of biofilms. Methods derived from quantitative image analysis are applied to digitised data from confocal laser scanning microscopes to obtain quantitative descriptions of volumetric, topological (and topographical properties of different compartments of the components under research. In addition to free-moving flocs, also biofilms attached to a substratum in an experimental environment are analysed. Growth form as well as interaction of components are quantitatively described. Classical measurements of volume and intensity (shape, distribution and distance dependent interaction measurements using methods from mathematical morphology are performed. Mainly image (volume processing methods are outlined. Segmented volumes are globally and individually (in terms of 3Dconnected components measured and used for distance mapping transform as well as for estimation of geodesic distances from the substrate. All transformations are applied on the 3D data set. Resulting distance distributions are quantified and related to information on the identity and activity of the probe-identified bacteria.

  3. Handbook of Microscopy for Nanotechnology

    Science.gov (United States)

    Yao, Nan; Wang, Zhong L.

    This handbook highlights various key microcopic techniques and their applications in this fast-growing field. Topics to be covered include the following: scanning near field optical microscopy, confocal optical microscopy, atomic force microscopy, magnetic force microscopy, scanning turning microscopy, high-resolution scanning electron microscopy, and many more.

  4. Small fiber neuropathy in women with fibromyalgia. An in vivo assessment using corneal confocal bio-microscopy.

    Science.gov (United States)

    Ramírez, Manuel; Martínez-Martínez, Laura-Aline; Hernández-Quintela, Everardo; Velazco-Casapía, Jorge; Vargas, Angélica; Martínez-Lavín, Manuel

    2015-10-01

    A consistent line of investigation suggests that fibromyalgia is a neuropathic pain syndrome. This outlook has been recently reinforced by several controlled studies that describe decreased small nerve fiber density in skin biopsies of patients with fibromyalgia. The cornea receives the densest small fiber innervation of the body. Corneal confocal bio-microscopy is a new noninvasive method to evaluate small nerve fiber morphology. Our objective was to assess corneal small nerve fiber morphology in patients with fibromyalgia, and to associate corneal nerve microscopic features with neuropathic pain descriptors and other fibromyalgia symptoms. We studied 17 female patients with fibromyalgia and 17 age-matched healthy control subjects. All the participants completed different questionnaires regarding the symptoms of fibromyalgia, including a neuropathic pain survey. A central corneal thickness scan was obtained with a confocal microscope. Nerve measurements were made by a single ophthalmologist without knowledge of the clinical diagnosis. Stromal nerve thickness was defined as the mean value between the widest and the narrowest portion of each analyzed stromal nerve. Corneal sub-basal plexus nerve density was also assessed. Patients with fibromyalgia had stromal nerve thickness of 5.0 ± 1.0 µm (mean ± standard deviation) significantly different from that of control's values (6.1 ± 1.3) p = 0.01. Patients also had decreased sub-basal plexus nerve density per square millimeter (85 ± 29) vs. 107 ± 26 of controls p = 0.02. When controls and patients were grouped together, there was an association between stromal nerve slenderness and neuropathic pain descriptors (Fisher's exact test p = 0.007). Women suffering from fibromyalgia have thinner corneal stromal nerves and diminished sub-basal plexus nerve density when compared to healthy controls. Nerve scarcity is associated with neuropathic pain descriptors. Small fiber neuropathy may play a role in the pathogenesis of

  5. Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy.

    Science.gov (United States)

    Talamás-Lara, Daniel; Talamás-Rohana, Patricia; Fragoso-Soriano, Rogelio Jaime; Espinosa-Cantellano, Martha; Chávez-Munguía, Bibiana; González-Robles, Arturo; Martínez-Palomo, Adolfo

    2015-10-01

    Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Automated detection of malignant features in confocal microscopy on superficial spreading melanoma versus nevi

    Science.gov (United States)

    Gareau, Dan; Hennessy, Ricky; Wan, Eric; Pellacani, Giovanni; Jacques, Steven L.

    2010-11-01

    In-vivo reflectance confocal microscopy (RCM) shows promise for the early detection of superficial spreading melanoma (SSM). RCM of SSM shows pagetoid melanocytes (PMs) in the epidermis and disarray at the dermal-epidermal junction (DEJ), which are automatically quantified with a computer algorithm that locates depth of the most superficial pigmented surface [DSPS(x,y)] containing PMs in the epidermis and pigmented basal cells near the DEJ. The algorithm uses 200 noninvasive confocal optical sections that image the superficial 200 μm of ten skin sites: five unequivocal SSMs and five nevi. The pattern recognition algorithm automatically identifies PMs in all five SSMs and finds none in the nevi. A large mean gradient ψ (roughness) between laterally adjacent points on DSPS(x,y) identifies DEJ disruption in SSM ψ = 11.7 +/- 3.7 [-] for n = 5 SSMs versus a small ψ = 5.5 +/- 1.0 [-] for n = 5 nevi (significance, p = 0.0035). Quantitative endpoint metrics for malignant characteristics make digital RCM data an attractive diagnostic asset for pathologists, augmenting studies thus far, which have relied largely on visual assessment.

  7. Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy.

    Science.gov (United States)

    Nooh, Mohammed M; Bahouth, Suleiman W

    2017-01-01

    G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β 1 -adrenergic receptor (β 1 -AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β 1 -AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review. © 2017 Elsevier Inc. All rights reserved.

  8. In-situ detection of drugs-of-abuse on clothing using confocal Raman microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ali, Esam M.A. [Raman Spectroscopy Group, University Analytical Centre, Division of Chemical and Forensic Sciences, University of Bradford, Bradford BD7 1DP (United Kingdom); Edwards, Howell G.M. [Raman Spectroscopy Group, University Analytical Centre, Division of Chemical and Forensic Sciences, University of Bradford, Bradford BD7 1DP (United Kingdom)], E-mail: h.g.m.edwards@bradford.ac.uk; Hargreaves, Michael D.; Scowen, Ian J. [Raman Spectroscopy Group, University Analytical Centre, Division of Chemical and Forensic Sciences, University of Bradford, Bradford BD7 1DP (United Kingdom)

    2008-05-12

    This study describes the application of confocal Raman microscopy to the detection and identification of drugs-of-abuse in situ on undyed natural synthetic fibres, and coloured textile specimens. Raman spectra were obtained from drug particles trapped between the fibres of the specimens. Pure samples of cocaine hydrochloride and N-methyl-3,4-methylenedioxy-amphetamine HCl (MDMA-HCl) were used in this study. Raman spectra were collected from drug particles of an average size in the range 5-15 {mu}m. Despite the presence of spectral bands arising from the natural and synthetic polymer and dyed textiles, the drugs could be identified by their characteristic Raman bands. If necessary, interfering bands could be successfully removed by spectral subtraction. Furthermore, Raman spectra were recorded from drug particles trapped between the fibres of highly fluorescent specimens. Interference from the fibres, including background fluorescence, was overcome by careful focusing of the confocal beam and the resulting spectra allow ready differentiation from interference from the fibres substrate bands. Spectra of several drugs-of-abuse on dyed and undyed clothing substrates were readily obtained within 3 min with little or no sample preparation and with no alteration of the evidential material.

  9. In-situ detection of drugs-of-abuse on clothing using confocal Raman microscopy.

    Science.gov (United States)

    Ali, Esam M A; Edwards, Howell G M; Hargreaves, Michael D; Scowen, Ian J

    2008-05-12

    This study describes the application of confocal Raman microscopy to the detection and identification of drugs-of-abuse in situ on undyed natural synthetic fibres, and coloured textile specimens. Raman spectra were obtained from drug particles trapped between the fibres of the specimens. Pure samples of cocaine hydrochloride and N-methyl-3,4-methylenedioxy-amphetamine HCl (MDMA-HCl) were used in this study. Raman spectra were collected from drug particles of an average size in the range 5-15 microm. Despite the presence of spectral bands arising from the natural and synthetic polymer and dyed textiles, the drugs could be identified by their characteristic Raman bands. If necessary, interfering bands could be successfully removed by spectral subtraction. Furthermore, Raman spectra were recorded from drug particles trapped between the fibres of highly fluorescent specimens. Interference from the fibres, including background fluorescence, was overcome by careful focusing of the confocal beam and the resulting spectra allow ready differentiation from interference from the fibres substrate bands. Spectra of several drugs-of-abuse on dyed and undyed clothing substrates were readily obtained within 3 min with little or no sample preparation and with no alteration of the evidential material.

  10. In vivo three-dimensional reconstruction of the cornea from confocal microscopy images.

    Science.gov (United States)

    Scarpa, Fabio; Fiorin, Diego; Ruggeri, Alfredo

    2007-01-01

    Confocal microscopy can provide sequences of images from all cornea layers in a rapid, in vivo and non invasive way. These images are useful to extract important clinical information on cornea state of health. We address the problem of obtaining a 3-dimensional (3D) reconstruction of the cornea starting from a confocal microscope sequence, from endothelium to epithelium. A registration procedure, based on normalized correlation, is applied to each image, because eye movements normally occur during acquisition of the sequence and shifts in x-y plane take place in the sequence of images. Information on shifts along x and y directions comes from registration process, shift along z direction comes from the instrument itself. A 2D image stack is reconstructed by taking into account shifts along x, y, and z directions. If data are missing, we reconstruct them by taking lines from adjacent images and interpolating them. After reconstruction, it is possible to display and analyze corneal structures in the 3D volume and obtain slices in the x, y, or z direction.

  11. The use of reflectance confocal microscopy for monitoring response to therapy of skin malignancies.

    Science.gov (United States)

    Ulrich, Martina; Lange-Asschenfeldt, Susanne; Gonzalez, Salvador

    2012-04-01

    Reflectance confocal microscopy (RCM) is a new non-invasive imaging technique that enables visualizing cells and structures in living skin in real-time with resolution close to that of histological analysis. RCM has been successfully implemented in the assessment of benign and malignant lesions. Most importantly, it also enables monitoring dynamic changes in the skin over time and in response to different therapies, e.g., imiquimod, photodynamic therapy, and others. Given the often traumatic nature of skin cancer that affects both the physiology and the psychology of the patients, it is crucial to have methods that enable monitoring the response to treatment but that minimize the distress and discomfort associated with such process. This article provides a very brief overview of the fundamentals of RCM and then focuses on its recent employment as a monitoring tool in skin cancer and other pathologies that may require frequent follow-up.

  12. Reflectance Confocal Microscopy: A Promising Tool to Identify Malignancy in Melanocytic Lesions Exhibiting a Dermoscopic Island.

    Science.gov (United States)

    Elosua-González, M; Gamo-Villegas, R; Floristán-Muruzábal, U; Pinedo-Moraleda, F; López-Estebaranz, J L

    2017-11-22

    The dermoscopic island is described as a well-defined area in a melanocytic lesion, with a different dermoscopic pattern from the rest of the lesion. It is predictive of melanoma, particularly when the pattern of the island is atypical. We present the reflectance confocal microscopy (RCM) findings in 3 lesions with dermoscopic islands: nevus-associated melanoma, melanocytic nevus, and in situ melanoma. The nevus-associated melanoma and in situ melanoma presented cellular atypia (atypical cells in isolation or forming nests) and architectural distortion on RCM. The nevus presented an island sign with a typical globular pattern with dense nests and no atypia on RCM. The island sign is mainly associated with in situ and nevus-associated melanomas. RCM offers good cellular resolution to the depth of the reticular dermis and is useful for diagnosing of melanomas presenting a dermoscopic island. Copyright © 2017 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

  13. Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

    Science.gov (United States)

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

    2010-01-01

    Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

  14. Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer

    Science.gov (United States)

    Jabbour, Joey M.; Cheng, Shuna; Malik, Bilal H.; Cuenca, Rodrigo; Jo, Javier A.; Wright, John; Cheng, Yi-Shing Lisa; Maitland, Kristen C.

    2013-04-01

    Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm2 tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

  15. In vivo reflectance-mode confocal microscopy in clinical dermatology and cosmetology.

    Science.gov (United States)

    González, S; Gilaberte-Calzada, Y

    2008-02-01

    In vivo reflectance confocal microscopy (RCM) is a non-invasive imaging tool that allows real-time visualization of cells and structures in living skin with near histological resolution. RCM has been used for the assessment of benign and malignant lesions, showing great potential for applications in basic skin research and clinical dermatology. RCM also reveals dynamic changes in the skin over time and in response to specific stimuli, like ultraviolet exposure, which makes it a promising tool in cosmetology, as it allows repetitive sampling without biopsy collection, causing no further damage to the areas under investigation. This review summarizes the latest advances in RCM, and its applications in the characterization of both normal and pathological skin.

  16. Confocal microscopy movies of fibrin clots during ultrasound-accelerated thrombolysis

    Science.gov (United States)

    Everbach, E. Carr; Chernysh, Irina N.; Weisel, John W.

    2005-04-01

    Blood clots made of human purified fibrin (white clots) were insonified with 1 MHz pulsed ultrasound during observation by fluorescence confocal microscopy. A deconvolution microscope allowed extremely thin sheets (0.2 μm) of fibrin to be viewed at a resolution of 0.2 μm per pixel, and the clot microstructure visualized. Acoustic pressure amplitudes from 0.1 to 0.8 MPa (peak-to-peak) were inferred using the image blur of 0.6-μm-diameter polystyrene spheres coated with FITC fluorecent label present in the clots. Acoustic pulse widths of 1 ms and pulse repetition frequencies of 125 Hz reduced clot heating to less than 3°C during each 30-minute exposure. Still 100 μm by 100 μm images were recorded every 10 seconds during pauses in insonificaiton, to produce time-lapse movies that are compared with movies made during sham ultrasound exposures.

  17. In-situ detection of single particles of explosive on clothing with confocal Raman microscopy.

    Science.gov (United States)

    Ali, Esam M A; Edwards, Howell G M; Scowen, Ian J

    2009-05-15

    Confocal Raman microscopy is shown to detect picogram quantities of explosives in-situ on undyed natural and synthetic fibres, and coloured textile specimens leaving potentially evidential materials unaltered. Raman spectra were obtained from pentaerythritol tetranitrate (PETN), trinitrotoluene (TNT), and ammonium nitrate particles trapped between the fibres of the specimens. Despite the presence of spectral bands arising from the natural and synthetic polymers and dyed textiles, the explosive substances could be identified by their characteristic Raman bands. Furthermore, Raman spectra were obtained from explosives particles trapped between highly fluorescent clothing fibres. Raman spectra were collected from explosives particles with maximum dimensions in the range 5-10 microm. Spectra of the explosives on dyed and undyed clothing substrates were readily obtained in-situ within 90 s and without sample preparation.

  18. Surface determination of 3D confocal Raman microscopy imaging of the skin

    Science.gov (United States)

    Schleusener, J.; Carrer, V.; Patzelt, A.; Lademann, J.; Darvin, M. E.

    2017-12-01

    A surface determination method for the application of 3D confocal Raman microscopy on inhomogeneous skin sections has been presented, which is based on depth profiles of the keratin contribution of the acquired Raman spectra. The method was compared to two similar auto-focusing methods that are based on the intensity of the reflected excitation light and Raman spectra, respectively. The measurements were performed on hair follicles containing skin sections of porcine ears ex vivo. The surface determination on such samples is especially challenging due to their different molecular composition and surface inhomogeneity. An advantage of this method is molecular sensitivity, whereby only the surface of the sample will be detected and not the substrate of the microscope slide, in the case of disruptions during the processing of samples. A disadvantage of the method is the increased overall acquisition time if only the surface spectra are to be applied for 2D mapping.

  19. Plant cell wall characterization using scanning probe microscopy techniques

    Science.gov (United States)

    Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

    2009-01-01

    Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

  20. Plant cell wall characterization using scanning probe microscopy techniques

    Directory of Open Access Journals (Sweden)

    Himmel Michael E

    2009-08-01

    Full Text Available Abstract Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy.

  1. Corneal confocal microscopy and dry eye findings in contact lens discomfort patients.

    Science.gov (United States)

    Dogan, Aysun Sanal; Gurdal, Canan; Arslan, Nese

    2017-08-16

    To evaluate the corneal confocal microscopy and dry eye findings in patients with contact lens discomfort. The study included 3 groups of participants: Contact lens wearers using silicone hydrogel soft contact lenses who are symptomatic (CLD, n=15) or asymptomatic (ACL, n=11) and non-wearers as controls (n=14). Duration of contact lens wear, Ocular Surface Disease Index (OSDI) questionnaire responses, fluorescein tear break-uptime (FBUT), and corneal confocal microscopy findings were recorded. Mean age was 25.7±8.2 years and male/female ratio was 7/33. Demographic findings were similar regarding the groups. CLD patients had a longer lens use history than ACL (median 5 vs 2 years, p<0.001). OSDI scores were higher in CLD group than ACL or controls (p<0.001, p=0.002). FBUT was significantly lowest in CLD group, compared to controls and ACL (p<0.001, p=0.039). FBUT was also lower in ACL patients compared to controls (p=0.036). There was no difference between basal epithelium cell counts between all 3 groups. Anterior stromal activated keratocyte numbers were similar between contact lens using groups but was lower in controls (p=0.005). However, dendritiform cells in the sub-basal nerve layer were higher in CLD group compared to controls but similar to ACL (p<0.001, p=0.058). Graded sub-basal nerve tortuosity was more prominent in CLD group than the ACL (p=0.014). Patients with CLD had been wearing contact lenses for longer than those without symptoms. OSDI and FBUT scores were worse in CLD patients. In contact lens discomfort patients, there were increased dendritiform cells, indicating intensified inflammatory status of the cornea. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  2. Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures

    Science.gov (United States)

    Tatini, Francesca; Rossi, Francesca; Coppi, Elisabetta; Magni, Giada; Fusco, Irene; Menabuoni, Luca; Pedata, Felicita; Pugliese, Anna Maria; Pini, Roberto

    2016-04-01

    The characterization of the ion channels in corneal endothelial cells and the elucidation of their involvement in corneal pathologies would lead to the identification of new molecular target for pharmacological treatments and to the clarification of corneal physiology. The corneal endothelium is an amitotic cell monolayer with a major role in preserving corneal transparency and in regulating the water and solute flux across the posterior surface of the cornea. Although endothelial cells are non-excitable, they express a range of ion channels, such as voltage-dependent Na+ channels and K+ channels, L-type Ca2 channels and many others. Interestingly, purinergic receptors have been linked to a variety of conditions within the eye but their presence in the endothelium and their role in its pathophysiology is still uncertain. In this study, we were able to extract endothelial cells from single human corneas, thus obtaining primary cultures that represent the peculiarity of each donor. Corneas were from tissues not suitable for transplant in patients. We characterized the endothelial cells by confocal microscopy, both within the intact cornea and in the primary endothelial cells cultures. We also studied the functional role of the purinergic system (adenosine, ATP and their receptors) by means of electrophysiological recordings. The experiments were performed by patch clamp recordings and confocal time-lapse microscopy and our results indicate that the application of purinergic compounds modulates the amplitude of outward currents in the isolated endothelial cells. These findings may lead to the proposal of new therapies for endothelium-related corneal diseases.

