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Sample records for glycosylation confocal images

  1. Confocal Imaging of porous media

    Science.gov (United States)

    Shah, S.; Crawshaw, D.; Boek, D.

    2012-12-01

    Carbonate rocks, which hold approximately 50% of the world's oil and gas reserves, have a very complicated and heterogeneous structure in comparison with sandstone reservoir rock. We present advances with different techniques to image, reconstruct, and characterize statistically the micro-geometry of carbonate pores. The main goal here is to develop a technique to obtain two dimensional and three dimensional images using Confocal Laser Scanning Microscopy. CLSM is used in epi-fluorescent imaging mode, allowing for the very high optical resolution of features well below 1μm size. Images of pore structures were captured using CLSM imaging where spaces in the carbonate samples were impregnated with a fluorescent, dyed epoxy-resin, and scanned in the x-y plane by a laser probe. We discuss the sample preparation in detail for Confocal Imaging to obtain sub-micron resolution images of heterogeneous carbonate rocks. We also discuss the technical and practical aspects of this imaging technique, including its advantages and limitation. We present several examples of this application, including studying pore geometry in carbonates, characterizing sub-resolution porosity in two dimensional images. We then describe approaches to extract statistical information about porosity using image processing and spatial correlation function. We have managed to obtain very low depth information in z -axis (~ 50μm) to develop three dimensional images of carbonate rocks with the current capabilities and limitation of CLSM technique. Hence, we have planned a novel technique to obtain higher depth information to obtain high three dimensional images with sub-micron resolution possible in the lateral and axial planes.

  2. Confocal imaging of butterfly tissue.

    Science.gov (United States)

    Brunetti, Craig R

    2014-01-01

    To understand the molecular events responsible for morphological change requires the ability to examine gene expression in a wide range of organisms in addition to model systems to determine how the differences in gene expression correlate with phenotypic differences. There are approximately 12,000 species of butterflies, most, with distinct patterns on their wings. The most important tool for studying gene expression in butterflies is confocal imaging of butterfly tissue by indirect immunofluorescence using either cross-reactive antibodies from closely related species such as Drosophila or developing butterfly-specific antibodies. In this report, we describe how indirect immunofluorescence protocols can be used to visualize protein expression patterns on the butterfly wing imaginal disc and butterfly embryo.

  3. Submillimeter Confocal Imaging Active Module

    Science.gov (United States)

    Hong, John; Mehdi, Imran; Siegel, Peter; Chattopadhyay, Goutam; Cwik, Thomas; Rowell, Mark; Hacker, John

    2009-01-01

    The term submillimeter confocal imaging active module (SCIAM) denotes a proposed airborne coherent imaging radar system that would be suitable for use in reconnaissance, surveillance, and navigation. The development of the SCIAM would include utilization and extension of recent achievements in monolithic microwave integrated circuits capable of operating at frequencies up to and beyond a nominal radio frequency of 340 GHz. Because the SCIAM would be primarily down-looking (in contradistinction to primarily side-looking), it could be useful for imaging shorter objects located between taller ones (for example, objects on streets between buildings). The SCIAM would utilize a confocal geometry to obtain high cross-track resolution, and would be amenable to synthetic-aperture processing of its output to obtain high along-track resolution. The SCIAM (see figure) would include multiple (two in the initial version) antenna apertures, separated from each other by a cross-track baseline of suitable length (e.g., 1.6 m). These apertures would both transmit the illuminating radar pulses and receive the returns. A common reference oscillator would generate a signal at a controllable frequency of (340 GHz + (Delta)f)/N, where (Delta)f is an instantaneous swept frequency difference and N is an integer. The output of this oscillator would be fed to a frequency- multiplier-and-power-amplifier module to obtain a signal, at 340 GHz + (Delta)f, that would serve as both the carrier signal for generating the transmitted pulses and a local-oscillator (LO) signal for a receiver associated with each antenna aperture. Because duplexers in the form of circulators or transmit/receive (T/R) switches would be lossy and extremely difficult to implement, the antenna apertures would be designed according to a spatial-diplexing scheme, in which signals would be coupled in and out via separate, adjacent transmitting and receiving feed horns. This scheme would cause the transmitted and received beams

  4. Aorta Fluorescence Imaging by Using Confocal Microscopy

    OpenAIRE

    Wang, Chun-Yang; Tsai, Jui-che; Chuang, Ching-Cheng; Hsieh, Yao-Sheng; Sun, Chia-Wei

    2011-01-01

    The activated leukocyte attacked the vascular endothelium and the associated increase in VEcadherin number was observed in experiments. The confocal microscopic system with a prism-based wavelength filter was used for multiwavelength fluorescence measurement. Multiwavelength fluorescence imaging based on the VEcadherin within the aorta segment of a rat was achieved. The confocal microscopic system capable of fluorescence detection of cardiovascular tissue is a useful tool for measuring the bi...