  3. Full information acquisition in scanning probe microscopy and spectroscopy

    Science.gov (United States)

    Jesse, Stephen; Belianinov, Alex; Kalinin, Sergei V.; Somnath, Suhas

    2017-04-04

    Apparatus and methods are described for scanning probe microscopy and spectroscopy based on acquisition of full probe response. The full probe response contains valuable information about the probe-sample interaction that is lost in traditional scanning probe microscopy and spectroscopy methods. The full probe response is analyzed post data acquisition using fast Fourier transform and adaptive filtering, as well as multivariate analysis. The full response data is further compressed to retain only statistically significant components before being permanently stored.

  4. Coin-shaped epithelial lesions following an acute attack of erythema multiforme minor with confocal microscopy findings.

    Science.gov (United States)

    Babu, Kalpana; Murthy, Vinay R; Akki, Veeresh P; Prabhakaran, Venkatesh C; Murthy, K R

    2010-01-01

    We report an interesting ocular finding of bilateral multiple coin-shaped epithelial lesions along with the confocal microscopy findings in a patient following an acute attack of erythema multiforme (EM) minor. A 30-year-old male presented with a history of watering and irritation in both eyes of three days duration. He was diagnosed to have EM minor and was on oral acyclovir. Slit-lamp examination revealed multiple coin-shaped epithelial lesions. Confocal microscopy showed a corresponding conglomerate of hyper-reflective epithelial lesions. The corneal lesions resolved over six weeks with oral steroids and acyclovir. An immunological mechanism is suspected.

  5. Coin-shaped epithelial lesions following an acute attack of erythema multiforme minor with confocal microscopy findings

    Directory of Open Access Journals (Sweden)

    Babu Kalpana

    2010-01-01

    Full Text Available We report an interesting ocular finding of bilateral multiple coin-shaped epithelial lesions along with the confocal microscopy findings in a patient following an acute attack of erythema multiforme (EM minor. A 30-year-old male presented with a history of watering and irritation in both eyes of three days duration. He was diagnosed to have EM minor and was on oral acyclovir. Slit-lamp examination revealed multiple coin-shaped epithelial lesions. Confocal microscopy showed a corresponding conglomerate of hyper-reflective epithelial lesions. The corneal lesions resolved over six weeks with oral steroids and acyclovir. An immunological mechanism is suspected.

  6. Local order in a supercooled colloidal fluid observed by confocal microscopy

    CERN Document Server

    Gasser, U; Weitz, D A

    2003-01-01

    The local order in a supercooled monodisperse colloidal fluid is studied by direct imaging of the particles with a laser scanning confocal microscope. The local structure is analysed with a bond order parameter method, which allows one to discern simple structures that are relevant in this system. As expected for samples that crystallize eventually, a large fraction of the particles are found to sit in surroundings with dominant face-centred cubic or hexagonally close-packed character. Evidence for local structures that contain fragments of icosahedra is found, and, moreover, the icosahedral character increases with volume fraction phi, which indicates that it might play an important role at volume fractions near the glass transition.

  7. Fluorescence microscopy.

    Science.gov (United States)

    Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

    2014-10-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

  8. Confocal fluorescence microscopy for rapid evaluation of invasive tumor cellularity of inflammatory breast carcinoma core needle biopsies.

    Science.gov (United States)

    Dobbs, Jessica; Krishnamurthy, Savitri; Kyrish, Matthew; Benveniste, Ana Paula; Yang, Wei; Richards-Kortum, Rebecca

    2015-01-01

    Tissue sampling is a problematic issue for inflammatory breast carcinoma, and immediate evaluation following core needle biopsy is needed to evaluate specimen adequacy. We sought to determine if confocal fluorescence microscopy provides sufficient resolution to evaluate specimen adequacy by comparing invasive tumor cellularity estimated from standard histologic images to invasive tumor cellularity estimated from confocal images of breast core needle biopsy specimens. Grayscale confocal fluorescence images of breast core needle biopsy specimens were acquired following proflavine application. A breast-dedicated pathologist evaluated invasive tumor cellularity in histologic images with hematoxylin and eosin staining and in grayscale and false-colored confocal images of cores. Agreement between cellularity estimates was quantified using a kappa coefficient. 23 cores from 23 patients with suspected inflammatory breast carcinoma were imaged. Confocal images were acquired in an average of less than 2 min per core. Invasive tumor cellularity estimated from histologic and grayscale confocal images showed moderate agreement by kappa coefficient: κ = 0.48 ± 0.09 (p fluorescence microscopy can be performed immediately following specimen acquisition and could indicate the need for additional biopsies at the initial visit.

  9. Towards Automated Nanomanipulation under Scanning Electron Microscopy

    Science.gov (United States)

    Ye, Xutao

    Robotic Nanomaterial Manipulation inside scanning electron microscopes (SEM) is useful for prototyping functional devices and characterizing one-dimensional nanomaterial's properties. Conventionally, manipulation of nanowires has been performed via teleoperation, which is time-consuming and highly skill-dependent. Manual manipulation also has the limitation of low success rates and poor reproducibility. This research focuses on a robotic system capable of automated pick-place of single nanowires. Through SEM visual detection and vision-based motion control, the system transferred individual silicon nanowires from their growth substrate to a microelectromechanical systems (MEMS) device that characterized the nanowires' electromechanical properties. The performances of the nanorobotic pick-up and placement procedures were quantified by experiments. The system demonstrated automated nanowire pick-up and placement with high reliability. A software system for a load-lock-compatible nanomanipulation system is also designed and developed in this research.

  10. Use of Corneal Confocal Microscopy to Detect Corneal Nerve Loss and Increased Dendritic Cells in Patients With Multiple Sclerosis.

    Science.gov (United States)

    Bitirgen, Gulfidan; Akpinar, Zehra; Malik, Rayaz A; Ozkagnici, Ahmet

    2017-07-01

    Multiple sclerosis (MS) is characterized by demyelination, axonal degeneration, and inflammation. Corneal confocal microscopy has been used to identify axonal degeneration in several peripheral neuropathies. To assess corneal subbasal nerve plexus morphologic features, corneal dendritic cell (DC) density, and peripapillary retinal nerve fiber layer (RNFL) thickness in patients with MS. This single-center, cross-sectional comparative study was conducted at a tertiary referral university hospital between May 27, 2016, and January 30, 2017. Fifty-seven consecutive patients with relapsing-remitting MS and 30 healthy, age-matched control participants were enrolled in the study. Corneal subbasal nerve plexus measures and DC density were quantified in images acquired with the laser scanning in vivo corneal confocal microscope, and peripapillary RNFL thickness was measured with spectral-domain optical coherence tomography. Corneal nerve fiber density, nerve branch density, nerve fiber length, DC density, peripapillary RNFL thickness, and association with the severity of neurologic disability as assessed by the Kurtzke Expanded Disability Status Scale (score range, 0-10; higher scores indicate greater disability) and Multiple Sclerosis Severity Score (score range, 0.01-9.99; higher scores indicate greater severity). Of the 57 participants with MS, 42 (74%) were female and the mean (SD) age was 35.4 (8.9) years; of the 30 healthy controls, 19 (63%) were female and the mean (SD) age was 34.8 (10.2) years. Corneal nerve fiber density (mean [SE] difference, -6.78 [2.14] fibers/mm2; 95% CI, -11.04 to -2.52; P = .002), nerve branch density (mean [SE] difference, -17.94 [5.45] branches/mm2; 95% CI, -28.77 to -7.10; P = .001), nerve fiber length (mean [SE] difference, -3.03 [0.89] mm/mm2; 95% CI, -4.81 to -1.25; P = .001), and the mean peripapillary RNFL thickness (mean [SE] difference, -17.06 [3.14] μm; 95% CI, -23.29 to -10.82; P < .001) were reduced in patients with MS compared

  11. Imaging of whole tumor cut sections using a novel scanning beam confocal fluorescence MACROscope

    Science.gov (United States)

    Constantinou, Paul; Vukovic, Vojislav; Haugland, Hans K.; Nicklee, Trudey; Hedley, David W.; Wilson, Brian C.

    2001-07-01

    Hypoxia caused by inadequate structure and function of the tumor vasculature has been found to negatively determine the prognosis of cancer patients. Hence, understanding the biological basis of tumor hypoxia is of significant clinical interest. To study solid tumor microenvironments in sufficient detail, large areas (several mm in diameter) need to be imaged at micrometers resolutions. We have used a novel confocal scanning laser MACROscopeTM (CSLM) capable of acquiring images over fields of view up to 2 cm X 2 cm. To demonstrate its performance, frozen sections from a cervical carcinoma xenograft were triple labeled for tissue hypoxia, blood vessels and hypoxia-inducible transcription factor 1 alpha (HIF-1(alpha) ), imaged using the CSLM and compared to images obtained using a standard epifluorescence microscope imaging system. The results indicate that the CSLM is a useful instrument for imaging tissue-based fluorescence at resolutions comparable to standard low-power microscope objectives.

  12. Real-time line-scanning reflectance confocal endoscope to enhance sectioning and reduce speckle for intraoral imaging

    Science.gov (United States)

    Glazowski, Christopher; Abeytunge, Sanjeewa; Rajadhyaksha, Milind

    2012-02-01

    The line-scanning confocal microscope is simpler than a point-scanning confocal microscope and allows for a smaller and lower cost footprint, making it attractive for endoscopic clinical use. The optical configuration affects image fidelity. Here, we present a benchtop version of an endoscopic line-scanning confocal microscope for intraoral imaging, with a divided pupil and optimal detection configuration (magnification, pixel-to-resolution ratio) to enhance image fidelity. Improved sectioning performance and reduction of "speckle" noise are demonstrated. A topology for use of a deformable MEMs mirror-based optical axial focus control for imaging in depth is presented. Preliminary images of human oral mucosa in vivo demonstrate feasibility for clinical application.

  13. Large area mapping of excised breast tissue by fluorescence confocal strip scanning: a preliminary feasibility study

    Science.gov (United States)

    Larson, Bjorg A.; Abeytunge, Sanjee; Murray, Melissa; Rajadhyaksha, Milind

    2013-03-01

    Lumpectomy, in conjunction with radiation and chemotherapy drugs, together comprise breast-conserving treatment as an alternative to total mastectomy for patients with breast tumors. The tumor is removed in surgery and sent for pathology processing to assess the margins, a process that takes at minimum several hours, and generally days. If the margins are not clear of tumor, the patient must undergo a second surgery to remove residual tumor. This re-excision rate varies by institution, but can be as high as 60%. Currently, no intraoperative microscopic technique is used routinely to examine tumor margins in breast tissue. A new technique for rapidly scanning large areas of tissue has been developed, called confocal strip scanning, which provides high resolution and seamless mosaics over large areas of intact tissue, with nuclear and cellular resolution and optical sectioning of about 2 microns. Up to 3.5 x 3.5 cm2 of tissue is imaged in 13 minutes at current stage speeds. This technique is demonstrated in freshly excised breast tissue, using a mobile confocal microscope stationed in our pathology laboratory. Twenty-five lumpectomy and mastectomy cases were used as a testing ground for reflectance and fluorescence contrast modes, resolution requirements and tissue fixturing configurations. It was concluded that fluorescent imaging provides the needed contrast to distinguish ducts and lobules from surrounding stromal tissue. Therefore the system was configured with 488 nm illumination, with acridine orange fluorescent dye for nuclear contrast, with the aim of building an image library of malignant and benign breast pathologies.

  14. New Applications of Scanning Tunneling Microscopy

    Science.gov (United States)

    Smith, Douglas Philip Edward

    This dissertation describes the application of the scanning tunneling microscope (STM) technique to four new fields of study: thin organic films, phonon spectroscopy of bulk surfaces, the vibrational spectroscopy of molecules, and the tribological forces which occur between STM tip and sample. Images with atomic resolution were obtained with speeds approaching video rates. Two types of microscopes were used: one operated at room temperature in air, another at 4.2K in liquid helium. At room temperature, the STM was able to image molecules of cadmium arachidate deposited onto graphite by the Langmuir-Blodgett technique. The packing of molecules in the lipid bilayer was found to be partially ordered, with density of 1 molecule per 19.4 square angstroms. At liquid-helium temperature, inelastic electron processes were detected, and it was possible to determine within an area of a few square angstroms where the vibrational excitations occurred. On a bare graphite substrate, phonons of the sample and tip caused step increases in the tunneling conductivity at the phonon energies. Molecules of sorbic acid could be resolved when deposited onto graphite, and these molecules caused spatially localized peaks in conductivity at the energies of the bond vibrations. Although the STM is usually considered a non-contact instrument, under certain circumstances the tip and sample exerted strong forces on each other. With a tungsten tip and a graphite sample, friction and mechanical deformations on the atomic scale were observed.

  15. Optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy in retinal nerve fiber layer measurements of glaucoma patients.

    Science.gov (United States)

    Fanihagh, Farsad; Kremmer, Stephan; Anastassiou, Gerasimos; Schallenberg, Maurice

    2015-01-01

    To determine the correlations and strength of association between different imaging systems in analyzing the retinal nerve fiber layer (RNFL) of glaucoma patients: optical coherence tomography (OCT), scanning laser polarimetry (SLP) and confocal scanning laser ophthalmoscopy (CSLO). 114 eyes of patients with moderate open angle glaucoma underwent spectral domain OCT (Topcon SD-OCT 2000 and Zeiss Cirrus HD-OCT), SLP (GDx VCC and GDx Pro) and CSLO (Heidelberg Retina Tomograph, HRT 3). Correlation coefficients were calculated between the structural parameters yielded by these examinations. The quantitative relationship between the measured RNFL thickness globally and for the four regions (superior, inferior, nasal, temporal) were evaluated with different regression models for all used imaging systems. The strongest correlation of RNFL measurements was found between devices using the same technology like GDx VCC and GDx Pro as well as Topcon OCT and Cirrus OCT. In glaucoma patients, the strongest associations (R²) were found between RNFL measurements of the two optical coherence tomography devices Topcon OCT and Cirrus OCT (R² = 0.513) and between GDx VCC and GDx Pro (R² = 0.451). The results of the OCTs and GDX Pro also had a strong quantitative relationship (Topcon OCT R² = 0.339 and Cirrus OCT R² = 0.347). GDx VCC and the OCTs showed a mild to moderate association (Topcon OCT R² = 0.207 and Cirrus OCT R² = 0.258). The confocal scanning laser ophthalmoscopy (HRT 3) had the lowest association to all other devices (Topcon OCT R² = 0.254, Cirrus OCT R² = 0.158, GDx Pro R² = 0.086 and GDx VCC R² = 0.1). The measurements of the RNFL in glaucoma patients reveal a high correlation of OCT and GDx devices because OCTs can measure all major retinal layers and SLP can detect nerve fibers allowing a comparison between the results of this devices. However, CSLO by means of HRT topography can only measure height values of the retinal surface but it cannot distinguish

  16. Open Source Scanning Probe Microscopy Control Software Package Gxsm

    Energy Technology Data Exchange (ETDEWEB)

    Zahl P.; Wagner, T.; Moller, R.; Klust, A.

    2009-08-10

    Gxsm is a full featured and modern scanning probe microscopy (SPM) software. It can be used for powerful multidimensional image/data processing, analysis, and visualization. Connected toan instrument, it is operating many different avors of SPM, e.g., scanning tunneling microscopy(STM) and atomic force microscopy (AFM) or in general two-dimensional multi channel data acquisition instruments. The Gxsm core can handle different data types, e.g., integer and oating point numbers. An easily extendable plug-in architecture provides many image analysis and manipulation functions. A digital signal processor (DSP) subsystem runs the feedback loop, generates the scanning signals and acquires the data during SPM measurements. The programmable Gxsm vector probe engine performs virtually any thinkable spectroscopy and manipulation task, such as scanning tunneling spectroscopy (STS) or tip formation. The Gxsm software is released under the GNU general public license (GPL) and can be obtained via the Internet.

  17. Evaluation of breast tissue with confocal strip-mosaicking microscopy: a test approach emulating pathology-like examination

    Science.gov (United States)

    Abeytunge, Sanjee; Larson, Bjorg; Peterson, Gary; Morrow, Monica; Rajadhyaksha, Milind; Murray, Melissa P.

    2017-03-01

    Confocal microscopy is an emerging technology for rapid imaging of freshly excised tissue without the need for frozen- or fixed-section processing. Initial studies have described imaging of breast tissue using fluorescence confocal microscopy with small regions of interest, typically 750×750 μm2. We present exploration with a microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 2×2 cm2 of tissue with cellular-level resolution in 10 min of excision. Using the CSM microscope, we imaged 34 fresh, human, large breast tissue specimens from 18 patients, blindly analyzed by a board-certified pathologist and subsequently correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM strip-mosaic images. Thirty specimens were concordant for image-to-histopathology correlation while four were discordant.

  18. Time-resolved measurements of DNA interactions in an electrowetting-on-dielectric system using confocal microscopy

    Science.gov (United States)

    Sparrenberg, Lorenz T.; Greiner, Benjamin; Mueller, Christian; Mathis, Harald P.

    2017-11-01

    To identify new drug candidates a deep and profound knowledge of molecule interactions is needed. In the current work, a combination of an electrowetting-on-dielectric (EWOD) system with a confocal microscopy is presented for the first time. The aim of this research is to attain time-resolved information about nucleic acid interactions at a single molecule level. Confocal microscopy is a promising technique for reaction analysis on a molecular scale. But the liquid handling of the needed tiny volumes of highly diluted solutions is very challenging. An EWOD based system for droplet handling can address this demand. In this paper the development of an EWOD system for droplet handling in nanoliter scale is discussed and the combination of the EWOD system with a confocal microscope, to investigate nucleic acid interactions, is evaluated.

  19. Vector sensor for scanning SQUID microscopy

    Science.gov (United States)

    Dang, Vu The; Toji, Masaki; Thanh Huy, Ho; Miyajima, Shigeyuki; Shishido, Hiroaki; Hidaka, Mutsuo; Hayashi, Masahiko; Ishida, Takekazu

    2017-07-01

    We plan to build a novel 3-dimensional (3D) scanning SQUID microscope with high sensitivity and high spatial resolution. In the system, a vector sensor consists of three SQUID sensors and three pick-up coils realized on a single chip. Three pick-up coils are configured in orthogonal with each other to measure the magnetic field vector of X, Y, Z components. We fabricated some SQUID chips with one uniaxial pick-up coil or three vector pick-up coils and carried out fundamental measurements to reveal the basic characteristics. Josephson junctions (JJs) of sensors are designed to have the critical current density J c of 320 A/cm2, and the critical current I c becomes 12.5 μA for the 2.2μm × 2.2μm JJ. We carefully positioned the three pickup coils so as to keep them at the same height at the centers of all three X, Y and Z coils. This can be done by arranging them along single line parallel to a sample surface. With the aid of multilayer technology of Nb-based fabrication, we attempted to reduce an inner diameter of the pickup coils to enhance both sensitivity and spatial resolution. The method for improving a spatial resolution of a local magnetic field image is to employ an XYZ piezo-driven scanner for controlling the positions of the pick-up coils. The fundamental characteristics of our SQUID sensors confirmed the proper operation of our SQUID sensors and found a good agreement with our design parameters.

  20. Detection of latent fingerprints using high-resolution 3D confocal microscopy in non-planar acquisition scenarios

    Science.gov (United States)

    Kirst, Stefan; Vielhauer, Claus

    2015-03-01

    In digitized forensics the support of investigators in any manner is one of the main goals. Using conservative lifting methods, the detection of traces is done manually. For non-destructive contactless methods, the necessity for detecting traces is obvious for further biometric analysis. High resolutional 3D confocal laser scanning microscopy (CLSM) grants the possibility for a detection by segmentation approach with improved detection results. Optimal scan results with CLSM are achieved on surfaces orthogonal to the sensor, which is not always possible due to environmental circumstances or the surface's shape. This introduces additional noise, outliers and a lack of contrast, making a detection of traces even harder. Prior work showed the possibility of determining angle-independent classification models for the detection of latent fingerprints (LFP). Enhancing this approach, we introduce a larger feature space containing a variety of statistical-, roughness-, color-, edge-directivity-, histogram-, Gabor-, gradient- and Tamura features based on raw data and gray-level co-occurrence matrices (GLCM) using high resolutional data. Our test set consists of eight different surfaces for the detection of LFP in four different acquisition angles with a total of 1920 single scans. For each surface and angles in steps of 10, we capture samples from five donors to introduce variance by a variety of sweat compositions and application influences such as pressure or differences in ridge thickness. By analyzing the present test set with our approach, we intend to determine angle- and substrate-dependent classification models to determine optimal surface specific acquisition setups and also classification models for a general detection purpose for both, angles and substrates. The results on overall models with classification rates up to 75.15% (kappa 0.50) already show a positive tendency regarding the usability of the proposed methods for LFP detection on varying surfaces in non

  1. Modeling of Fibrin Gels Based on Confocal Microscopy and Light-Scattering Data

    Science.gov (United States)

    Magatti, Davide; Molteni, Matteo; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

    2013-01-01

    Fibrin gels are biological networks that play a fundamental role in blood coagulation and other patho/physiological processes, such as thrombosis and cancer. Electron and confocal microscopies show a collection of fibers that are relatively monodisperse in diameter, not uniformly distributed, and connected at nodal points with a branching order of ∼3–4. Although in the confocal images the hydrated fibers appear to be quite straight (mass fractal dimension Dm = 1), for the overall system 1gels made of cylindrical sticks of diameter d, density ρ, and average length 〈L〉, joined at randomly distributed nodal points. The resulting 3D network strikingly resembles real fibrin gels and can be sketched as an assembly of densely packed fractal blobs, i.e., regions of size ξ, where the fiber concentration is higher than average. The blobs are placed at a distance ξ0 between their centers of mass so that they are overlapped by a factor η = ξ/ξ0 and have Dm ∼1.2–1.6. The in silico gels’ structure is quantitatively analyzed by its 3D spatial correlation function g3D(r) and corresponding power spectrum I(q) = FFT3D[g3D(r)], from which ρ, d, Dm, η, and ξ0 can be extracted. In particular, ξ0 provides an excellent estimate of the gel mesh size. The in silico gels’ I(q) compares quite well with real gels’ elastic light-scattering measurements. We then derived an analytical form factor for accurately fitting the scattering data, which allowed us to directly recover the gels’ structural parameters. PMID:23473498

  2. Depth-profiling by confocal Raman microscopy (CRM): data correction by numerical techniques.

    Science.gov (United States)

    Tomba, J Pablo; Eliçabe, Guillermo E; Miguel, María de la Paz; Perez, Claudio J

    2011-03-01

    The data obtained in confocal Raman microscopy (CRM) depth profiling experiments with dry optics are subjected to significant distortions, including an artificial compression of the depth scale, due to the combined influence of diffraction, refraction, and instrumental effects that operate on the measurement. This work explores the use of (1) regularized deconvolution and (2) the application of simple rescaling of the depth scale as methodologies to obtain an improved, more precise, confocal response. The deconvolution scheme is based on a simple predictive model for depth resolution and the use of regularization techniques to minimize the dramatic oscillations in the recovered response typical of problem inversion. That scheme is first evaluated using computer simulations on situations that reproduce smooth and sharp sample transitions between two materials and finally it is applied to correct genuine experimental data, obtained in this case from a sharp transition (planar interface) between two polymeric materials. It is shown that the methodology recovers very well most of the lost profile features in all the analyzed situations. The use of simple rescaling appears to be only useful for correcting smooth transitions, particularly those extended over distances larger than those spanned by the operative depth resolution, which limits the strategy to the study of profiles near the sample surface. However, through computer simulations, it is shown that the use of water immersion objectives may help to reduce optical distortions and to expand the application window of this simple methodology, which could be useful, for instance, to safely monitor Fickean sorption/desorption of penetrants in polymer films/coatings in a nearly noninvasive way.

  3. Improving axial resolution in confocal microscopy with new high refractive index mounting media.

    Directory of Open Access Journals (Sweden)

    Coralie Fouquet

    Full Text Available Resolution, high signal intensity and elevated signal to noise ratio (SNR are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF, a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.

  4. Improving axial resolution in confocal microscopy with new high refractive index mounting media.

    Science.gov (United States)

    Fouquet, Coralie; Gilles, Jean-François; Heck, Nicolas; Dos Santos, Marc; Schwartzmann, Richard; Cannaya, Vidjeacoumary; Morel, Marie-Pierre; Davidson, Robert Stephen; Trembleau, Alain; Bolte, Susanne

    2015-01-01

    Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.

  5. Raman confocal microscopy and AFM combined studies of cancerous cells treated with Paclitaxel

    Science.gov (United States)

    Derely, L.; Collart Dutilleul, P.-Y.; Michotte de Welle, Sylvain; Szabo, V.; Gergely, C.; Cuisinier, F. J. G.

    2011-03-01

    Paclitaxel interferes with the normal function of microtubule breakdown, induces apoptosis in cancer cells and sequesters free tubulin. As this drug acts also on other cell mechanisms it is important to monitor its accumulation in the cell compartments. The intracellular spreading of the drug was followed using a WITEC 300R confocal Raman microscope equipped with a CCD camera. Hence Atomic force microscopy (an MFP3D- Asylum Research AFM) in imaging and force mode was used to determine the morphological and mechanical modifications induced on living cells. These studies were performed on living epithelial MCF-7 breast cancer cells. Paclitaxel was added to cell culture media for 3, 6 and 9 hours. Among the specific paclitaxel Raman bands we selected the one at 1670 cm-1 because it is not superposed by the spectrum of the cells. Confocal Raman images are formed by monitoring this band, the NH2 and the PO4 band. Paclitaxel slightly accumulates in the nucleus forming patches. The drug is also concentrated in the vicinity of the cell membrane and in an area close to the nucleus where proteins accumulate. Our AFM images reveal that the treated cancerous MCF-7 cells keep the same size as the non treated ones, but their shape becomes more oval. Cell's elasticity is also modified: a difference of 2 kPa in the Young Modulus characterizes the treated MCF-7 mammary cancerous cell. Our observations demonstrate that paclitaxel acts not only on microtubules but accumulates also in other cell compartments (nucleus) where microtubules are absent.

  6. Spatiotemporal closure of fractional laser-ablated channels imaged by optical coherence tomography and reflectance confocal microscopy

    NARCIS (Netherlands)

    Banzhaf, Christina A.; Wind, Bas S.; Mogensen, Mette; Meesters, Arne A.; Paasch, Uwe; Wolkerstorfer, Albert; Haedersdal, Merete

    2016-01-01

    Optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) offer high-resolution optical imaging of the skin, which may provide benefit in the context of laser-assisted drug delivery. We aimed to characterize postoperative healing of ablative fractional laser (AFXL)-induced

  7. Reflectance confocal microscopy for monitoring the density of Demodex mites in patients with rosacea before and after treatment.

    Science.gov (United States)

    Sattler, E C; Hoffmann, V S; Ruzicka, T; Braunmühl, T V; Berking, C

    2015-07-01

    Demodex mites seem to serve as a pathogenic trigger in many Demodex-associated diseases such as rosacea. In facial skin of patients with rosacea significantly higher numbers of Demodex mites have been shown compared with healthy controls. Reflectance confocal microscopy (RCM) allows the detection and quantification of Demodex mites in vivo noninvasively. It is hypothesized that a reduction of Demodex mites under rosacea therapy can be monitored by RCM. To use RCM to monitor the density of Demodex mites in patients with rosacea before and after treatment. In 25 patients with facial rosacea RCM was performed before and after therapy. Mosaics of 5 × 5 mm(2) and 8 × 8 mm(2) were scanned, and the total numbers of mites per follicle and per area were counted, along with the number of follicles per area. In all patients Demodex folliculorum could be detected and quantified using RCM. RCM showed significant differences pre- and post-treatment (P = 0.0053 for 5 × 5 mm(2) and P rosacea under therapy, correlating to clinical improvement, can be quantified and monitored noninvasively. Possible reasons for this therapeutic effect are discussed. © 2015 British Association of Dermatologists.

  8. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    Energy Technology Data Exchange (ETDEWEB)

    Estrela-Lopis, I; Donath, E [Institute of Medical Physics and Biophysics, Leipzig University, Haertelstrasse 16, 04107 Leipzig (Germany); Romero, G; Rojas, E; Moya, S E, E-mail: Irina.Estrela-Lopis@medizin.uni-leipzig.de [CIC biomaGUNE, Paseo Miramon 182 Edificio Empresarial C, 20009 San Sebastian, Gipuzkoa (Spain)

    2011-07-06

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al{sub 2}O{sub 3} NPs were found in the cytoplasm. CeO{sub 2} NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  9. Quantitative Confocal Microscopy Analysis as a Basis for Search and Study of Potassium Kv1.x Channel Blockers

    Science.gov (United States)

    Feofanov, Alexey V.; Kudryashova, Kseniya S.; Nekrasova, Oksana V.; Vassilevski, Alexander A.; Kuzmenkov, Alexey I.; Korolkova, Yuliya V.; Grishin, Eugene V.; Kirpichnikov, Mikhail P.

    Artificial KcsA-Kv1.x (x = 1, 3) receptors were recently designed by transferring the ligand-binding site from human Kv1.x voltage-gated potassium channels into corresponding domain of the bacterial KscA channel. We found that KcsA-Kv1.x receptors expressed in E. coli cells are embedded into cell membrane and bind ligands when the cells are transformed to spheroplasts. We supposed that E. coli spheroplasts with membrane-embedded KcsA-Kv1.x and fluorescently labeled ligand agitoxin-2 (R-AgTx2) can be used as elements of an advanced analytical system for search and study of Kv1-channel blockers. To realize this idea, special procedures were developed for measurement and quantitative treatment of fluorescence signals obtained from spheroplast membrane using confocal laser scanning microscopy (CLSM). The worked out analytical "mix and read" systems supported by quantitative CLSM analysis were demonstrated to be reliable alternative to radioligand and electrophysiology techniques in the search and study of selective Kv1.x channel blockers of high scientific and medical importance.

  10. Characterization of pars intermedia connections in amphibians by biocytin tract tracing and immunofluorescence aided by confocal microscopy.

    Science.gov (United States)

    Jansen, K; Fabro, C; Artero, C; Feuilloley, M; Vaudry, H; Fasolo, A; Franzoni, M F

    1997-02-01

    Biocytin, recently introduced in neuroanatomical studies, was used as a retrograde tract tracer in combination with immunofluorescence in order to analyse the neurochemical characters of some central neuronal projections to the pars intermedia in two amphibian species, the anuran Rana esculenta and the urodele Triturus carnifex. After biocytin insertions in the pars intermedia, neurons became retrogradely labelled in the suprachiasmatic hypothalamus and the locus coeruleus of the brainstem in both species. Some scattered biocytin-labelled neurons were observed in the preoptic area. Moreover, working on the same sections, immunofluorescence revealed a number of codistributions and, in some cases, colocalization in the same neurons of biocytin labellings and immunopositivity for (1) tyrosine hydroxylase in the suprachiasmatic hypothalamus and the locus coeruleus of Rana and Triturus, (2) gamma-aminobutyric acid in the suprachiasmatic hypothalamus of Rana and Triturus and (3) neuropeptide Y in the suprachiasmatic hypothalamus of Rana. The specificity of such colocalizations was fully confirmed using dual-channel confocal laser scanning microscopy analysis.

  11. In Vivo Reflectance Confocal Microscopy for the Diagnosis of Melanoma and Melanotic Macules of the Lip.

    Science.gov (United States)

    Uribe, Pablo; Collgros, Helena; Scolyer, Richard A; Menzies, Scott W; Guitera, Pascale

    2017-09-01

    Benign melanotic macules (MAC) are the most frequent cause of lip pigmentation and sometimes difficult to differentiate from lip melanoma (MEL). To report in vivo reflectance confocal microscopy (RCM) features of normal lips of different phototypes and to identify features that assist in distinguishing MEL from MAC using dermoscopy and RCM. For this retrospective observational study, 2 groups of patients from 2 tertiary referral centers for melanoma (Sydney Melanoma Diagnostic Centre and Melanoma Institute Australia) were recruited between June 2007 and January 2015. Group 1 included patients with normal lips and different phototypes, and Group 2 consisted of patients with MAC and MEL; RCM and dermoscopy were used for lips analysis. Overall, 92 RCM features were correlated with clinical history, dermoscopic images, and histopathology in all patients with MEL and 5 patients with MAC. Images from the vermillion and/or mucosal part of the lip were recorded from 10 patients with clinically normal lips (mean [SD] age, 34.5 [6.1] years), 16 patients with MAC (mean [SD] age, 49.6 [17.9] years), and 5 patients with 6 cases of MEL (1 patient had a recurrent lesion; mean [SD] age, 56.2 [15.5] years). In normal lips, the draped pattern-a previously described MAC RCM feature-was identified in all cases. In MEL, the following findings were frequent and significantly different from MAC: epidermal disarray; pagetoid infiltration of dendritic and/or round cells; a nonspecific architectural pattern at the dermoepidermal junction (DEJ); nonhomogenously distributed papillae; continuous (lentiginous) proliferation of cells with marked atypia at the DEJ, especially in interpapillary spaces; a higher number of dendritic cells (especially roundish); and atypical round cells at the DEJ. The cellular body area of dendritic cells was about the double in MEL compared with MAC. An RCM lip algorithm was developed that provided 100% sensitivity and 88% specificity for the diagnosis of MEL of

  12. Simultaneous pH measurement in endocytic and cytosolic compartments in living cells using confocal microscopy.

    Science.gov (United States)

    Lucien, Fabrice; Harper, Kelly; Pelletier, Pierre-Paul; Volkov, Leonid; Dubois, Claire M

    2014-04-28

    Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported(1). Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases(2). Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells(3). This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis(3,4). Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can

  13. Precision automation of cell type classification and sub-cellular fluorescence quantification from laser scanning confocal images

    Directory of Open Access Journals (Sweden)

    Hardy Craig Hall

    2016-02-01

    Full Text Available While novel whole-plant phenotyping technologies have been successfully implemented into functional genomics and breeding programs, the potential of automated phenotyping with cellular resolution is largely unexploited. Laser scanning confocal microscopy has the potential to close this gap by providing spatially highly resolved images containing anatomic as well as chemical information on a subcellular basis. However, in the absence of automated methods, the assessment of the spatial patterns and abundance of fluorescent markers with subcellular resolution is still largely qualitative and time-consuming. Recent advances in image acquisition and analysis, coupled with improvements in microprocessor performance, have brought such automated methods within reach, so that information from thousands of cells per image for hundreds of images may be derived in an experimentally convenient time-frame. Here, we present a MATLAB-based analytical pipeline to 1 segment radial plant organs into individual cells, 2 classify cells into cell type categories based upon random forest classification, 3 divide each cell into sub-regions, and 4 quantify fluorescence intensity to a subcellular degree of precision for a separate fluorescence channel. In this research advance, we demonstrate the precision of this analytical process for the relatively complex tissues of Arabidopsis hypocotyls at various stages of development. High speed and robustness make our approach suitable for phenotyping of large collections of stem-like material and other tissue types.

  14. Applications of orientation mapping by scanning and transmission electron microscopy

    DEFF Research Database (Denmark)

    Juul Jensen, D.

    1997-01-01

    The potentials of orientation mapping techniques (in the following referred to as OIM) for studies of thermomechanical processes are analysed. Both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) based OIM techniques are considered. Among the thermomechanical processes...... information is achieved when the results of OIM and these various techniques are combined. Examples hereof are given to illustrate the potentials of OIM techniques. Finally, limitations of TEM and SEM based OIM for specific applications are discussed....