  5. Confocal microlaparoscope for imaging the fallopian tube

    Science.gov (United States)

    Wu, Tzu-Yu; Rouse, Andrew R.; Chambers, Setsuko K.; Hatch, Kenneth D.; Gmitro, Arthur F.

    2014-11-01

    Recent evidence suggests that ovarian cancer can originate in the fallopian tube. Unlike many other cancers, poor access to the ovary and fallopian tubes has limited the ability to study the progression of this deadly disease and to diagnosis it during the early stage when it is most amenable to therapy. A rigid confocal microlaparoscope system designed to image the epithelial surface of the ovary in vivo was previously reported. A new confocal microlaparoscope with an articulating distal tip has been developed to enable in vivo access to human fallopian tubes. The new microlaparoscope is compatible with 5-mm trocars and includes a 2.2-mm-diameter articulating distal tip consisting of a bare fiber bundle and an automated dye delivery system for fluorescence confocal imaging. This small articulating device should enable the confocal microlaparoscope to image early stage ovarian cancer arising inside the fallopian tube. Ex vivo images of animal tissue and human fallopian tube using the new articulating device are presented along with in vivo imaging results using the rigid confocal microlaparoscope system.

  6. Microsphere imaging with confocal microscopy and two photon microscopy

    International Nuclear Information System (INIS)

    Chun, Hyung Su; An, Kyung Won; Lee, Jai Hyung

    2002-01-01

    We have acquired images of polystyrene and fused-silica microsphere by using conventional optical microscopy, confocal microscopy and two-photon microscopy, and performed comparative analysis of these images. Different from conventional optical microscopy, confocal and two-photon microscopy had good optical sectioning capability. In addition, confocal microscopy and two-photon microscopy had better lateral resolution than conventional optical microscopy. These results are attributed to confocality and nonlinearity of confocal microscopy and two photon microscopy, respectively.

  7. Fluorescence confocal endomicroscopy in biological imaging

    Science.gov (United States)

    Delaney, Peter; Thomas, Steven; Allen, John; McLaren, Wendy; Murr, Elise; Harris, Martin

    2007-02-01

    In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices. There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo microscopy systems. A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system). Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204 sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of a high-density optical fibre bundle (~30,000 fibres) of small animal organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and sub-cellular details could be readily visualised in vivo at high

  8. 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.

  9. 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...... the concentration of solutes and the diffusive properties of the biofilm matrix....

  10. An invertebrate embryologist's guide to routine processing of confocal images.

    Science.gov (United States)

    von Dassow, George

    2014-01-01

    It is almost impossible to use a confocal microscope without encountering the need to transform the raw data through image processing. Adherence to a set of straightforward guidelines will help ensure that image manipulations are both credible and repeatable. Meanwhile, attention to optimal data collection parameters will greatly simplify image processing, not only for convenience but for quality and credibility as well. Here I describe how to conduct routine confocal image processing tasks, including creating 3D animations or stereo images, false coloring or merging channels, background suppression, and compressing movie files for display.

  11. Improvement in volume estimation from confocal sections after image deconvolution

    Czech Academy of Sciences Publication Activity Database

    Difato, Francesco; Mazzone, F.; Scaglione, S.; Fato, M.; Beltrame, F.; Kubínová, Lucie; Janáček, Jiří; Ramoino, P.; Vicidomini, G.; Diaspro, A.

    2004-01-01

    Roč. 64, č. 2 (2004), s. 151-155 ISSN 1059-910X Institutional research plan: CEZ:AV0Z5011922 Keywords : confocal microscopy * image deconvolution * point spread function Subject RIV: EA - Cell Biology Impact factor: 2.609, year: 2004

  12. Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.