  15. Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Talamás-Lara, Daniel, E-mail: daniel_talamas@hotmail.com [Department of Infectomics and Molecular Pathogenesis, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico City (Mexico); Talamás-Rohana, Patricia, E-mail: ptr@cinvestav.mx [Department of Infectomics and Molecular Pathogenesis, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico City (Mexico); Fragoso-Soriano, Rogelio Jaime, E-mail: rogelio@fis.cinvestav.mx [Department of Physics, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico City (Mexico); Espinosa-Cantellano, Martha, E-mail: mespinosac@cinvestav.mx [Department of Infectomics and Molecular Pathogenesis, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico City (Mexico); Chávez-Munguía, Bibiana, E-mail: bchavez@cinvestav.mx [Department of Infectomics and Molecular Pathogenesis, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico City (Mexico); González-Robles, Arturo, E-mail: goroa@cinvestav.mx [Department of Infectomics and Molecular Pathogenesis, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico City (Mexico); Martínez-Palomo, Adolfo, E-mail: amartine@cinvestav.mx [Department of Infectomics and Molecular Pathogenesis, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico City (Mexico)

    2015-10-01

    Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica. - Highlights: • Striking differences in adhesion to FN between E. histolytica and E. dispar. • A greater degree of cell stiffness in E. histolytica with respect to E. dispar. • E. histolytica but not E. dispar forms regions of close contact with FN. • The actin cytoskeleton is involved in the pathogenicity of E. histolytica.

  16. System and method for compressive scanning electron microscopy

    Science.gov (United States)

    Reed, Bryan W

    2015-01-13

    A scanning transmission electron microscopy (STEM) system is disclosed. The system may make use of an electron beam scanning system configured to generate a plurality of electron beam scans over substantially an entire sample, with each scan varying in electron-illumination intensity over a course of the scan. A signal acquisition system may be used for obtaining at least one of an image, a diffraction pattern, or a spectrum from the scans, the image, diffraction pattern, or spectrum representing only information from at least one of a select subplurality or linear combination of all pixel locations comprising the image. A dataset may be produced from the information. A subsystem may be used for mathematically analyzing the dataset to predict actual information that would have been produced by each pixel location of the image.

  17. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks.

    Science.gov (United States)

    Hames, Samuel C; Ardigò, Marco; Soyer, H Peter; Bradley, Andrew P; Prow, Tarl W

    2016-01-01

    Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20-30 and 50-70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin.

  18. Modeling enzymatic hydrolysis of lignocellulosic substrates using fluorescent confocal microscopy II: pretreated biomass.

    Science.gov (United States)

    Luterbacher, Jeremy S; Moran-Mirabal, Jose M; Burkholder, Eric W; Walker, Larry P

    2015-01-01

    In this study, we extend imaging and modeling work that was done in Part I of this report for a pure cellulose substrate (filter paper) to more industrially relevant substrates (untreated and pretreated hardwood and switchgrass). Using confocal fluorescence microscopy, we are able to track both the structure of the biomass particle via its autofluorescence, and bound enzyme from a commercial cellulase cocktail supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A. Imaging was performed throughout hydrolysis at temperatures relevant to industrial processing (50°C). Enzyme bound predominantly to areas with low autofluorescence, where structure loss and lignin removal had occurred during pretreatment; this confirms the importance of these processes for successful hydrolysis. The overall shape of both untreated and pretreated hardwood and switchgrass particles showed little change during enzymatic hydrolysis beyond a drop in autofluorescence intensity. The permanence of shape along with a relatively constant bound enzyme signal throughout hydrolysis was similar to observations previously made for filter paper, and was consistent with a modeling geometry of a hollowing out cylinder with widening pores represented as infinite slits. Modeling estimates of available surface areas for pretreated biomass were consistent with previously reported experimental results. © 2014 Wiley Periodicals, Inc.

  19. Progress in reflectance confocal microscopy for imaging oral tissues in vivo

    Science.gov (United States)

    Peterson, Gary; Zanoni, Daniella K.; Migliacci, Jocelyn; Cordova, Miguel; Rajadhyaksha, Milind; Patel, Snehal

    2016-02-01

    We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and "video-mosaicing", extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.

  20. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

    Directory of Open Access Journals (Sweden)

    Antonio M. Fea

    2016-01-01

    Full Text Available Purpose. To determine the effectiveness of autologous platelet lysate (APL eye drops in patients with primary Sjögren syndrome (SS dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM. Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI, best corrected visual acuity (BCVA, Schirmer test, fluorescein score, and breakup time (BUT. A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n=20 patients mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n=10 patients. The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p<0.05. Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

  1. Confocal Raman microscopy supported by optical clearing treatment of the skin—influence on collagen hydration

    Science.gov (United States)

    Sdobnov, Anton Yu; Tuchin, Valery V.; Lademann, Juergen; E Darvin, Maxim

    2017-07-01

    Confocal Raman microscopy (CRM) is employed to study the skin physiology, drug permeation and skin disease monitoring. In order to increase the depth of investigations, the effect of optical clearing was observed on porcine ear skin ex vivo. The optical clearing agents (OCAs) glycerol and iohexol (Omnipaque™) were applied to the porcine ear skin and investigated by CRM after 30 and 60 min of treatment. The extent of optical clearing by utilizing concentrations of 70% glycerol and 100% Omnipaque™ was evaluated. The intensity of the skin-related Raman peaks significantly increased starting from the depth 160 µm for Omnipaque™ and 40 µm for glycerol (p  ⩽  0.05) after 60 min of treatment. The OCAs’ influence on the collagen hydration in the deep-located dermis was investigated. Both OCAs induce skin dehydration, but the effect of glycerol treatment (30 min and 60 min) is stronger. The obtained results demonstrate that with increasing the treatment time, both glycerol and Omnipaque™ solutions improve the optical clearing of porcine skin making the deep-located dermal regions able for investigations. At the used concentrations and time intervals, glycerol is more effective than Omnipaque™. However, Omnipaque™ is more promising than glycerol for future in vivo applications as it is an already approved pharmaceutic substance without any known impact on the skin structure.

  2. Validating Intravascular Imaging with Serial Optical Coherence Tomography and Confocal Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Pier-Luc Tardif

    2016-12-01

    Full Text Available Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF, the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D histology was performed combining optical coherence tomography (OCT and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

  3. Skeletal remodeling dynamics: New approaches with imaging instrumentation. [Laser confocal microscopy:a2

    Energy Technology Data Exchange (ETDEWEB)

    Parks, N.J.; Pinkerton, K.E.; Seibert, J.A.; Pool, R.R.

    1991-01-01

    This report of progress and future objectives timetable is based on an included schematic of goals and objectives and the project abstract which is included as Appendix 1. Five matters are summarized in the order of (1) novel methods of calcified bone confocal microscopy and reconstruction image analysis of decalcified beagle and human cortical bone serial sections, (2) macroscopic cross-correlation of beagle and human cortical and cancellous bone fractions with CT analysis, (3) guidance to the most radiobiologically important skeletal regions of interest with the just completed {sup 90}Sr bone tumor map from life time beagle studies, (4) deposition patterns of radioactive agents that participate in apatite crystal nucleation processes in bone and leave radiation-excited electrons trapped in bone mineral, and (5) the budget period timetable. The discovery that beta particles from {sup 166}Ho (T{sub {1/2}} =26 hr, {beta}{sub max} = 1.8 MeV) phosphonic acid bone agents leave detectable, long-lived, electron paramagnetic resonance signals in bone is included in Appendix 2 as a joint report.

  4. Investigation of domain walls in PPLN by confocal raman microscopy and PCA analysis

    Science.gov (United States)

    Shur, Vladimir Ya.; Zelenovskiy, Pavel; Bourson, Patrice

    2017-07-01

    Confocal Raman microscopy (CRM) is a powerful tool for investigation of ferroelectric domains. Mechanical stresses and electric fields existed in the vicinity of neutral and charged domain walls modify frequency, intensity and width of spectral lines [1], thus allowing to visualize micro- and nanodomain structures both at the surface and in the bulk of the crystal [2,3]. Stresses and fields are naturally coupled in ferroelectrics due to inverse piezoelectric effect and hardly can be separated in Raman spectra. PCA is a powerful statistical method for analysis of large data matrix providing a set of orthogonal variables, called principal components (PCs). PCA is widely used for classification of experimental data, for example, in crystallization experiments, for detection of small amounts of components in solid mixtures etc. [4,5]. In Raman spectroscopy PCA was applied for analysis of phase transitions and provided critical pressure with good accuracy [6]. In the present work we for the first time applied Principal Component Analysis (PCA) method for analysis of Raman spectra measured in periodically poled lithium niobate (PPLN). We found that principal components demonstrate different sensitivity to mechanical stresses and electric fields in the vicinity of the domain walls. This allowed us to separately visualize spatial distribution of fields and electric fields at the surface and in the bulk of PPLN.

  5. A confocal microscopy-based atlas of tissue architecture in the tapeworm Hymenolepis diminuta.

    Science.gov (United States)

    Rozario, Tania; Newmark, Phillip A

    2015-11-01

    Tapeworms are pervasive and globally distributed parasites that infect millions of humans and livestock every year, and are the causative agents of two of the 17 neglected tropical diseases prioritized by the World Health Organization. Studies of tapeworm biology and pathology are often encumbered by the complex life cycles of disease-relevant tapeworm species that infect hosts such as foxes, dogs, cattle, pigs, and humans. Thus, studies of laboratory models can help overcome the practical, ethical, and cost-related difficulties faced by tapeworm parasitologists. The rat intestinal tapeworm Hymenolepis diminuta is easily reared in the laboratory and has the potential to enable modern molecular-based experiments that will greatly contribute to our understanding of multiple aspects of tapeworm biology, such as growth and reproduction. As part of our efforts to develop molecular tools for experiments on H. diminuta, we have characterized a battery of lectins, antibodies, and common stains that label different tapeworm tissues and organ structures. Using confocal microscopy, we have assembled an "atlas" of H. diminuta organ architecture that will be a useful resource for helminthologists. The methodologies we describe will facilitate characterization of loss-of-function perturbations using H. diminuta. This toolkit will enable a greater understanding of fundamental tapeworm biology that may elucidate new therapeutic targets toward the eradication of these parasites. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Fluorescence and confocal microscopy studies of the ice surface - antifreeze protein interactions.

    Science.gov (United States)

    Pertaya, N.; Thomson, E.; Davies, P. L.; Braslavsky, I.

    2005-03-01

    Biomineralization is a phenomenon in which biological material influences mineral growth on the molecular level. A compelling example involves antifreeze proteins (AFPs) known to prevent fish and insects from freezing. AFPs have many potential applications in agriculture, biomedical science, and can be used as a model platform to understand biomineralization processes for future nanotechnology applications. Here we describe a new approach to study the interaction between AFPs and ice using fluorescence and confocal microscopy combined with a unique ice growth cell. After conjugating green fluorescent protein (GFP) to Type III AFP, we imaged the fluorescence signal around and inside of the ice crystals that emerged from the cooled AFP-GFP solution, and have observed an enhanced fluorescence signal at the edge of the ice crystal. In a second cell we observed a dramatic change in the ice growth morphology when AFPs were introduced into an initially pure system. Further developments of these methods will permit the direct imaging of the location and concentration of the AFPs on ice surfaces and enable a better understanding of their operation. Supported by CIHR, the Bosack and Kruger Foundation, Ohio and Yale Universities.

  7. Validating Intravascular Imaging with Serial Optical Coherence Tomography and Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Tardif, Pier-Luc; Bertrand, Marie-Jeanne; Abran, Maxime; Castonguay, Alexandre; Lefebvre, Joël; Stähli, Barbara E; Merlet, Nolwenn; Mihalache-Avram, Teodora; Geoffroy, Pascale; Mecteau, Mélanie; Busseuil, David; Ni, Feng; Abulrob, Abedelnasser; Rhéaume, Éric; L'Allier, Philippe; Tardif, Jean-Claude; Lesage, Frédéric

    2016-12-15

    Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

  8. In Vivo Confocal Microscopy of Corneal Nerves in Health and Disease.

    Science.gov (United States)

    Cruzat, Andrea; Qazi, Yureeda; Hamrah, Pedram

    2017-01-01

    In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. Enabling the dissection of corneal architecture at a cellular level, this technique offers fast and noninvasive in vivo imaging of the cornea with images comparable to those of ex vivo histochemical techniques. Corneal nerves bear substantial relevance to clinicians and scientists alike, given their pivotal roles in regulation of corneal sensation, maintenance of epithelial integrity, as well as proliferation and promotion of wound healing. Thus, IVCM offers a unique method to study corneal nerve alterations in a myriad of conditions, such as ocular and systemic diseases and following corneal surgery, without altering the tissue microenvironment. Of particular interest has been the correlation of corneal subbasal nerves to their function, which has been studied in normal eyes, contact lens wearers, and patients with keratoconus, infectious keratitis, corneal dystrophies, and neurotrophic keratopathy. Longitudinal studies have applied IVCM to investigate the effects of corneal surgery on nerves, demonstrating their regenerative capacity. IVCM is increasingly important in the diagnosis and management of systemic conditions such as peripheral diabetic neuropathy and, more recently, in ocular diseases. In this review, we outline the principles and applications of IVCM in the study of corneal nerves in various ocular and systemic diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Effectiveness of in vivo confocal microscopy in detecting filamentous fungi during clinical course of fungal keratitis.

    Science.gov (United States)

    Takezawa, Yuki; Shiraishi, Atsushi; Noda, Eriko; Hara, Yuko; Yamaguchi, Masahiko; Uno, Toshihiko; Ohashi, Yuichi

    2010-12-01

    To determine the effectiveness of laser confocal microscopy in detecting filamentous fungi in the cornea of patients with fungal keratitis (FK) and in evaluating the effectiveness of the treatment. The corneas of 6 patients clinically diagnosed with FK were examined with the Heidelberg Retina Tomograph II-Rostock Cornea Module (HRT II-RCM). Three of these patients were also monitored periodically with the HRT II-RCM after antifungal treatment. The HRT II-RCM examination showed interlocking and branching, white, septated, hyphae-like lines in the cornea of all patients. All 6 patients had positive corneal smears and/or laboratory cultures. Three patients were monitored with HRT II-RCM after antifungal treatment. One patient, whose initial smear was negative, was diagnosed by HRT II-RCM before the positive culture results. In another case, the epithelial regeneration was impaired even 3 weeks after the initial treatment and HRT II-RCM revealed a mass of hyphae in the corneal ulcerated lesion. These findings indicated the necessity of surgical debridement. After the surgical debridement, the corneal epithelial defect was healed. HRT II-RCM was able to detect the morphological changes of hyphae after antifungal treatment and helped in the treatment modifications during the clinical course in all 3 patients. These results indicate that HRT II-RCM can be used to diagnose FK and to monitor the effect of therapy on FK.

  10. Evaluation of keratic precipitates and corneal endothelium in Fuchs' heterochromic cyclitis by in vivo confocal microscopy.

    Science.gov (United States)

    Labbé, A; Dupas, B; Offret, H; Baudouin, C; Labetoulle, M

    2009-05-01

    To analyse keratic precipitates in Fuchs' heterochromic cyclitis (FHC) by in-vivo confocal microscopy (IVCM). A retrospective chart review of 13 consecutive patients with FHC was conducted. Data collection included medical and ophthalmological history, age, age at diagnosis, gender, detailed slit-lamp examination and IVCM images. The IVCM characteristics of keratic precipitates and of the endothelium were analysed. Large hyperreflective deposits corresponding to keratic precipitates were observed on the endothelium of all FHC eyes and showed a great consistency among the different patients. These infiltrating keratic precipitates had a dendritic shape, with a small central core with numerous thin pseudopodia sometimes making connection between different keratic precipitates. The mean density of these keratic precipitates was 16.01/mm(2) (SD 6.54). The mean size of the largest dimension of these keratic precipitates was 127.31 microm (SD 41.49; range 66.16-201.4 microm). Hyporeflective round defects were observed at the level of the endothelium at contact or in the close vicinity of keratic precipitates or smaller hyperreflective deposits. All contralateral (non-affected eyes) had no keratic precipitates nor endothelial abnormalities. By providing high resolution images of corneal endothelium and keratic precipitates, IVCM could help the diagnosis and understanding of complex forms of intraocular inflammation such as FHC.

  11. Reflectance confocal microscopy vs. standardized skin surface biopsy for measuring the density of Demodex mites.

    Science.gov (United States)

    Turgut Erdemir, A; Gurel, M S; Koku Aksu, A E; Bilgin Karahalli, F; Incel, P; Kutlu Haytoğlu, N S; Falay, T

    2014-11-01

    Reflectance confocal microscopy (RCM) has been recently shown to be effective for measuring the Demodex mite density. To compare and demonstrate the advantages and disadvantages of standardized skin surface biopsy (SSSB) and RCM for measuring the density of Demodex mites. Forty-eight patients (30 female, 18 male) and 47 healthy controls (30 female, 17 male) were enrolled in the study. The patients diagnoses were pityriasis folliculorum (n = 40), papulopustulary rosecea (n = 7) and erythema-telengiectatic rosacea (n = 1). The area with the most intense erythema on the right cheek was selected for imaging with RCM (VivaScope 3000) and SSSB. Forty-two patients demonstrated high Demodex density [(Dd) > 5 mites/cm(2) ] with SSSB (85.7%). RCM identified demodicosis in 48 patients (100%). The mean Dd measured with RCM (409.8 ± 209.2) was significantly higher than SSSB (15.33 ± 18.1) (P Demodex-associated diseases and it is superior to SSSB for Demodex mite detection. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Implementation of fluorescence confocal mosaicking microscopy by ``early adopter'' Mohs surgeons and dermatologists: recent progress

    Science.gov (United States)

    Jain, Manu; Rajadhyaksha, Milind; Nehal, Kishwer

    2017-02-01

    Confocal mosaicking microscopy (CMM) enables rapid imaging of large areas of fresh tissue ex vivo without the processing that is necessary for conventional histology. When performed in fluorescence mode using acridine orange (nuclear specific dye), it enhances nuclei-to-dermis contrast that enables detection of all types of basal cell carcinomas (BCCs), including micronodular and thin strands of infiltrative types. So far, this technique has been mostly validated in research settings for the detection of residual BCC tumor margins with high sensitivity of 89% to 96% and specificity of 99% to 89%. Recently, CMM has advanced to implementation and testing in clinical settings by "early adopter" Mohs surgeons, as an adjunct to frozen section during Mohs surgery. We summarize the development of CMM guided imaging of ex vivo skin tissues from bench to bedside. We also present its current state of application in routine clinical workflow not only for the assessment of residual BCC margins in the Mohs surgical setting but also for some melanocytic lesions and other skin conditions in clinical dermatology settings. Last, we also discuss the potential limitations of this technology as well as future developments. As this technology advances further, it may serve as an adjunct to standard histology and enable rapid surgical pathology of skin cancers at the bedside.