    Science.gov (United States)

    Hayashi, Shinichi; Okada, Yasushi

    2015-05-01

    Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro-tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30-100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging. © 2015 Hayashi and Okada. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  13. Embryonic Heart Morphogenesis from Confocal Microscopy Imaging and Automatic Segmentation

    Directory of Open Access Journals (Sweden)

    Hongda Mao

    2013-01-01

    Full Text Available Embryonic heart morphogenesis (EHM is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. This process is of great biological and clinical interest but is still poorly understood for two main reasons. On the one hand, the existing imaging modalities for investigating EHM suffered from either limited penetration depth or limited spatial resolution. On the other hand, current works typically adopted manual segmentation, which was tedious, subjective, and time consuming considering the complexity of developing heart geometry and the large size of images. In this paper, we propose to utilize confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for EHM study. The imaging method is able to produce high spatial resolution images and achieve large penetration depth at the same time. Furthermore, we propose a novel convex active contour model for automatic image segmentation. The model has the ability to deal with intensity fall-off in depth which is characterized by confocal microscopy images. We acquired the images of embryonic quail hearts from day 6 to day 14 of incubation for EHM study. The experimental results were promising and provided us with an insight view of early heart growth pattern and also paved the road for data-driven heart growth modeling.

  14. 3D confocal imaging in CUBIC-cleared mouse heart

    Energy Technology Data Exchange (ETDEWEB)

    Nehrhoff, I.; Bocancea, D.; Vaquero, J.; Vaquero, J.J.; Lorrio, M.T.; Ripoll, J.; Desco, M.; Gomez-Gaviro, M.V.

    2016-07-01

    Acquiring high resolution 3D images of the heart enables the ability to study heart diseases more in detail. Here, the CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) clearing protocol was adapted for thick mouse heart sections to increase the penetration depth of the confocal microscope lasers into the tissue. The adapted CUBIC clearing of the heart lets the antibody penetrate deeper into the tissue by a factor of five. The here shown protocol enables deep 3D highresolution image acquisition in the heart. This allows a much more accurate assessment of the cellular and structural changes that underlie heart diseases. (Author)

  15. 3D confocal imaging in CUBIC-cleared mouse heart

    International Nuclear Information System (INIS)

    Nehrhoff, I.; Bocancea, D.; Vaquero, J.; Vaquero, J.J.; Lorrio, M.T.; Ripoll, J.; Desco, M.; Gomez-Gaviro, M.V.

    2016-01-01

    Acquiring high resolution 3D images of the heart enables the ability to study heart diseases more in detail. Here, the CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) clearing protocol was adapted for thick mouse heart sections to increase the penetration depth of the confocal microscope lasers into the tissue. The adapted CUBIC clearing of the heart lets the antibody penetrate deeper into the tissue by a factor of five. The here shown protocol enables deep 3D highresolution image acquisition in the heart. This allows a much more accurate assessment of the cellular and structural changes that underlie heart diseases. (Author)

  16. Multi-spectral confocal microendoscope for in-vivo imaging

    Science.gov (United States)

    Rouse, Andrew Robert

    The concept of in-vivo multi-spectral confocal microscopy is introduced. A slit-scanning multi-spectral confocal microendoscope (MCME) was built to demonstrate the technique. The MCME employs a flexible fiber-optic catheter coupled to a custom built slit-scan confocal microscope fitted with a custom built imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The design and performance of the miniature objective and focus assembly are discussed. The 3mm diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope. The confocal nature of the system provides optical sectioning with 3mum lateral resolution and 30mum axial resolution. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible chromatic range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 7nm to 18nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersive power of the prism. The MCME is designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. The primary applications for the system include diagnosis of disease in the gastro-intestinal tract and female reproductive system. Recent data from the grayscale imaging mode are presented. Preliminary multi-spectral results from phantoms, cell cultures, and excised human tissue are presented to demonstrate the potential of in-vivo multi-spectral imaging.

  17. 3D Image Analysis of Geomaterials using Confocal Microscopy

    Science.gov (United States)

    Mulukutla, G.; Proussevitch, A.; Sahagian, D.

    2009-05-01

    Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the

  18. Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    TANG Zhilie; XING Da; LIU Songhao

    2004-01-01

    The imaging theory of nonlinear second harmonic generation (SHG) and third harmonic generation (THG) in confocal microscopy is presented in this paper. The nonlinear effect of SHG and THG on the imaging properties of confocal microscopy has been analyzed in detail by the imaging theory. It is proved that the imaging process of SHG and THG in confocal microscopy, which is different from conventional coherent imaging or incoherent imaging, can be divided into two different processes of coherent imaging. The three-dimensional point spread functions (3D-PSF) of SHG and THG confocal microscopy are derived based on the nonlinear principles of SHG and THG. The imaging properties of SHG and THG confocal microscopy are discussed in detail according to its 3D-PSF. It is shown that the resolution of SHG and THG confocal microscopy is higher than that of single-and two-photon confocal microscopy.

  19. Using Photoshop with images created by a confocal system.

    Science.gov (United States)

    Sedgewick, Jerry

    2014-01-01

    Many pure colors and grayscales tones that result from confocal imaging are not reproducible to output devices, such as printing presses, laptop projectors, and laser jet printers. Part of the difficulty in predicting the colors and tones that will reproduce lies in both the computer display, and in the display of unreproducible colors chosen for fluorophores. The use of a grayscale display for confocal channels and a LUT display to show saturated (clipped) tonal values aids visualization in the former instance and image integrity in the latter. Computer monitors used for post-processing in order to conform the image to the output device can be placed in darkened rooms, and the gamma for the display can be set to create darker shadow regions, and to control the display of color. These conditions aid in visualization of images so that blacks are set to grayer values that are more amenable to faithful reproduction. Preferences can be set in Photoshop for consistent display of colors, along with other settings to optimize use of memory. The Info window is opened so that tonal information can be shown via readouts. Images that are saved as indexed color are converted to grayscale or RGB Color, 16-bit is converted to 8-bit when desired, and colorized images from confocal software is returned to grayscale and re-colorized according to presented methods so that reproducible colors are made. Images may also be sharpened and noise may be reduced, or more than one image layered to show colocalization according to specific methods. Images are then converted to CMYK (Cyan, Magenta, Yellow and Black) for consequent assignment of pigment percentages for printing presses. Changes to single images and multiple images from image stacks are automated for efficient and consistent image processing changes. Some additional changes are done to those images destined for 3D visualization to better separate regions of interest from background. Files are returned to image stacks, saved and

  20. In vitro confocal imaging of the rabbit cornea.

    Science.gov (United States)

    Masters, B R; Paddock, S

    1990-05-01

    We were able to observe in vitro the fine structure of the rabbit cornea using a laser scanning confocal microscope, especially in the regions between Descemet's membrane and the epithelial basal lamina. We observed submicrometre filaments throughout the stroma with high concentrations adjacent to Descemet's membrane, and found extensive interconnecting processes between stromal keratocytes. There are numerous regions containing nerve plexuses in the stroma. We found a deeply convoluted basal lamina adjacent to the epithelium, and observed regions containing junctions between endothelial cells in fluorescent images of rabbit corneas stained with the actin-specific compound fluorescein phalloidin.

  1. Confocal stereology and image analysis: methods for estimating geometrical characteristics of cells and tissues from three-dimensional confocal images

    Czech Academy of Sciences Publication Activity Database

    Kubínová, Lucie; Janáček, Jiří; Karen, Petr; Radochová, Barbora; Difato, Francesco; Krekule, Ivan

    2004-01-01

    Roč. 53, Suppl.1 (2004), s. S47-S55 ISSN 0862-8408 R&D Projects: GA ČR GA304/01/0257; GA ČR GA310/02/1470; GA AV ČR KJB6011309; GA AV ČR KJB5039302 Grant - others:SI - CZ(CZ) KONTAKT 001/2001 Institutional research plan: CEZ:AV0Z5011922 Keywords : confocal microscopy * image analysis * stereology Subject RIV: EA - Cell Biology Impact factor: 1.140, year: 2004

  2. Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment

    Science.gov (United States)

    Nimchuk, Zachary L.; Perdue, Tony D.

    2017-01-01

    Live imaging of above ground meristems can lead to new insights in plant development not possible from static imaging of fixed tissue. The use of an upright confocal microscope offers several technical and biological advantages for live imaging floral or shoot meristems. However, many departments and core facilities possess only inverted confocal microscopes and lack the funding for an additional upright confocal microscope. Here we show that imaging of living apical meristems can be performed on existing inverted confocal microscopes with the use of an affordable and detachable InverterScope accessory. PMID:28579995

  3. Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment.

    Science.gov (United States)

    Nimchuk, Zachary L; Perdue, Tony D

    2017-01-01

    Live imaging of above ground meristems can lead to new insights in plant development not possible from static imaging of fixed tissue. The use of an upright confocal microscope offers several technical and biological advantages for live imaging floral or shoot meristems. However, many departments and core facilities possess only inverted confocal microscopes and lack the funding for an additional upright confocal microscope. Here we show that imaging of living apical meristems can be performed on existing inverted confocal microscopes with the use of an affordable and detachable InverterScope accessory.