  13. Confocal microscopy for the histological fluorescence pattern of a recurrent atypical meningioma: case report.

    Science.gov (United States)

    Whitson, Wesley J; Valdes, Pablo A; Harris, Brent T; Paulsen, Keith D; Roberts, David W

    2011-06-01

    Fluorescence-guided resection with 5-aminolevulinic acid (5-ALA), which has shown promising results in the resection of malignant gliomas, has been used for meningioma resection in an attempt to more clearly delineate the tumor margin. However, no article has investigated the fluorescence pattern of meningiomas on a histological level. Understanding the microscopic pattern of fluorescence could help assess the precision and utility of using 5-ALA for these tumors. We present the case of a recurrent atypical meningioma operated on with 5-ALA fluorescence-guided resection for delineation of tumor tissue from surrounding uninvolved dura. A 53-year-old woman presented with recurrent atypical meningioma of the falx. Prior treatment included surgical resection 6 years earlier with subsequent fractionated radiation therapy and radiosurgery for tumor progression. The patient was given 5-ALA 20 mg/kg body weight dissolved in 100 mL water 3 hours before induction of anesthesia. Intraoperative fluorescence was coregistered with preoperative imaging. Neuropathological analysis of the resected falx with confocal microscopy enabled correlation of fluorescence with the extent of tumor on a histological level. Fluorescence guidance allowed clear intraoperative delineation of tumor tissue from adjacent, uninvolved dura. On a microscopic level, there was a very close correlation of fluorescence with tumor, but some tumor cells did not fluoresce. Copyright © 2011 by the Congress of Neurological Surgeons

  14. Intraoperative imaging during Mohs surgery with reflectance confocal microscopy: initial clinical experience

    Science.gov (United States)

    Flores, Eileen S.; Cordova, Miguel; Kose, Kivanc; Phillips, William; Rossi, Anthony; Nehal, Kishwer; Rajadhyaksha, Milind

    2015-06-01

    Mohs surgery for the removal of nonmelanoma skin cancers (NMSCs) is performed in stages, while being guided by the examination for residual tumor with frozen pathology. However, preparation of frozen pathology at each stage is time consuming and labor intensive. Real-time intraoperative reflectance confocal microscopy (RCM), combined with video mosaicking, may enable rapid detection of residual tumor directly in the surgical wounds on patients. We report our initial experience on 25 patients, using aluminum chloride for nuclear contrast. Imaging was performed in quadrants in the wound to simulate the Mohs surgeon's examination of pathology. Images and videos of the epidermal and dermal margins were found to be of clinically acceptable quality. Bright nuclear morphology was identified at the epidermal margin and detectable in residual NMSC tumors. The presence of residual tumor and normal skin features could be detected in the peripheral and deep dermal margins. Intraoperative RCM imaging may enable detection of residual tumor directly on patients during Mohs surgery, and may serve as an adjunct for frozen pathology. Ultimately, for routine clinical utility, a stronger tumor-to-dermis contrast may be necessary, and also a smaller microscope with an automated approach for imaging in the entire wound in a rapid and controlled manner.

  15. Feasibility of intraoperative imaging during Mohs surgery with reflectance confocal microscopy

    Science.gov (United States)

    Flores, Eileen S.; Cordova, Miguel; Kose, Kivanc; Phillips, William; Nehal, Kishwer; Rajadhyaksha, Milind

    2014-03-01

    Mohs surgery for the removal of non-melanoma skin cancers (NMSCs) is performed in stages, while being guided by the examination for residual tumor with frozen pathology. However, preparation of frozen pathology at each stage is timeconsuming and labor-intensive. Real-time intraoperative reflectance confocal microscopy (RCM) may enable rapid detection of residual tumor directly in surgical wounds on patients. We report initial feasibility on twenty-one patients, using 35% AlCl3 for nuclear contrast. Imaging was performed in quadrants in the wound, to simulate the Mohs surgeon's examination of pathology. Images and videos of the epidermal and dermal margins were found to be of clinically acceptable quality. Bright nuclear morphology was identified at the epidermal margin. The presence of residual BCC/SCC tumor and normal skin features could be detected in the peripheral and deep dermal margins. Nuclear morphology was detectable in residual BCC/SCC tumors. Intraoperative RCM imaging may enable detection of residual tumor, directly on Mohs patients, and may serve as an adjunct for frozen pathology. However, a stronger source of contrast will be necessary, and also a smaller device with an automated approach for imaging in the entire wound in a rapid and controlled manner for clinical utility.

  16. Identifying dynamic membrane structures with atomic-force microscopy and confocal imaging.

    Science.gov (United States)

    Timmel, Tobias; Schuelke, Markus; Spuler, Simone

    2014-04-01

    Combining the biological specificity of fluorescence microscopy with topographical features revealed by atomic force microscopy (AFM) provides new insights into cell biology. However, the lack of systematic alignment capabilities especially in scanning-tip AFM has limited the combined application approach as AFM drift leads to increasing image mismatch over time. We present an alignment correction method using the cantilever tip as a reference landmark. Since the precise tip position is known in both the fluorescence and AFM images, exact re-alignment becomes possible. We used beads to demonstrate the validity of the method in a complex artificial sample. We then extended this method to biological samples to depict membrane structures in fixed and living human fibroblasts. We were able to map nanoscale membrane structures, such as clathrin-coated pits, to their respective fluorescent spots. Reliable alignment between fluorescence signals and topographic structures opens possibilities to assess key biological processes at the cell surface such as endocytosis and exocytosis.

  17. Structural examination of lithium niobate ferroelectric crystals by combining scanning electron microscopy and atomic force microscopy

    Science.gov (United States)

    Efremova, P. V.; Ped'ko, B. B.; Kuznecova, Yu. V.

    2016-02-01

    The structure of lithium niobate single crystals is studied by a complex technique that combines scanning electron microscopy and atomic force microscopy. By implementing the piezoresponse force method on an atomic force microscope, the domain structure of lithium niobate crystals, which was not revealed without electron beam irradiation, is visualized

  18. Polarization contrast in photon scanning tunnelling microscopy combined with atomic force microscopy

    NARCIS (Netherlands)

    Propstra, K.; Propstra, K.; van Hulst, N.F.

    1995-01-01

    Photon scanning tunnelling microscopy combined with atomic force microscopy allows simultaneous acquisition and direct comparison of optical and topographical images, both with a lateral resolution of about 30 nm, far beyond the optical diffraction limit. The probe consists of a modified

  19. MRT letter: A unified accelerated maximum likelihood technique for widefield, confocal, and super-resolution 4Pi microscopy.

    Science.gov (United States)

    Chelur K, Rasmi; Kanhirodan, Rajan; Mondal, Partha Pratim

    2015-05-01

    We propose an algorithmic technique for accelerating maximum likelihood (ML) algorithm for image reconstruction in fluorescence microscopy. This is made possible by integrating Biggs-Andrews (BA) method with ML approach. The results on widefield, confocal, and super-resolution 4Pi microscopy reveal substantial improvement in the speed of 3D image reconstruction (the number of iterations has reduced by approximately one-half). Moreover, the quality of reconstruction obtained using accelerated ML closely resembles with nonaccelerated ML method. The proposed technique is a step closer to realize real-time reconstruction in 3D fluorescence microscopy. © 2015 Wiley Periodicals, Inc.

  20. Confocal microscopy evaluation of the effect of irrigants on Enterococcus faecalis biofilm: An in vitro study.

    Science.gov (United States)

    Flach, Nicole; Böttcher, Daiana Elisabeth; Parolo, Clarissa Cavalcanti Fatturi; Firmino, Luciana Bitello; Malt, Marisa; Lammers, Marcelo Lazzaron; Grecca, Fabiana Soares

    2016-01-01

    The purpose of this study was to evaluate in vitro the effectiveness of two endodontic irrigants and their association against Enterococcus faecalis (E. faecalis) by confocal laser scanning microscope (CLSM). Twenty-four bovine incisors were inoculated in a monoculture of E. faecalis for 21 days. After this period, the teeth were divided into three test groups (n = 5) according to the chemical used. Group 1: 2.5% sodium hypochlorite (NaOCl), group 2: 2% chlorhexidine gel (CHX), group 3: 2.5% NaOCl + 2% CHX gel, and two control groups (n = 3): negative control group (NCG)-sterile and without root canals preparation and positive control group (PCG)-saline. Then, the samples were stained with SYTO9 and propidium iodide and subjected to analysis by CLSM. Bacterial viability was quantitatively analyzed by the proportions of dead and live bacteria in the biofilm remnants. Statistical analysis was performed by the One-way ANOVA test (p = 0.05). No statistical differences were observed to bacterial viability. According to CLSM analysis, none of the tested substances could completely eliminate E. faecalis from the root canal space. Until now, there are no irrigant solutions able to completely eliminate E. faecalis from the root canal. In this regard, the search for irrigants able to intensify the antimicrobial action is of paramount importance. © Wiley Periodicals, Inc.

  1. In situ Observation of Calcium Oxide Treatment of Inclusions in Molten Steel by Confocal Microscopy

    Science.gov (United States)

    Khurana, Bharat; Spooner, Stephen; Rao, M. B. V.; Roy, Gour Gopal; Srirangam, Prakash

    2017-06-01

    Calcium treatment of aluminum killed steel was observed in situ using high-temperature confocal scanning laser microscope (HT-CSLM). This technique along with a novel experimental design enables continuous observation of clustering behavior of inclusions before and after the calcium treatment. Results show that the increase in average inclusion size in non-calcium-treated condition was much faster compared to calcium-treated condition. Results also show that the magnitude of attractive capillary force between inclusion particles in non-treated condition was about 10-15 N for larger particles (10 µm) and 10-16 N for smaller particles (5 µm) and acting length of force was about 30 µm. In the case of calcium-treated condition, the magnitude and acting length of force was reduced to 10-16 N and 10 µm, respectively, for particles of all sizes. This change in attractive capillary attractive force is due to change in inclusion morphology from solid alumina disks to liquid lens particles during calcium treatment.

  2. An endolithic microbial community in dolomite rock in central Switzerland: characterization by reflection spectroscopy, pigment analyses, scanning electron microscopy, and laser scanning microscopy.

    Science.gov (United States)

    Horath, T; Neu, T R; Bachofen, R

    2006-04-01

    A community of endolithic microorganisms dominated by phototrophs was found as a distinct band a few millimeters below the surface of bare exposed dolomite rocks in the Piora Valley in the Alps. Using in situ reflectance spectroscopy, we detected chlorophyll a (Chl a), phycobilins, carotenoids, and an unknown type of bacteriochlorophyll-like pigment absorbing in vivo at about 720 nm. In cross sections, the data indicated a defined distribution of different groups of organisms perpendicular to the rock surface. High-performance liquid chromatography analyses of pigments extracted with organic solvents confirmed the presence of two types of bacteriochlorophylls besides chlorophylls and various carotenoids. Spherical organisms of varying sizes and small filaments were observed in situ with scanning electron microscopy and confocal laser scanning microscopy (one- and two-photon technique). The latter allowed visualization of the distribution of phototrophic microorganisms by the autofluorescence of their pigments within the rock. Coccoid cyanobacteria of various sizes predominated over filamentous ones. Application of fluorescence-labeled lectins demonstrated that most cyanobacteria were embedded in an exopolymeric matrix. Nucleic acid stains revealed a wide distribution of small heterotrophs. Some biological structures emitting a green autofluorescence remain to be identified.

  3. Normative values for corneal nerve morphology assessed using corneal confocal microscopy: a multinational normative data set.

    Science.gov (United States)

    Tavakoli, Mitra; Ferdousi, Maryam; Petropoulos, Ioannis N; Morris, Julie; Pritchard, Nicola; Zhivov, Andrey; Ziegler, Dan; Pacaud, Danièle; Romanchuk, Kenneth; Perkins, Bruce A; Lovblom, Leif E; Bril, Vera; Singleton, J Robinson; Smith, Gordon; Boulton, Andrew J M; Efron, Nathan; Malik, Rayaz A

    2015-05-01

    Corneal confocal microscopy is a novel diagnostic technique for the detection of nerve damage and repair in a range of peripheral neuropathies, in particular diabetic neuropathy. Normative reference values are required to enable clinical translation and wider use of this technique. We have therefore undertaken a multicenter collaboration to provide worldwide age-adjusted normative values of corneal nerve fiber parameters. A total of 1,965 corneal nerve images from 343 healthy volunteers were pooled from six clinical academic centers. All subjects underwent examination with the Heidelberg Retina Tomograph corneal confocal microscope. Images of the central corneal subbasal nerve plexus were acquired by each center using a standard protocol and analyzed by three trained examiners using manual tracing and semiautomated software (CCMetrics). Age trends were established using simple linear regression, and normative corneal nerve fiber density (CNFD), corneal nerve fiber branch density (CNBD), corneal nerve fiber length (CNFL), and corneal nerve fiber tortuosity (CNFT) reference values were calculated using quantile regression analysis. There was a significant linear age-dependent decrease in CNFD (-0.164 no./mm(2) per year for men, P < 0.01, and -0.161 no./mm(2) per year for women, P < 0.01). There was no change with age in CNBD (0.192 no./mm(2) per year for men, P = 0.26, and -0.050 no./mm(2) per year for women, P = 0.78). CNFL decreased in men (-0.045 mm/mm(2) per year, P = 0.07) and women (-0.060 mm/mm(2) per year, P = 0.02). CNFT increased with age in men (0.044 per year, P < 0.01) and women (0.046 per year, P < 0.01). Height, weight, and BMI did not influence the 5th percentile normative values for any corneal nerve parameter. This study provides robust worldwide normative reference values for corneal nerve parameters to be used in research and clinical practice in the study of diabetic and other peripheral neuropathies. © 2015 by the American Diabetes Association

  4. Exophiala phaeomuriformis Fungal Keratitis: Case Report and In Vivo Confocal Microscopy Findings.

    Science.gov (United States)

    Aggarwal, Shruti; Yamaguchi, Takefumi; Dana, Reza; Hamrah, Pedram

    2017-03-01

    Corneal infections, particularly fungal keratitis due to rare fungal species, pose a diagnostic and therapeutic challenge because of difficulty in identification and varying susceptibility profiles. In this study, we report the first case of fungal keratitis because of Exophiala phaeomuriformis. We report the clinical findings and microbial identification techniques of a case of fungal keratitis due to E. phaeomuriformis. An 84-year-old woman presented with redness, pain, and itching in the left eye for 2 weeks. Slit-lamp biomicroscopy revealed one broken suture from previous penetrating keratoplasty (PKP), black infiltrates at the 4-o'clock position, without an overlying epithelial defect and hypopyon. Microbial identification was based cultures on Sabouraud dextrose agar and DNA sequencing and correlations to laser in vivo confocal microscopy (IVCM; Heidelberg Retinal Tomograph 3/Rostock Cornea Module, Heidelberg Engineering) and multiphoton microscopy (Ultima Microscope; Prairie Technologies) images. Slit-lamp biomicroscopy revealed one broken suture from previous PKP, black infiltrates at the 4-o'clock position, without an overlying epithelial defect and hypopyon. Based on a clinical suspicion of fungal keratitis, antifungals and fortified antibiotics were started. However, the patient did not respond to therapy and required urgent PKP. After surgery, the patient was maintained on topical and systemic voriconazole and also topical 2% cyclosporine for 5 months because of possibility of scleral involvement noticed during surgery. At the end of the treatment period, her vision improved from hand motion to 20/40, with no recurrence observed in a follow-up period of 1 year. Results of diagnostic tests were supported by fungal elements in stroma on IVCM. Culture from the infiltrate grew black yeast. DNA sequencing led to the diagnosis of E. phaeomuriformis keratitis. Antifungal susceptibility testing revealed sensitivity to voriconazole. This is, to our knowledge

  5. Influences of edges and steep slopes in 3D interference and confocal microscopy

    Science.gov (United States)

    Xie, Weichang; Hagemeier, Sebastian; Woidt, Carsten; Hillmer, Harmut; Lehmann, Peter

    2016-04-01

    Optical measurement techniques are widely applied in high-resolution contour, topography and roughness measurement. In this context vertical scanning white-light interferometers and confocal microscopes have become mature instruments over the last decades. The accuracy of measurement results is highly related not only to the type and physical properties of the measuring instruments, but also to the measurement object itself. This contribution focuses on measurement effects occurring at edges and height steps using white-light interferometers of different numerical apertures. If the edge is perfectly perpendicular, batwing effects appear at height steps. These batwings show maximum height if the height-to-wavelength-ratio (HWR) is about one forth or three forth, and they disappear if the HWR value is about an integer multiple of one half. The wavelength that is relevant in this context is the effective wavelength, i.e. the center wavelength of the illuminating light multiplied by a correction factor known as the numerical aperture correction. However, in practice the edges are usually not perfectly perpendicular. In this case, the measurement results depend also on the derivative of the surface height function and they may differ from theory and the prediction according to the HWR value. Measurements of such steps show systematical effects depending on the lateral resolution of the instrument. In this context, a Linnik interferometer with a magnification of 100x and NA = 0.9 is used to characterize the three dimensional topography of more or less rectangular calibration specimens and quasi-perpendicular structures produced by the nanoimprint technology. The Linnik interferometer is equipped with LED light sources emitting at different wavelengths, so that the HWR value can be changed. This is possible since the high NA objective lenses show a rather limited depth of focus such that the temporal coherence gating may be replaced by focal gating in this particular

  6. Scanning electron microscopy-energy dispersive X-ray spectrometer ...

    African Journals Online (AJOL)

    The distribution of arsenic (As) and cadmium (Cd) in himematsutake was analyzed using scanning electron microscopy-energy dispersive X-ray spectrometer (SEM-EDX). The atomic percentage of the metals was confirmed by inductively coupled plasma-mass spectrometer (ICP-MS). Results show that the accumulation of ...

  7. Challenges of scanning hall microscopy using batch fabricated probes

    NARCIS (Netherlands)

    Hatakeyama, Kodai

    2016-01-01

    Scanning Hall probe microscopy is a widely used technique for quantitative high resolution imaging of magnetic stray fields. Up to now probes with nanometer spatial resolution have only been realized by electron beam lithography, which is a slow and expensive fabrication technique. In this thesis,

  8. Nanochannel alignment analysis by scanning transmission ion microscopy

    DEFF Research Database (Denmark)

    Rajta, I.; Gál, G.A.B.; Szilasi, S.Z.

    2010-01-01

    In this paper a study on the ion transmission ratio of a nanoporous alumina sample is presented. The sample was investigated by scanning transmission ion microscopy (STIM) with different beam sizes. The hexagonally close-packed AlO nanocapillary array, realized as a suspended membrane of 15 νm...