  4. Confocal imaging of protein distributions in porous silicon optical structures

    International Nuclear Information System (INIS)

    De Stefano, Luca; D'Auria, Sabato

    2007-01-01

    The performances of porous silicon optical biosensors depend strongly on the arrangement of the biological probes into their sponge-like structures: it is well known that in this case the sensing species do not fill the pores but instead cover their internal surface. In this paper, the direct imaging of labelled proteins into different porous silicon structures by using a confocal laser microscope is reported. The distribution of the biological matter in the nanostructured material follows a Gaussian behaviour which is typical of the diffusion process in the porous media but with substantial differences between a porous silicon monolayer and a multilayer such as a Bragg mirror. Even if semi-quantitative, the results can be very useful in the design of the porous silicon based biosensing devices

  5. Smartphone confocal microscopy for imaging cellular structures in human skin in vivo.

    Science.gov (United States)

    Freeman, Esther E; Semeere, Aggrey; Osman, Hany; Peterson, Gary; Rajadhyaksha, Milind; González, Salvador; Martin, Jeffery N; Anderson, R Rox; Tearney, Guillermo J; Kang, Dongkyun

    2018-04-01

    We report development of a low-cost smartphone confocal microscope and its first demonstration of in vivo human skin imaging. The smartphone confocal microscope uses a slit aperture and diffraction grating to conduct two-dimensional confocal imaging without using any beam scanning devices. Lateral and axial resolutions of the smartphone confocal microscope were measured as 2 and 5 µm, respectively. In vivo confocal images of human skin revealed characteristic cellular structures, including spinous and basal keratinocytes and papillary dermis. Results suggest that the smartphone confocal microscope has a potential to examine cellular details in vivo and may help disease diagnosis in resource-poor settings, where conducting standard histopathologic analysis is challenging.

  6. Comparison of mouse mammary gland imaging techniques and applications: Reflectance confocal microscopy, GFP Imaging, and ultrasound

    International Nuclear Information System (INIS)

    Tilli, Maddalena T; Parrish, Angela R; Cotarla, Ion; Jones, Laundette P; Johnson, Michael D; Furth, Priscilla A

    2008-01-01

    Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary

  7. 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...

  8. 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

  9. Intravital Confocal and Two-photon Imaging of Dual-color Cells and Extracellular Matrix Mimics

    Science.gov (United States)

    Bal, Ufuk; Andresen, Volker; Baggett, Brenda; Utzinger, Urs

    2013-01-01

    To optimize imaging of cells in three dimensional culture we studied confocal backscattering, Second Harmonic Generation (SHG) and autofluorescence as source of contrast in extracellular matrix (ECM) mimics and evaluated the attenuation as well as bleaching of endogenous cellular fluorescence signals. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence while still providing good reflectance to detect voids in the embedding medium. We labeled breast cancer cells’ outline with DsRed2 and nucleus with eGFP. DsRed2 can be excited with confocal imaging at 568nm, and with two photon excitation (TPE) in the red and longer NIR. eGFP was excited at 488nm for confocal and in the NIR for TPE. While there is small difference in the bleaching rate for eGFP between confocal and TPE we observed significant difference for DsRed2 where bleaching is strongest during TPE in the red wavelengths and smallest during confocal imaging. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence becomes twice as strong compared to confocal imaging. PMID:23380006

  10. RELIABILITY OF CONFOCAL MICROSCOPY SPECTRAL IMAGING SYSTEMS: USE OF MULTISPECTRAL BEADS

    Science.gov (United States)

    Background: There is a need for a standardized, impartial calibration, and validation protocol on confocal spectral imaging (CSI) microscope systems. To achieve this goal, it is necessary to have testing tools to provide a reproducible way to evaluate instrument performance. ...

  11. Research and application on imaging technology of line structure light based on confocal microscopy

    Science.gov (United States)

    Han, Wenfeng; Xiao, Zexin; Wang, Xiaofen

    2009-11-01

    In 2005, the theory of line structure light confocal microscopy was put forward firstly in China by Xingyu Gao and Zexin Xiao in the Institute of Opt-mechatronics of Guilin University of Electronic Technology. Though the lateral resolution of line confocal microscopy can only reach or approach the level of the traditional dot confocal microscopy. But compared with traditional dot confocal microscopy, it has two advantages: first, by substituting line scanning for dot scanning, plane imaging only performs one-dimensional scanning, with imaging velocity greatly improved and scanning mechanism simplified, second, transfer quantity of light is greatly improved by substituting detection hairline for detection pinhole, and low illumination CCD is used directly to collect images instead of photoelectric intensifier. In order to apply the line confocal microscopy to practical system, based on the further research on the theory of the line confocal microscopy, imaging technology of line structure light is put forward on condition of implementation of confocal microscopy. Its validity and reliability are also verified by experiments.

  12. Portable oral cancer detection using a miniature confocal imaging probe with a large field of view

    Science.gov (United States)

    Wang, Youmin; Raj, Milan; McGuff, H. Stan; Bhave, Gauri; Yang, Bin; Shen, Ting; Zhang, Xiaojing

    2012-06-01

    We demonstrate a MEMS micromirror enabled handheld confocal imaging probe for portable oral cancer detection, where a comparatively large field of view (FOV) was generated through the programmable Lissajous scanning pattern of the MEMS micromirror. Miniaturized handheld MEMS confocal imaging probe was developed, and further compared with the desktop confocal prototype under clinical setting. For the handheld confocal imaging system, optical design simulations using CODE VR® shows the lateral and axial resolution to be 0.98 µm and 4.2 µm, where experimental values were determined to be 3 µm and 5.8 µm, respectively, with a FOV of 280 µm×300 µm. Fast Lissajous imaging speed up to 2 fps was realized with improved Labview and Java based real-time imaging software. Properties such as 3D imaging through autofocusing and mosaic imaging for extended lateral view (6 mm × 8 mm) were examined for carcinoma real-time pathology. Neoplastic lesion tissues of giant cell fibroma and peripheral ossifying fibroma, the fibroma inside the paraffin box and ex vivo gross tissues were imaged by the bench-top and handheld imaging modalities, and further compared with commercial microscope imaging results. The MEMS scanner-based handheld confocal imaging probe shows great promise as a potential clinical tool for oral cancer diagnosis and treatment.

  13. Portable oral cancer detection using a miniature confocal imaging probe with a large field of view

    International Nuclear Information System (INIS)

    Wang, Youmin; Raj, Milan; Bhave, Gauri; Yang, Bin; Zhang, Xiaojing; McGuff, H. Stan; Shen, Ting

    2012-01-01

    We demonstrate a MEMS micromirror enabled handheld confocal imaging probe for portable oral cancer detection, where a comparatively large field of view (FOV) was generated through the programmable Lissajous scanning pattern of the MEMS micromirror. Miniaturized handheld MEMS confocal imaging probe was developed, and further compared with the desktop confocal prototype under clinical setting. For the handheld confocal imaging system, optical design simulations using CODE V R® shows the lateral and axial resolution to be 0.98 µm and 4.2 µm, where experimental values were determined to be 3 µm and 5.8 µm, respectively, with a FOV of 280 µm×300 µm. Fast Lissajous imaging speed up to 2 fps was realized with improved Labview and Java based real-time imaging software. Properties such as 3D imaging through autofocusing and mosaic imaging for extended lateral view (6 mm × 8 mm) were examined for carcinoma real-time pathology. Neoplastic lesion tissues of giant cell fibroma and peripheral ossifying fibroma, the fibroma inside the paraffin box and ex vivo gross tissues were imaged by the bench-top and handheld imaging modalities, and further compared with commercial microscope imaging results. The MEMS scanner-based handheld confocal imaging probe shows great promise as a potential clinical tool for oral cancer diagnosis and treatment. (paper)

  14. Nonlinear Image Restoration in Confocal Microscopy : Stability under Noise

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.

    1995-01-01

    In this paper we study the noise stability of iterative algorithms developed for attenuation correction in Fluorescence Confocal Microscopy using FT methods. In each iteration the convolution of the previous estimate is computed. It turns out that the estimators are robust to noise perturbation.

  15. Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

    Science.gov (United States)

    Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki

    2013-07-29

    We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

  16. Lateral resolution testing of a novel developed confocal microscopic imaging system

    Science.gov (United States)

    Zhang, Xin; Zhang, Yunhai; Chang, Jian; Huang, Wei; Xue, Xiaojun; Xiao, Yun

    2015-10-01

    Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths 405nm 488nm 561nm and 638nm which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.