  9. Scanning electron microscopy of Dermatobia hominis reveals cutaneous anchoring features.

    Science.gov (United States)

    Möhrenschlager, Matthias; Mempel, Martin; Weichenmeier, Ingrid; Engst, Reinhard; Ring, Johannnes; Behrendt, Heidrun

    2007-10-01

    We report the case of a 45-year-old Caucasian woman suffering from cutaneous myiasis. With the use of scanning electron microscopy, we placed special focus on the mechanisms by which Dermatobia hominis can fasten securely within the human skin.

  10. Ultrafast terahertz scanning tunneling microscopy with atomic resolution

    DEFF Research Database (Denmark)

    Jelic, Vedran; Iwaszczuk, Krzysztof; Nguyen, Peter H.

    2016-01-01

    We demonstrate that ultrafast terahertz scanning tunneling microscopy (THz-STM) can probe single atoms on a silicon surface with simultaneous sub-nanometer and sub-picosecond spatio-temporal resolution. THz-STM is established as a new technique for exploring high-field non-equilibrium tunneling...

  11. Characterization of Polycaprolactone Films Biodeterioration by Scanning Electron Microscopy

    Czech Academy of Sciences Publication Activity Database

    Hrubanová, Kamila; Voberková, S.; Hermanová, S.; Krzyžánek, Vladislav

    2014-01-01

    Roč. 20, S3 (2014), s. 1950-1951 ISSN 1431-9276 R&D Projects: GA MŠk EE.2.3.20.0103; GA MŠk(CZ) LO1212 Institutional support: RVO:68081731 Keywords : polycaprolactone films * biodeterioration * scanning electron microscopy Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 1.877, year: 2014

  12. Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

    Science.gov (United States)

    Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

    2017-07-01

    Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

  13. High-speed adaptive optics line scan confocal retinal imaging for human eye.

    Science.gov (United States)

    Lu, Jing; Gu, Boyu; Wang, Xiaolin; Zhang, Yuhua

    2017-01-01

    Continuous and rapid eye movement causes significant intraframe distortion in adaptive optics high resolution retinal imaging. To minimize this artifact, we developed a high speed adaptive optics line scan confocal retinal imaging system. A high speed line camera was employed to acquire retinal image and custom adaptive optics was developed to compensate the wave aberration of the human eye's optics. The spatial resolution and signal to noise ratio were assessed in model eye and in living human eye. The improvement of imaging fidelity was estimated by reduction of intra-frame distortion of retinal images acquired in the living human eyes with frame rates at 30 frames/second (FPS), 100 FPS, and 200 FPS. The device produced retinal image with cellular level resolution at 200 FPS with a digitization of 512×512 pixels/frame in the living human eye. Cone photoreceptors in the central fovea and rod photoreceptors near the fovea were resolved in three human subjects in normal chorioretinal health. Compared with retinal images acquired at 30 FPS, the intra-frame distortion in images taken at 200 FPS was reduced by 50.9% to 79.7%. We demonstrated the feasibility of acquiring high resolution retinal images in the living human eye at a speed that minimizes retinal motion artifact. This device may facilitate research involving subjects with nystagmus or unsteady fixation due to central vision loss.

  14. Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope

    Science.gov (United States)

    Dubra, Alfredo; Sulai, Yusufu; Norris, Jennifer L.; Cooper, Robert F.; Dubis, Adam M.; Williams, David R.; Carroll, Joseph

    2011-01-01

    The rod photoreceptors are implicated in a number of devastating retinal diseases. However, routine imaging of these cells has remained elusive, even with the advent of adaptive optics imaging. Here, we present the first in vivo images of the contiguous rod photoreceptor mosaic in nine healthy human subjects. The images were collected with three different confocal adaptive optics scanning ophthalmoscopes at two different institutions, using 680 and 775 nm superluminescent diodes for illumination. Estimates of photoreceptor density and rod:cone ratios in the 5°–15° retinal eccentricity range are consistent with histological findings, confirming our ability to resolve the rod mosaic by averaging multiple registered images, without the need for additional image processing. In one subject, we were able to identify the emergence of the first rods at approximately 190 μm from the foveal center, in agreement with previous histological studies. The rod and cone photoreceptor mosaics appear in focus at different retinal depths, with the rod mosaic best focus (i.e., brightest and sharpest) being at least 10 μm shallower than the cones at retinal eccentricities larger than 8°. This study represents an important step in bringing high-resolution imaging to bear on the study of rod disorders. PMID:21750765

  15. Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope.

    Science.gov (United States)

    Mukai, Rie; Shirai, Yasuhito; Saito, Naoaki; Yoshida, Ken-Ichi; Ashida, Hitoshi

    2009-04-01

    Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning fluorescence microscope and mouse hepatoma Hepa-1c1c7 cells. Five flavonol aglycones showed autofluorescence in the cells under the conditions (Ex. 488 nm to Em. 515-535 nm), whereas three flavonol glycosides and eight compounds belonging to other flavonoid subclasses, i.e., flavones, flavanones, and catechins, did not. The autofluorescence of galangin and kaempferol appeared stronger in the nucleus than cytoplasm, suggesting that they are incorporated into the cells and accumulated in the nucleus. The proposed method provided evidence that flavonol aglycones are incorporated into, and accumulated in the nucleus of, hepatocytes.

  16. Sarcoglycan immunoreactivity is lacking in infantile hypertrophic pyloric stenosis. A confocal laser scanning microscopic study.

    Science.gov (United States)

    Romeo, C; Santoro, G; Impellizzeri, P; Manganaro, A; Cutroneo, G; Trimarchi, E; Antonuccio, P; Anastasi, G; Zuccarello, B

    2007-01-01

    The Dystrophin-Glycoprotein Complex (DGC) is a large multisubunit complex that plays a crucial role in maintaining the structural integrity and physiology of muscle fibers. Dystrophin has been reported to be absent in the pyloric muscle of infantile hypertrophic pyloric stenosis (IHPS) patients. The present study was designed to investigate the other two patterns of DGC (dystroglycan and sarcoglycan complexes) in normal pyloric muscle and their possible modifications in IHPS patients. Ten pyloric muscle biopsies were obtained from babies operated for IHPS and five control pylorus biopsy taken at autopsy from cases without gastrointestinal disease. The DGC sub-complexes (beta-dystroglican and beta, delta- sarcoglycans) were localized immunohistochemically using specific monoclonal antibodies. The results were evaluated using a confocal laser scanning microscope. Positive immunolocalization of the two DGC sub complexes was demonstrated in the smooth muscle cells (SMCs) of the pyloric region of control patients. Similarly, a positive immune expression of beta-dystroglican was observed in the pyloric SMCs of IHPS patients. On the other hand a negative immunoreaction for sarcoglycans was recorded within the full thickness of the pyloric SMCs of these patients. The absence of sarcoglycans within the hypertrophied pyloric muscle may be a predisposing factor in the pathogenesis of IHPS since it could alter the normal physiology of SMCs through the modifications of structural integrity of sarcolemma and signaling between the extracellular and intracellular compartment.

  17. Cost-effectiveness analysis of confocal scan laser ophthalmoscope (HRT II) versus GDX for diagnosing glaucoma.

    Science.gov (United States)

    Mokhtari-Payam, Mahdi; Moradi-Lakeh, Maziar; Yaghoubi, Mohsen; Moradijou, Mohammad

    2015-01-01

    The aim of this study was to assess the cost-effectiveness of confocal scan laser ophthalmoscopy (HRT II) and compare it with scanning laser polarimetry (GDx) for diagnosing glaucoma. A cost-effectiveness analysis was performed at two eye hospitals in Iran. The outcome was measured as the proportion of correctly diagnosed patients based on systematic review and Meta analysis. Costs were estimated at two hospitals that used the HRT II (Noor Hospital) and current diagnostic testing technology GDx (Farabi Hospital) from the perspective of the healthcare provider. The incremental cost-effectiveness ratio (ICER) was estimated on the base scenario. Annual average costs were estimated as 12.70 USD and 13.59 USD per HRT II and GDx test in 2012, respectively. It was assumed that 80% of the maximum feasible annual tests in a work shift would be performed using HRT II and GDx and that the glaucoma-positive (Gl+) proportion would be 56% in the referred eyes; the estimated diagnostic accuracies were 0.753 and 0.737 for GDx and HRT II, respectively. The incremental cost-effectiveness ratio (ICER) was estimated at USD44.18 per additional test accuracy. In a base sensitivity sampling analysis, we considered different proportions of Gl+ patients (30%-85%), one or two work shifts, and efficiency rate (60%-100%), and found that the ICER ranged from USD29.45to USD480.26, the lower and upper values in all scenarios. Based on ICER, HRT II as newer diagnostic technology is cost-effective according to the World Health Organization threshold of <1 Gross Domestic Product (GDP) per capita in Iran in 2012 (USD7228). Although GDx is more accurate and costly, the average cost-effectiveness ratio shows that HRT II provided diagnostic accuracy at a lower cost than GDx.

  18. Scanning tunneling microscopy III theory of STM and related scanning probe methods

    CERN Document Server

    Güntherodt, Hans-Joachim

    1996-01-01

    Scanning Tunneling Microscopy III provides a unique introduction to the theoretical foundations of scanning tunneling microscopy and related scanning probe methods. The different theoretical concepts developed in the past are outlined, and the implications of the theoretical results for the interpretation of experimental data are discussed in detail. Therefore, this book serves as a most useful guide for experimentalists as well as for theoreticians working in the filed of local probe methods. In this second edition the text has been updated and new methods are discussed.

  19. Fast evaluation of 69 basal cell carcinomas with ex vivo fluorescence confocal microscopy: criteria description, histopathological correlation, and interobserver agreement.

    Science.gov (United States)

    Bennàssar, Antoni; Carrera, Cristina; Puig, Susana; Vilalta, Antoni; Malvehy, Josep

    2013-07-01

    Fluorescence confocal microscopy (FCM) represents a first step toward a rapid "bedside pathology" in the Mohs surgery setting and in other fields of general pathology. To describe and validate FCM criteria for the main basal cell carcinoma (BCC) subtypes and to demonstrate the overall agreement with classic pathologic analysis of hematoxylin-eosin-stained samples. DESIGN A total of 69 BCCs from 66 patients were prospectively imaged using ex vivo FCM. Confocal mosaics were evaluated in real time and compared with classic pathologic analysis. Department of Dermatology, Hospital Clínic of Barcelona, Barcelona, Spain, between November 2010 and July 2011. Patients with BCC attending the Mohs Surgery Unit. Presence or absence of BCC and histological subtype (superficial, nodular, and infiltrating) in the confocal mosaics. Eight criteria for BCC were described, evaluated, and validated. Although there were minor differences among BCC subtypes, the most BCC-defining criteria were peripheral palisading, clefting, nuclear pleomorphism, and presence of stroma. These criteria were validated with independent observers (κ values >0.7 [corrected] for most criteria). We herein propose, describe, and validate FCM criteria for BCC diagnosis. Fluorescence confocal microscopy is an attractive alternative to histopathologic analysis of frozen sections during Mohs surgery because large areas of freshly excised tissue can be assessed in real time without the need for tissue processing while minimizing labor and costs.

  20. [Use of the confocal laser scanning method for determining corneal topography and corneal tissue effects in refractive corneal surgery].

    Science.gov (United States)

    Koop, N; Brinkmann, R; Schirner, G

    1996-06-01

    Refraction of the cornea head been generally measured with ophthalmometers or computer disk keratometers. We therefore used a confocal laser scanning system for measurement of the corneal topography. Enucleated tonicized pig eyes were measured before and after laser thermokeratoplasty (LTK). The topographical data were used to determine refraction and refractive change; the data were stored digitally. The single images and their differences were displayed on a PC. Unlike conventional ophthalmometry, confocal laser scanning can demonstrate the topographical shape, showing the overall topography of the cornea and local corneal effects, e.g., coagulation, mechanical lesions or high-energy laser effects. Topographical laser scanning has proven to be a generally useful method of determining refraction and surface alterations in corneal refractive surgery.

  1. Risk Factors Associated With Corneal Nerve Alteration in Type 1 Diabetes in the Absence of Neuropathy: A Longitudinal In Vivo Corneal Confocal Microscopy Study.

    Science.gov (United States)

    Dehghani, Cirous; Pritchard, Nicola; Edwards, Katie; Russell, Anthony W; Malik, Rayaz A; Efron, Nathan

    2016-06-01

    The aim of this study was to determine alterations to the corneal subbasal nerve plexus (SNP) over 4 years using in vivo corneal confocal microscopy in participants with type 1 diabetes and to identify significant risk factors associated with these alterations. A cohort of 108 individuals with type 1 diabetes and no evidence of peripheral neuropathy at enrollment underwent laser-scanning in vivo corneal confocal microscopy, ocular screening, and health and metabolic assessment at baseline, and the examinations continued for 4 subsequent annual visits. At each annual visit, 8 central corneal images of the SNP were selected and analyzed to quantify corneal nerve fiber density, corneal nerve branch density and corneal nerve fiber length. Linear mixed model approaches were fitted to examine the relationship between risk factors and corneal nerve parameters. A total of 96 participants completed the final visit and 91 participants completed all visits. No significant relationships were found between corneal nerve parameters and time, sex, duration of diabetes, smoking, alcohol consumption, blood pressure, or body mass index. However, corneal nerve fiber density was negatively associated with glycated hemoglobin (β = -0.76, P high-density lipids (β = 2.01, P = 0.03). Higher glycated hemoglobin (β = -1.58, P = 0.04) and age (β = -0.23, P high-density lipid, and age have significant effects on SNP structure. These findings highlight the importance of diabetic management to prevent corneal nerve damage and the capability of in vivo corneal confocal microscopy for monitoring subclinical alterations in the corneal SNP in diabetes.

  2. Core/shell nanofiber characterization by Raman scanning microscopy

    Science.gov (United States)

    Sfakis, Lauren; Sharikova, Anna; Tuschel, David; Costa, Felipe Xavier; Larsen, Melinda; Khmaladze, Alexander; Castracane, James

    2017-01-01

    Core/shell nanofibers are becoming increasingly popular for applications in tissue engineering. Nanofibers alone provide surface topography and increased surface area that promote cellular attachment; however, core/shell nanofibers provide the versatility of incorporating two materials with different properties into one. Such synthetic materials can provide the mechanical and degradation properties required to make a construct that mimics in vivo tissue. Many variations of these fibers can be produced. The challenge lies in the ability to characterize and quantify these nanofibers post fabrication. We developed a non-invasive method for the composition characterization and quantification at the nanoscale level of fibers using Confocal Raman microscopy. The biodegradable/biocompatible nanofibers, Poly (glycerol-sebacate)/Poly (lactic-co-glycolic) (PGS/PLGA), were characterized as a part of a fiber scaffold to quickly and efficiently analyze the quality of the substrate used for tissue engineering. PMID:28271000

  3. [Revealing the Cell Structure and Formation of Bamboo with Confocal Raman Microscopy].

    Science.gov (United States)

    Li, Xiao-li; Zhou, Bin-xiong; Zhang, Yi; Yao, Yan-ming; He, Yong

    2016-02-01

    Parenchyma cell (PAC), transition tissue between parenchyma cell and fiber cell (TC) and fibre cell (FC) of bamboo were studied by confocal Raman microscopy in this paper. Partial least squares regression was applied to establish a quantitative differentiation model for the three types of cells. The result showed that the determination coefficients (R²) of calibration and validation were respectively 0.810 and 0.800, and the root mean square error (RMSE) were respectively 0.323 and 0.332. What's more, three raman bands of 1,095, 1,319 and 1,636 cm⁻¹, verified to the characteristic peaks of pectin, hemicellulose and lignin, were found to be the important bands for the differentiation. Subsequently, these three raman bands were used to establish a multiple linear regression (MLR) model, and the determination coefficients (R²) of calibration and validation of the model were respectively 0.644 and 0.643, and the root mean square error (RMSE) were respectively 0.442 and 0.443. This result showed that there existed obvious difference among the three types of cells in these three raman bands. Finally, the raman spectral signal processed by wavelet transform to eliminate baseline were used to chemical imaging analysis. These results showed a rather large microfibril angle between cellulose fibrils and fibre axis, which contributed to higher modulus and hardness of cells. Hemicellulose and cellulose have similar distribution in the raman chemical image, due to the connection of hemicellulose and cellulose microfiber through hydrogen bond and the closely combination under the action of van der Waals force. The cell corners (CC) and compound middle lamella (CML) were heavily lignified, and a gradual decrease of lignification from the outer layer to the inner layer of the three cells indicate that lignification was first occurred at the CC and CML, and the lignification was not fully completed.

  4. Integration of reflectance confocal microscopy in sequential dermoscopy follow-up improves melanoma detection accuracy.

    Science.gov (United States)

    Stanganelli, I; Longo, C; Mazzoni, L; Magi, S; Medri, M; Lanzanova, G; Farnetani, F; Pellacani, G

    2015-02-01

    Successful treatment of melanoma depends on early diagnosis, but its varied clinical presentation means that no single noninvasive method or criterion can provide reliable detection in all cases. To determine whether combining sequential dermoscopy imaging with reflectance confocal microscopy (RCM) can improve melanoma detection and reduce the burden of unnecessary excisions. We conducted a retrospective study with median follow-up of 25 months. We included equivocal pigmented lesions that lacked clear dermoscopy criteria for melanoma at baseline but were excised subsequently because of changes during digital monitoring. RCM imaging was performed before excision. Main melanoma dermoscopy features, seven-point checklist score at baseline, and changes in structure and/or colour, and development of new melanoma-specific criteria at follow-up (scored as major, moderate or minor) were considered. Main melanoma RCM criteria were evaluated and diagnosis was made. Histopathological diagnosis was the reference standard for defining parameter frequency and diagnostic accuracy. Seventy lesions were included. Major changes were more frequently correlated with melanoma diagnosis, although one-third (four of 12) of melanomas showed moderate or minor changes. Cytological atypia and architectural disarrangement on RCM were correlated with melanoma diagnosis. A correct melanoma diagnosis was achieved with RCM in almost all cases (11 of 12, 92%). Referring for excision only those lesions with RCM-positive features and/or presenting major changes at digital dermoscopy follow-up, theoretically 27 of 58 naevi could be saved from surgery. Integration of RCM in the clinical and instrumental strategy for managing difficult pigmented lesions provided additional diagnostic information useful in the decision-making process. © 2014 British Association of Dermatologists.