  17. Confocal Microscope Alignment of Nanocrystals for Coherent Diffraction Imaging

    International Nuclear Information System (INIS)

    Beitra, Loren; Watari, Moyu; Matsuura, Takashi; Shimamoto, Naonobu; Harder, Ross; Robinson, Ian

    2010-01-01

    We have installed and tested an Olympus LEXT confocal microscope at the 34-ID-C beamline of the Advanced Photon Source (APS). The beamline is for Coherent X-ray Diffraction (CXD) experiments in which a nanometre-sized crystal is aligned inside a focussed X-ray beam. The microscope was required for three-dimensional (3D) sample alignment to get around sphere-of-confusion issues when locating Bragg peaks in reciprocal space. In this way, and by use of strategic sample preparations, we have succeeded in measuring six Bragg peaks from a single 200 nm gold crystal and obtained six projections of its internal displacement field. This enables the clear identification of stacking-fault bands within the crystal. The confocal alignment method will allow a full determination of the strain tensor provided three or more Bragg reflections from the same crystal are found.

  18. 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).

  19. Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

    International Nuclear Information System (INIS)

    Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

    2008-01-01

    Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes

  20. 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.

  1. Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium

    International Nuclear Information System (INIS)

    Saloma, Caesar; Palmes-Saloma, Cynthia; Kondoh, Hisato

    1998-01-01

    Normally transparent biological structures in a turbid medium are imaged using a laser confocal microscope and multiwavelength site-specific fluorescence labelling. The spatial filtering capability of the detector pinhole in the confocal microscope limits the number of scattered fluorescent photons that reach the photodetector. Simultaneous application of different fluorescent markers on the same sample site minimizes photobleaching by reducing the excitation time for each marker. A high-contrast grey-level image is also produced by summing confocal images of the same site taken at different fluorescence wavelengths. Monte Carlo simulations are performed to obtain the quantitative behaviour of confocal fluorescence imaging in turbid media. Confocal images of the following samples were also obtained: (i) 15 μm diameter fluorescent spheres placed 1.16 mm deep beneath an aqueous suspension of 0.0823 μm diameter polystyrene latex spheres, and (ii) hindbrain of a whole-mount mouse embryo (age 10 days) that was stained to fluoresce at 515 nm and 580 nm peak wavelengths. Expression of RNA transcripts of a gene within the embryo hindbrain was detected by a fluorescence-based whole-mount in situ hybridization procedure that we recently tested. (author)

  2. Confocal scanning microscopy with multiple optical probes for high speed measurements and better imaging

    Science.gov (United States)

    Chun, Wanhee; Lee, SeungWoo; Gweon, Dae-Gab

    2008-02-01

    Confocal scanning microscopy (CSM) needs a scanning mechanism because only one point information of specimen can be obtained. Therefore the speed of the confocal scanning microscopy is limited by the speed of the scanning tool. To overcome this limitation from scanning tool we propose another scanning mechanism. We make three optical probes in the specimen under confocal condition of each point. Three optical probes are moved by beam scanning mechanism with shared resonant scanning mirror (RM) and galvanometer driven mirror (GM). As each optical probe scan allocated region of the specimen, information from three points is obtained simultaneously and image acquisition time is reduced. Therefore confocal scanning microscopy with multiple optical probes is expected to have three times faster speed of the image acquisition than conventional one. And as another use, multiple optical probes to which different light wavelength is applied can scan whole same region respectively. It helps to obtain better contrast image in case of specimens having different optical characteristics for specific light wavelength. In conclusion confocal scanning microscopy with multiple optical probes is useful technique for views of image acquisition speed and image quality.

  3. Cellular features of psoriatic skin: imaging and quantification using in vivo reflectance confocal microscopy

    NARCIS (Netherlands)

    Wolberink, E.A.W.; Erp, P.E.J. van; Teussink, M.M.; Kerkhof, P.C.M. van de; Gerritsen, M.J.P.

    2011-01-01

    BACKGROUND: In vivo reflectance confocal microscopy (RCM) is a novel, exciting imaging technique. It provides images of cell-and tissue structures and dynamics in situ, in real time, without the need for ex vivo tissue samples. RCM visualizes the superficial part of human skin up to a depth of 250

  4. A method for analysis of lipid vesicle domain structure from confocal image data

    DEFF Research Database (Denmark)

    Husen, Peter Rasmussen; Fidorra, Matthias; Hartel, Steffen

    2012-01-01

    Quantitative characterization of the lateral structure of curved membranes based on fluorescence microscopy requires knowledge of the fluorophore distribution on the surface. We present an image analysis approach for extraction of the fluorophore distribution on a spherical lipid vesicle from...... confocal imaging stacks. The technique involves projection of volumetric image data onto a triangulated surface mesh representation of the membrane, correction of photoselection effects and global motion of the vesicle during image acquisition and segmentation of the surface into domains using histograms...