  5. In vivo confocal microscopy appearance of Fusarium and Aspergillus species in fungal keratitis.

    Science.gov (United States)

    Chidambaram, Jaya Devi; Prajna, Namperumalsamy Venkatesh; Larke, Natasha; Macleod, David; Srikanthi, Palepu; Lanjewar, Shruti; Shah, Manisha; Lalitha, Prajna; Elakkiya, Shanmugam; Burton, Matthew J

    2017-08-01

    Clinical outcomes in fungal keratitis vary between Fusarium and Aspergillus spp, therefore distinguishing between species using morphological features such as filament branching angles, sporulation along filaments (adventitious sporulation) or dichotomous branching may be useful. In this study, we assessed these three features within Heidelberg Retina Tomograph 3 in vivo confocal microscopy (IVCM) images from culture-positive Fusarium and Aspergillus spp keratitis participants. Prospective observational cohort study in Aravind Eye Hospital (February 2011-February 2012). Eligibility criteria: age ≥18 years, stromal infiltrate ≥3 mm diameter, Fusarium or Aspergillus spp culture-positive. previous/current herpetic keratitis, visual acuity 80% corneal thinning. IVCM was performed and images analysed for branch angle, presence/absence of adventitious sporulation or dichotomous branching by a grader masked to the microbiological diagnosis. 98 participants were included (106 eligible, 8 excluded as no measurable branch angles); 68 were positive for Fusarium spp, 30 for Aspergillus spp. Mean branch angle for Fusarium spp was 59.7° (95% CI 57.7° to 61.8°), and for Aspergillus spp was 63.3° (95% CI 60.8° to 65.8°), p=0.07. No adventitious sporulation was detected in Fusarium spp ulcers. Dichotomous branching was detected in 11 ulcers (7 Aspergillus spp, 4 Fusarium spp). There was very little difference in the branching angle of Fusarium and Aspergillus spp. Adventitious sporulation was not detected and dichotomous branching was infrequently seen. Although IVCM remains a valuable tool to detect fungal filaments in fungal keratitis, it cannot be used to distinguish Fusarium from Aspergillus spp and culture remains essential to determine fungal species. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  6. Feasibility and reliability of pancreatic cancer staging using fiberoptic confocal fluorescence microscopy in rats.

    Science.gov (United States)

    Ignat, Mihaela; Aprahamian, Marc; Lindner, Veronique; Altmeyer, Anaïs; Perretta, Silvana; Dallemagne, Bernard; Mutter, Didier; Marescaux, Jacques

    2009-11-01

    Surgical management of pancreatic cancer depends on tumor resectability and staging. This study evaluated a new in vivo technique, fiberoptic confocal fluorescence microscopy (FCFM), for detection and staging of pancreatic tumors in rats. FCFM was used with a protease-activated fluorescent marker (ProSense; VisEn Medical Inc, Woburn, MA) for in vivo imaging of solid organs (1.8-microm resolution) in a rat model of pancreatic ductal adenocarcinoma. A preliminary study described the FCFM rendering of normal and pathologic tissues. Subsequently, 2 double-blind studies compared FCFM to standard histology in (1) detection of tumors in rat models of cancer and controls and (2) detection of nodal involvement (splenic, celiac, mesenteric, and colic) 4, 5, and 6 weeks after tumor induction vs controls. Tumor cells displayed a fluorescent ductal pattern compared with non-fluorescent normal pancreas or normal follicular pattern of lymph nodes (LNs). FCFM detected all the pancreatic tumors (1.7-mm mean diameter) and identified 23 LNs that contained metastases of 99 LNs examined. Standard histologic analyses resulted in 1 false-negative result in tumor detection and 2 false negatives in LN detection, whereas FCFM produced no false-negative results. Additional serial sectioning confirmed all tumors and 16 metastatic LNs; FCFM had a negative predictive value of 100% and a positive predictive value of 69.6%. Real-time "virtual biopsy" using FCFM detects tumors and LN metastases with 100% sensitivity and 92.2% specificity in rats, making it a reliable technique for detection and staging of pancreatic cancer.

  7. Waterproofing in Arabidopsis: Following phenolics and lipids in situ by Confocal Raman Microscopy

    Science.gov (United States)

    Prats Mateu, Batirtze; Hauser, Marie-Theres; Heredia, Antonio; Gierlinger, Notburga

    2016-02-01

    Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm) at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299 cm-1, 1600 cm-1 and 3400 cm-1 band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobising changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum) as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol) and phenolic acids were acquired. Keywords: Arabidopsis thaliana, lignin, cutin, wax, Raman, cuticle, waterproofing

  8. Reflectance confocal microscopy-guided laser ablation of basal cell carcinomas: initial clinical experience

    Science.gov (United States)

    Sierra, Heidy; Yélamos, Oriol; Cordova, Miguel; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

    2017-08-01

    Laser ablation offers a procedure for precise, fast, and minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs). However, the lack of histopathological confirmation has been a limitation toward widespread use in the clinic. A reflectance confocal microscopy (RCM) imaging-guided approach offers cellular-level histopathology-like feedback directly on the patient, which may then guide and help improve the efficacy of the ablation procedure. Following an ex vivo benchtop study (reported in our earlier papers), we performed an initial study on 44 BCCs on 21 patients in vivo, using a pulsed erbium:ytterbium aluminum garnet laser and a contrast agent (aluminum chloride). In 10 lesions on six patients, the RCM imaging-guided detection of either presence of residual tumor or complete clearance was immediately confirmed with histopathology. Additionally, 34 BCCs on 15 patients were treated with RCM imaging-guided laser ablation, with immediate confirmation for clearance of tumor (no histopathology), followed by longer-term monitoring, currently in progress, with follow-up imaging (again, no histopathology) at 3, 6, and 18 months. Thus far, the imaging resolution appears to be sufficient and consistent for monitoring efficacy of ablation in the wound, both immediately postablation and subsequently during recovery. The efficacy results appear to be promising, with observed clearance in 19 cases of 22 cases with follow-ups ranging from 6 to 21 months. An additional 12 cases with 1 to 3 months of follow-ups has shown clearance of tumor but a longer follow-up time is required to establish conclusive results. Further instrumentation development will be necessary to cover larger areas with a more automatically controlled instrument for more uniform, faster, and deeper imaging of margins.

  9. In vivo confocal microscopy evaluation of ocular and cutaneous alterations in patients with rosacea.

    Science.gov (United States)

    Liang, Hong; Randon, Matthieu; Michee, Sylvain; Tahiri, Rachid; Labbe, Antoine; Baudouin, Christophe

    2017-03-01

    The physiopathology of rosacea and the correlation between ocular and cutaneous rosacea remains unclear. This study analysed ocular and cutaneous rosacea with in vivo confocal microscopy (IVCM). Thirty-four eyes of 34 patients with confirmed rosacea-associated meibomian gland dysfunction-related evaporative dry eye were enrolled in the study. The ophthalmological investigations included dry eye ocular surface disease index (OSDI), the Schirmer test, tear osmolarity, tear break up time, the Oxford score, infrared meibography for meibomian gland (MG) analysis and IVCM investigation for cornea, MG and skin analysis (cheek, hand). Presences of Demodex in the MG and in the cheek were also investigated. We established scores for quantifying the MG alterations in the MG (IVCM-MG) and cheek (IVCM-Cheek), and scores for Demodex quantification in the MG and cheek (IVCM-MG-Dex and IVCM-Cheek-Dex). IVCM was relevant for analysing the cornea and MG structures and was also suitable for cutaneous analysis. Exposed skin explorations presented the epidermal and dermal layers clearly. In patients with rosacea, the IVCM-MG alteration scores were correlated with IVCM-Cheek (R 2 =0.27 and p=0.0006) and IVCM-MG-Dex was correlated with IVCM-Cheek-Dex (R 2 =0.70 and prosacea combined with quantification of Demodex infections. As a valuable tool for investigating the pathophysiology of the disease, it could be used to assess the effectiveness of therapy. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  10. In vivo confocal microscopy of brachycephalic dogs with and without superficial corneal pigment.

    Science.gov (United States)

    Vallone, Lucien V; Enders, Andrew M; Mohammed, Hussni O; Ledbetter, Eric C

    2017-07-01

    To characterize canine superficial corneal pigment (SCP) in brachycephalic dogs using in vivo confocal microscopy (IVCM). Fifty-seven client-owned brachycephalic dogs from breeds predisposed to SCP (i.e., Boston Terrier, Lhasa Apso, Pekingese, Pug, and Shih Tzu). Complete ocular examination, including slit-lamp biomicroscopy, was used to determine presence or absence of SCP, and IVCM examinations were then performed. Clinical and IVCM abnormalities were recorded using a standardized scoring system and statistically compared between dogs with and without SCP. Dogs were split into two groups for analysis; Pugs and non-Pug breeds. Of the 57 dogs examined, 32 were Pugs and 25 were non-Pug breeds. Twenty-three Pugs (71.8%) and 10 non-Pugs (40%) displayed SCP. Six of 13 evaluated IVCM features were significantly (P Pugs and non-Pugs and included superficial epithelial pigment, basal epithelial pigment, Langerhans cells, anterior stromal dendritic cells, epithelial disorganization, and vascularization. Additionally, superficial epithelial leukocytes and anterior stromal dendritic cells were significantly associated with SCP in Pugs only. Many of the IVCM features associated with SCP were also observed in SCP unaffected dogs; however, they were present at a reduced frequency and confined to the perilimbal region of the cornea. By IVCM, SCP in dogs is characterized by microscopic features of chronic inflammation. Abnormalities were superficial and largely confined to the corneal epithelium. Superficial pigment in brachycephalic dogs appears morphologically as a centripetal corneal migration of microanatomic features normally confined to the perilimbal region of the cornea. © 2016 American College of Veterinary Ophthalmologists.

  11. Compressive sensing in reflectance confocal microscopy of skin images: a preliminary comparative study

    Science.gov (United States)

    Arias, Fernando X.; Sierra, Heidy; Rajadhyaksha, Milind; Arzuaga, Emmanuel

    2016-03-01

    Compressive Sensing (CS)-based technologies have shown potential to improve the efficiency of acquisition, manipulation, analysis and storage processes on signals and imagery with slight discernible loss in data performance. The CS framework relies on the reconstruction of signals that are presumed sparse in some domain, from a significantly small data collection of linear projections of the signal of interest. As a result, a solution to the underdetermined linear system resulting from this paradigm makes it possible to estimate the original signal with high accuracy. One common approach to solve the linear system is based on methods that minimize the L1-norm. Several fast algorithms have been developed for this purpose. This paper presents a study on the use of CS in high-resolution reflectance confocal microscopy (RCM) images of the skin. RCM offers a cell resolution level similar to that used in histology to identify cellular patterns for diagnosis of skin diseases. However, imaging of large areas (required for effective clinical evaluation) at such high-resolution can turn image capturing, processing and storage processes into a time consuming procedure, which may pose a limitation for use in clinical settings. We present an analysis on the compression ratio that may allow for a simpler capturing approach while reconstructing the required cellular resolution for clinical use. We provide a comparative study in compressive sensing and estimate its effectiveness in terms of compression ratio vs. image reconstruction accuracy. Preliminary results show that by using as little as 25% of the original number of samples, cellular resolution may be reconstructed with high accuracy.

  12. Waterproofing in Arabidopsis: Following phenolics and lipids in situ by Confocal Raman Microscopy

    Directory of Open Access Journals (Sweden)

    Batirtze ePrats Mateu

    2016-02-01

    Full Text Available Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299 cm-1, 1600 cm-1 and 3400 cm-1 band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobising changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol and phenolic acids were acquired. Keywords: Arabidopsis thaliana, lignin, cutin, wax, Raman

  13. The reflectance confocal microscopy features of sebaceous adenoma in a case of Muir Torre syndrome

    Directory of Open Access Journals (Sweden)

    Esma İnan Yüksel

    2015-03-01

    Full Text Available Muir-Torre syndrome (MTS is a rare autosomal dominant genodermatosis characterized by the occurrence of sebaceous gland neoplasms and/or keratoacanthomas associated with visceral malignancies. It is considered as a subtype of hereditary nonpolyposis colorectal cancer syndrome. Characteristic sebaceous gland neoplasms include sebaceous adenoma, sebaceous carcinoma, sebaceoma, and keratoacanthoma with sebaceous differentiation. The most common visceral malignancies are colorectal and genitourinary tumors. CASE: A 47year-old male patient admitted to our clinic complaining of two lesions on the nose. Dermatological examination revealed a plaque in 1 cm diameter consisting of bright yellowish-white coloured papules with slightly umblicated appearance and telangiectasias on the left site of the nose and had a dome shaped papule in 3 mm diameter with hyperkeratotic plug on the tip of the nose. He had personal history of partial colon resection because of colon cancer and familial Lynch 2 syndrome. On dermoscopic examination of sebaceous adenoma, a few yellow comedo-like globules and branching arborizing vessels were detected. Reflectance confocal microscopy (RCM revealed a good histopathologic correlation. Sebaceous lobules were composed by clusters of ovoid cells with hyporefractile dark nuclei and bright, hyperrefractile glistening cytoplasm. Numerous roundish to ovoid dark spaces corresponding to sebaceous ducts were detected. The diagnosis of MTS was established based on the personal and family history, dermoscopic, RCM and histopathologic findings. CONCLUSIONS: MTS evaluation is required in patients with biopsy-proven sebaceous adenoma. Early diagnosis may be lifesaving in patients with MTS. A better characterization of RCM features of sebaceous tumors will allow early diagnosis of the patients with MTS.

  14. In vivo confocal microscopy and tear cytokine analysis in post-LASIK ectasia.

    Science.gov (United States)

    Pahuja, Natasha Kishore; Shetty, Rohit; Deshmukh, Rashmi; Sharma, Anupam; Nuijts, Rudy M M A; Jhanji, Vishal; Sethu, Swaminathan; Ghosh, Arkasubhra

    2017-04-27

    Corneal keratectasia is one of the complications associated with laser in situ keratomileusis (LASIK) that results in vision impairment. The pathogenesis of post-LASIK ectasia (PLE) remains underexplored. We report the tear cytokine profile and in vivo confocal microscopy (IVCM) findings in eyes with PLE. This retrospective study included age-matched 7 (14 eyes) post-LASIK controls (PLCs) and 6 (12 eyes) PLE subjects. Corneal topography was used to categorise the subjects into PLC and PLE groups. Ocular Surface Disease Index (OSDI) scores obtained were based on standard questionnaire and IVCM images were used to determine corneal dendritic cells density (DCD) and sub-basal nerve plexus morphology. Inflammatory cytokines/chemokines in the tears were quantified using flow cytometry based cytometric bead array. Pentacam-based scores, OSDI scores and corneal DCD were significantly (pPLE compared with PLC. Discomfort-related subscale of OSDI score exhibited a positive correlation with total corneal DCD in the PLE cohort. The fold difference of chemokine (C-C motif) ligand/monocyte chemotactic protein-1 (CCL2/MCP1) (3.4±0.6) was found to be significantly (pPLE cohorts and a positive correlation between CCL2/MCP1 levels and total corneal DCD was also observed in the PLE cohort. The current study found a significant difference in the tear film cytokine profile between normal and PLE eyes. Presence of increased corneal dendritic cells and altered tear cytokines suggests an ongoing inflammatory response in PLE. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  15. Analysis of Microstructure of the Cardiac Conduction System Based on Three-Dimensional Confocal Microscopy.

    Directory of Open Access Journals (Sweden)

    Daniel Romero

    Full Text Available The specialised conducting tissues present in the ventricles are responsible for the fast distribution of the electrical impulse from the atrio-ventricular node to regions in the subendocardial myocardium. Characterisation of anatomical features of the specialised conducting tissues in the ventricles is highly challenging, in particular its most distal section, which is connected to the working myocardium via Purkinje-myocardial junctions. The goal of this work is to characterise the architecture of the distal section of the Purkinje network by differentiating Purkinje cells from surrounding tissue, performing a segmentation of Purkinje fibres at cellular scale, and mathematically describing its morphology and interconnections. Purkinje cells from rabbit hearts were visualised by confocal microscopy using wheat germ agglutinin labelling. A total of 16 3D stacks including labeled Purkinje cells were collected, and semi-automatically segmented. State-of-the-art graph metrics were applied to estimate regional and global features of the Purkinje network complexity. Two types of cell types, tubular and star-like, were characterised from 3D segmentations. The analysis of 3D imaging data confirms the previously suggested presence of two types of Purkinje-myocardium connections, a 2D interconnection sheet and a funnel one, in which the narrow side of a Purkinje fibre connect progressively to muscle fibres. The complex network analysis of interconnected Purkinje cells showed no small-world connectivity or assortativity properties. These results might help building more realistic computational PK systems at high resolution levels including different cell configurations and shapes. Better knowledge on the organisation of the network might help in understanding the effects that several treatments such as radio-frequency ablation might have when the PK system is disrupted locally.

  16. Usefulness of confocal microscopy in distinguishing between basal cell carcinoma and intradermal melanocytic nevus on the face.

    Science.gov (United States)

    Gamo, R; Floristan, U; Pampín, A; Caro, D; Pinedo, F; López-Estebaranz, J L

    2015-10-01

    The clinical distinction between basal cell carcinoma (BCC) and intradermal melanocytic nevus lesions on the face can be difficult, particularly in young patients or patients with multiple nevi. Dermoscopy is a useful tool for analyzing characteristic dermoscopic features of BCC, such as cartwheel structures, maple leaf-like areas, blue-gray nests and dots, and ulceration. It also reveals arborizing telangiectatic vessels and prominent curved vessels, which are typical of BCC, and comma vessels, which are typical of intradermal melanocytic nevi. It is, however, not always easy to distinguish between these 2 conditions, even when dermoscopy is used. We describe 2 facial lesions that posed a clinical and dermoscopic challenge in two 38-year-old patients; confocal microscopy showed separation between tumor nests and stroma and polarized nuclei, which are confocal microscopy features of basal cell carcinoma. Copyright © 2014 Elsevier España, S.L.U. y AEDV. All rights reserved.

  17. ATP concentration as possible marker of liver damage at leukaemia treatment: confocal microscopy-based experimental study and numerical simulations

    Science.gov (United States)

    Malashchenko, V.; Zyubin, A.; Babak, S.; Lavrova, A.

    2017-04-01

    We consider the method of confocal microscopy as a convenient instrument for determination of chemical compounds in biological tissues and cells. In particular, we study the dynamics of adenosine triphosphate (ATP) concentration that could be used as a bio-marker of energy metabolism pathologies at the treatment of acute lymphoblastic leukaemia (ALL). On the basis of data obtained by the confocal microscopy, the values of ATP concentration have been calculated for each case. Possible correlations with other characteristics of pathology processes obtained from plasma of leukemia patients show that ATP value could be a prognostic factor of the treatment success. The role of ATP in the drug metabolism switching is also discussed within the context of kinetic modelling of metabolism processes leading to the production of 6-Thioguanosine monophosphate, which is a principal acting agent in chemotherapy.

  18. Cryo scanning electron microscopy of Plasmodium falciparum-infected erythrocytes.

    Science.gov (United States)

    Hempel, Casper

    2017-07-01

    Plasmodium falciparum invades erythrocytes as an essential part of their life cycle. While living inside erythrocytes, the parasite remodels the cell's intracellular organization as well as its outer surface. Late trophozoite-stage parasites and schizonts introduce numerous small protrusions on the erythrocyte surface, called knobs. Current methods for studying these knobs include atomic force microscopy and electron microscopy. Standard electron microscopy methods rely on chemical fixation and dehydration modifying cell size. Here, a novel method is presented using rapid freezing and scanning electron microscopy under cryogenic conditions allowing for high resolution and magnification of erythrocytes. This novel technique can be used for precise estimates of knob density and for studies on cytoadhesion. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  19. In vivo laser confocal microscopy of Bowman's layer of the cornea.

    Science.gov (United States)

    Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

    2006-12-01

    To investigate in vivo microstructures of Bowman's layer in normal human subjects using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT2-RCM). Single-center, prospective, observational case series. Nineteen normal volunteers (10 male, 9 female; mean age, 46.2+/-21.7 years [range, 18-77]). The central and peripheral cornea, specifically the epithelium, Bowman's layer, and its subjacent stroma, were examined using the HRT2-RCM. Selected images of the corneal layers were evaluated qualitatively for the shape and degree of light reflection of the microstructures. In all subjects, normal epithelial (superficial, wing, basal) cells, subbasal nerve plexus, Bowman's layer, and its subjacent stoma were observed clearly. However, in all subjects, polymorphic structures composed of fibrillar materials with less reflectivity than corneal nerves were observed beneath Bowman's layer. After application of pressure by a Tomo-cap, we observed numerous ridges that protruded into the epithelial basal and wing cell layers. Superficial stromal striae were also observed. These ridges and striae corresponded exactly to the orientation of the fibrous structures located beneath the epithelial cells. We report for the first time, the presence of polymorphic structures composed of fibrillar materials (K-structures) beneath Bowman's layer in normal human subjects, detected by HRT2-RCM. We surmise that these microstructures may correspond to the modified and condensed anterior stromal collagen fibers/lamellae that merge into Bowman's layer and that these fibrillar materials may be responsible for the formation of the anterior corneal mosaic. Further investigation of these microstructures in diseased eyes may provide insights into their pathophysiologic role in Bowman's layer.

  20. Advanced chemical imaging and comparison of human and porcine hair follicles for drug delivery by confocal Raman microscopy

    Science.gov (United States)

    Franzen, Lutz; Mathes, Christiane; Hansen, Steffi; Windbergs, Maike

    2013-06-01

    Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery.

  1. Autologous Serum Tears for Treatment of Photoallodynia in Patients with Corneal Neuropathy: Efficacy and Evaluation with In Vivo Confocal Microscopy.

    Science.gov (United States)

    Aggarwal, Shruti; Kheirkhah, Ahmad; Cavalcanti, Bernardo M; Cruzat, Andrea; Colon, Clara; Brown, Emma; Borsook, David; Prüss, Harald; Hamrah, Pedram

    2015-07-01

    Patients suffering from corneal neuropathy may present with photoallodynia; i.e., increased light sensitivity, frequently with a normal slit-lamp examination. This study aimed to evaluate the efficacy of autologous serum tears (AST) for treatment of severe photoallodynia in corneal neuropathy and to correlate clinical findings with corneal subbasal nerve alterations by in vivo confocal microscopy (IVCM). Retrospective case control study with 16 patients with neuropathy-induced severe photoallodynia compared to 16 normal controls. Symptom severity, clinical examination and bilateral corneal IVCM scans were recorded. All patients suffered from extreme photoallodynia (8.8±1.1) with no concurrent ocular surface disease. Subbasal nerves were significantly decreased at baseline in patients compared to controls; total nerve length (9208±1264 vs 24714±1056 μm/mm(2); P<.0001) and total nerve number (9.6±1.4 vs 28.6±2.0; P<.0001), respectively. Morphologically, significantly increased reflectivity (2.9±0.2 vs 1.8±0.1; P<.0001), beading (in 93.7%), and neuromas (in 62.5%) were seen. AST (3.6±2.1 months) resulted in significantly decreased symptom severity (1.6±1.7; P=.02). IVCM demonstrated significantly improved nerve parameters (P<.005), total nerve length (15451±1595 μm/mm(2)), number (13.9±2.1), and reflectivity (1.9±0.1). Beading and neuromas were seen in only 56.2% and 7.6% of patients. Patients with corneal neuropathy-induced photoallodynia show profound alterations in corneal nerves. AST restores nerve topography through nerve regeneration, and this correlated with improvement in patient-reported photoallodynia. The data support the notion that corneal nerve damage results in alterations in afferent trigeminal pathways to produce photoallodynia. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Video-mosaicking of in vivo reflectance confocal microscopy images for noninvasive examination of skin lesion (Conference Presentation)

    Science.gov (United States)

    Kose, Kivanc; Gou, Mengran; Yelamos, Oriol; Cordova, Miguel A.; Rossi, Anthony; Nehal, Kishwer S.; Camps, Octavia I.; Dy, Jennifer G.; Brooks, Dana H.; Rajadhyaksha, Milind

    2017-02-01

    In this report we describe a computer vision based pipeline to convert in-vivo reflectance confocal microscopy (RCM) videos collected with a handheld system into large field of view (FOV) mosaics. For many applications such as imaging of hard to access lesions, intraoperative assessment of MOHS margins, or delineation of lesion margins beyond clinical borders, raster scan based mosaicing techniques have clinically significant limitations. In such cases, clinicians often capture RCM videos by freely moving a handheld microscope over the area of interest, but the resulting videos lose large-scale spatial relationships. Videomosaicking is a standard computational imaging technique to register, and stitch together consecutive frames of videos into large FOV high resolution mosaics. However, mosaicing RCM videos collected in-vivo has unique challenges: (i) tissue may deform or warp due to physical contact with the microscope objective lens, (ii) discontinuities or "jumps" between consecutive images and motion blur artifacts may occur, due to manual operation of the microscope, and (iii) optical sectioning and resolution may vary between consecutive images due to scattering and aberrations induced by changes in imaging depth and tissue morphology. We addressed these challenges by adapting or developing new algorithmic methods for videomosaicking, specifically by modeling non-rigid deformations, followed by automatically detecting discontinuities (cut locations) and, finally, applying a data-driven image stitching approach that fully preserves resolution and tissue morphologic detail without imposing arbitrary pre-defined boundaries. We will present example mosaics obtained by clinical imaging of both melanoma and non-melanoma skin cancers. The ability to combine freehand mosaicing for handheld microscopes with preserved cellular resolution will have high impact application in diverse clinical settings, including low-resource healthcare systems.

  3. Surface morphology of Trichinella spiralis by scanning electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kim, C.W. (State Univ. of New York, Stony Brook); Ledbetter, M.C.

    1980-02-01

    The surface morphology of larval and adult Trichinella spiralis was studied by scanning electron microscopy (SEM) of fixed, dried, and metal-coated specimens. The results are compared with those found earlier by various investigators using light and transmission electron microscopy. Some morphological features reported here are revealed uniquely by SEM. These include the pores of the cephalic sense organs, the character of secondary cuticular folds, variations of the hypodermal gland cell openings or pores, and the presence of particles on the copulatory bell.

  4. Scanning conductance microscopy investigations on fixed human chromosomes

    DEFF Research Database (Denmark)

    Clausen, Casper Hyttel; Lange, Jacob Moresco; Jensen, Linda Boye

    2008-01-01

    Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device...... optimization. The phase shift caused by the electrostatic forces, together with geometrical measurements of the atomic force microscopy (AFM) cantilever and the chromosomes were used to estimate a value,for the dielectric constant of different human chromosomes....

  5. Ultrafast Photon Counting Applied to Resonant Scanning STED Microscopy

    Science.gov (United States)

    Wu, Xundong; Toro, Ligia; Stefani, Enrico; Wu, Yong

    2014-01-01

    Summary To take full advantage of fast resonant scanning in super-resolution STimulated Emission Depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multi-giga-sample per second analog-to-digital conversion (ADC) chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (~50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time-gated continuous wave (CW) STED technology to the usage of resonant scanning with hardware based time-gating. The assembled system provides superb signal-to-noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant-scanning CW-STED microscopy with on-line time-gated detection. PMID:25227160

  6. Performance of confocal scanning laser tomograph Topographic Change Analysis (TCA) for assessing glaucomatous progression.

    Science.gov (United States)

    Bowd, Christopher; Balasubramanian, Madhusudhanan; Weinreb, Robert N; Vizzeri, Gianmarco; Alencar, Luciana M; O'Leary, Neil; Sample, Pamela A; Zangwill, Linda M

    2009-02-01

    To determine the sensitivity and specificity of confocal scanning laser ophthalmoscope's Topographic Change Analysis (TCA; Heidelberg Retina Tomograph [HRT]; Heidelberg Engineering, Heidelberg, Germany) parameters for discriminating between progressing glaucomatous and stable healthy eyes. The 0.90, 0.95, and 0.99 specificity cutoffs for various (n=70) TCA parameters were developed by using 1000 permuted topographic series derived from HRT images of 18 healthy eyes from Moorfields Eye Hospital, imaged at least four times. The cutoffs were then applied to topographic series from 36 eyes with known glaucomatous progression (by optic disc stereophotograph assessment and/or standard automated perimetry guided progression analysis, [GPA]) and 21 healthy eyes from the University of California, San Diego (UCSD) Diagnostic Innovations in Glaucoma Study (DIGS), all imaged at least four times, to determine TCA sensitivity and specificity. Cutoffs also were applied to 210 DIGS patients' eyes imaged at least four times with no evidence of progression (nonprogressed) by stereophotography or GPA. The TCA parameter providing the best sensitivity/specificity tradeoff using the 0.90, 0.95, and 0.99 cutoffs was the largest clustered superpixel area within the optic disc margin (CAREA(disc) mm(2)). Sensitivities/specificities for classifying progressing (by stereophotography and/or GPA) and healthy eyes were 0.778/0.809, 0.639/0.857, and 0.611/1.00, respectively. In nonprogressing eyes, specificities were 0.464, 0.570, and 0.647 (i.e., lower than in the healthy eyes). In addition, TCA parameter measurements of nonprogressing eyes were similar to those of progressing eyes. TCA parameters can discriminate between progressing and longitudinally observed healthy eyes. Low specificity in apparently nonprogressing patients' eyes suggests early progression detection using TCA.

  7. Feasibility and reliability of pancreatic cancer staging using a new confocal non-fluorescent microscopy probe: a double-blind study in rats.

    Science.gov (United States)

    Akladios, Cherif; De Ruijter, Vivian; Perretta, Sylvana; Aprahamian, Marc; Ignat, Mihaela; Lindner, Veronique; Averous, Gerlinde; Dallemagne, Bernard; Marescaux, Jacques

    2017-02-01

    Surgical management of pancreatic cancer depends on tumor resectability and staging. Lymph node (LN) metastases represent an important decision-making factor when it comes to surgical treatment. To evaluate a new in vivo, endoscopic confocal microscopy (CM) system not requiring fluorescence markers, for detection and staging of pancreatic cancer in rats. A confocal system consisting of a confocal scanning laser operating in reflection mode and a dedicated rigid Hopkins rod-lens endoscope were used for in vivo imaging in a rat model of pancreatic ductal adenocarcinoma. A double-blind study compared CM to standard histology in (1) the detection of tumors in rat bearing cancer (n = 11) and controls (n = 6), and (2) in the detection of local nodal involvement at 3 and 6 weeks after tumor induction. CM detected all pancreatic tumors with 100 % sensitivity and specificity and identified 15 metastatic LNs with an average adenocarcinoma nodule diameter of 2.3 mm (range from 1 to 4.2 mm) out of the 66 examined. CM demonstrated a sensitivity of 87.5 % and a specificity of 98 % in LN detection. The Spearman's rank correlation/rho calculator was of 0.87. CM demonstrated a negative predictive value of 96.1 % and a positive predictive value of 93.3 % in the detection of metastatic LNs. Interpretation of confocal images has a high concurrence rate with histopathology examination for primary tumor and lymphatic involvement detection making it a promising technique for in vivo real-time detection and staging of pancreatic cancer. Larger studies are warranted to confirm these preliminary results.

  8. A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors.

    Science.gov (United States)

    Kempen, Paul J; Kircher, Moritz F; de la Zerda, Adam; Zavaleta, Cristina L; Jokerst, Jesse V; Mellinghoff, Ingo K; Gambhir, Sanjiv S; Sinclair, Robert

    2015-01-01

    The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Confocal laser scanning microscopic analysis of ectopic sublingual gland-like tissue inside the hamster submandibular gland.

    Science.gov (United States)

    Moriguchi, Keiichi; Utsumi, Michiya; Ohno, Norikazu

    2013-12-01

    Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3'-diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO-positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous-like cells were found in resin-embedded semi-thin sections of the DAB-reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance-mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity-negative secretory granules and seromucous cells with PO activity-positive spot-like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron-dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron-dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland-like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Copyright © 2013 Wiley Periodicals, Inc.

  10. Sub-Kelvin scanning tunneling microscopy on magnetic molecules

    OpenAIRE

    Zhang, Lei

    2012-01-01

    Magnetic molecules have attracted lots interest. In this work, an ultra-stable and low noise scanning tunneling microscopy operating at 400 mK using He-3 (930 mK using He-4) has been developed. The magnetic behavior of different magnetic molecules on substrates, especially the exchange interaction between the magnetic ions, the magnetic anisotropy on the surface, the magnetic excitations as well as the Kondo effect, were studied by using STM.

  11. Scanning Electron Microscopy of Cristispira Species in Chesapeake Bay Oysters

    OpenAIRE

    Tall, Ben D.; Nauman, Robert K.

    1981-01-01

    Scanning electron microscopy was employed to observe the physical interactions between Cristispira spp. and the crystalline style of the Chesapeake Bay oyster (Crassostrea virginica Gmelin 1791). Cristispira organisms were found associated with both the inner and outer layers of the posterior two-thirds of the style. The spirochetes possessed blunt-tipped ends, a cell diameter range of 0.6 to 0.8 μm, and distended spirochetal envelopes which followed the contour of the cells. Transmission ele...

  12. Playing peekaboo with graphene oxide: a scanning electrochemical microscopy investigation.

    Science.gov (United States)

    Rapino, Stefania; Treossi, Emanuele; Palermo, Vincenzo; Marcaccio, Massimo; Paolucci, Francesco; Zerbetto, Francesco

    2014-11-07

    Scanning electrochemical microscopy (SECM) can image graphene oxide (GO) flakes on insulating and conducting substrates. The contrast between GO and the substrate is controlled by the electrostatic interactions that are established between the charges of the molecular redox mediator and the charges present in the sheet/substrate. SECM also allows quantitative measurement - at the nano/microscale - of the charge transfer kinetics between single monolayer sheets and agent molecules.

  13. Scanning gate microscopy of ultra clean carbon nanotube quantum dots

    OpenAIRE

    Xue, Jiamin; Dhall, Rohan; Cronin, Stephen B.; LeRoy, Brian J.

    2015-01-01

    We perform scanning gate microscopy on individual suspended carbon nanotube quantum dots. The size and position of the quantum dots can be visually identified from the concentric high conductance rings. For the ultra clean devices used in this study, two new effects are clearly identified. Electrostatic screening creates non-overlapping multiple sets of Coulomb rings from a single quantum dot. In double quantum dots, by changing the tip voltage, the interactions between the quantum dots can b...

  14. Clinical features and in vivo confocal microscopy assessment in 12 patients with ocular cicatricial pemphigoid

    Directory of Open Access Journals (Sweden)

    Qin Long

    2016-05-01

    Full Text Available AIM: To describe the clinical features and microstructural characteristics assessed by in vivo confocal microscopy (IVCM in patients with ocular cicatricial pemphigoid (OCP. METHODS: A descriptive, uncontrolled case series study. Patients diagnosed with OCP were examined by clinical history, slit-lamp biomicroscopy features and IVCM images. The results of direct immunofluorescence (DIF biopsies and indirect immunofluorescence (IIF were also recorded. Local and systemic immunosuppressive therapy were administered and adjusted according to response. RESULTS: A total of 12 consecutive OCP patients (7 male, 5 female; mean age 60.42±10.39y were recruited. All patients exhibited bilateral progressive conjunctival scarring and recurrent chronic conjunctivitis was the most frequent clinical pattern. The mean duration of symptoms prior to diagnosis of OCP was 2.95±2.85y (range: 5mo to 10y. The Foster classification varied from stage I to IV and 20 eyes (83% were within or greater than Foster stage Ⅲ on presentation. Two of the 12 patients (17% demonstrated positive DIF; 3 of the 12 (25% patients reported positive IIF. The mean duration of the follow-up period was 20.17±11.88mo (range: 6 to 48mo. IVCM showed variable degrees of abnormality in the conjuctiva-cornea and conjuctival scarring was detected in all the involved eyes. Corneal stromal cell activation and dendritic cell infiltration presented as ocular surface inflammation, ocular surface keratinization along with the destroyed Vogt palisades was noted in eyes with potential limbal stem cell deficiency. After treatment, remission of ocular surface inflammation was achieved in all the patients, 18 eyes (75% remained stable, 6 eyes (25% had recurrent conjunctivitis and cicatrization in 2 eyes (8% was progressing.  CONCLUSION: As an autoimmune disease, OCP manifests as variable degrees of clinical and laboratory abnormalities with both local and systemic immunosuppressive treatment playing

  15. Abrasion of 6 dentifrices measured by vertical scanning interference microscopy

    Science.gov (United States)

    PASCARETTI-GRIZON, Florence; MABILLEAU, Guillaume; CHAPPARD, Daniel

    2013-01-01

    Objectives The abrasion of dentifrices is well recognized to eliminate the dental plaque. The aims of this study were to characterize the abrasive powders of 6 dentifrices (3 toothpastes and 3 toothpowders) and to measure the abrasion on a test surface by Vertical Scanning Interference