  5. 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.

  6. Three-dimensional imaging of porous media using confocal laser scanning microscopy.

    Science.gov (United States)

    Shah, S M; Crawshaw, J P; Boek, E S

    2017-02-01

    In the last decade, imaging techniques capable of reconstructing three-dimensional (3-D) pore-scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO 2 storage potential. CLSM has a unique capability of producing 3-D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z-dimension) that can be imaged in porous materials. In this study, we introduce a 'grind and slice' technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3-D confocal volumetric data into pores and grains. Finally, we use the resulting 3-D pore-scale binarized confocal data obtained to quantitatively determine petrophysical pore-scale properties such as total porosity, macro- and microporosity and single-phase permeability using lattice Boltzmann (LB) simulations, validated by experiments. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  7. Effects of Glycosylation on Biodistribution and Imaging Quality of Necrotic Myocardium of Iodine-131-Labeled Sennidins.

    Science.gov (United States)

    Li, Ling; Zhang, Dongjian; Yang, Shengwei; Song, Shaoli; Li, Jindian; Wang, Qin; Wang, Cong; Feng, Yuanbo; Ni, Yicheng; Zhang, Jian; Liu, Wei; Yin, Zhiqi

    2016-12-01

    Sennidins are necrosis-avid agents for noninvasive assessment of myocardial viability which is important for patients with myocardial infarction (MI). However, high accumulation of radioactivity in the liver interferes with the assessment of myocardial viability. In this study, we compared sennidins with sennosides to investigate the effects of glycosylation on biodistribution and imaging quality of sennidins. Sennidin A (SA), sennidin B (SB), sennoside A (SSA), and sennoside B (SSB) were labeled with I-131. In vitro binding to necrotic cells and hepatic cells and in vivo biodistribution in rats with muscular necrosis were evaluated by gamma counting, autoradiography, and histopathology. Single photon emission computed tomography/computed tomography (SPECT/CT) images were acquired in rats with acute MI. The uptake of [ 131 I]SA, [ 131 I]SSA, [ 131 I]SB, and [ 131 I]SSB in necrotic cells was significantly higher than that in viable cells (p sennosides than those with [ 131 I]sennidins (p < 0.01). Autoradiography showed preferential accumulation of these four radiotracers in necrotic areas of muscle, confirmed by histopathology. SPECT/CT imaging studies showed better image quality with [ 131 I]SSB than with [ 131 I]SB due to less liver interference. Glycosylation significantly decreased the liver uptake and improved the quality of cardiac imaging. [ 131 I]SSB may serve as a promising necrosis-avid agent for noninvasive assessment of myocardial viability.

  8. Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells.

    Science.gov (United States)

    Okuno, Masanari; Hamaguchi, Hiro-o

    2010-12-15

    We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

  9. Diagnostic accuracy of confocal microscopy imaging vs. punch biopsy for diagnosing and subtyping basal cell carcinoma

    NARCIS (Netherlands)

    Kadouch, D. J.; Leeflang, M. M.; Elshot, Y. S.; Longo, C.; Ulrich, M.; van der Wal, A. C.; Wolkerstorfer, A.; Bekkenk, M. W.; de Rie, M. A.

    2017-01-01

    BackgroundIn vivo reflectance confocal microscopy (RCM) is a promising non-invasive skin imaging technique that could facilitate early diagnosis of basal cell carcinoma (BCC) instead of routine punch biopsies. However, the clinical value and utility of RCM vs. a punch biopsy in diagnosing and

  10. Super-resolution for everybody: An image processing workflow to obtain high-resolution images with a standard confocal microscope.

    Science.gov (United States)

    Lam, France; Cladière, Damien; Guillaume, Cyndélia; Wassmann, Katja; Bolte, Susanne

    2017-02-15

    In the presented work we aimed at improving confocal imaging to obtain highest possible resolution in thick biological samples, such as the mouse oocyte. We therefore developed an image processing workflow that allows improving the lateral and axial resolution of a standard confocal microscope. Our workflow comprises refractive index matching, the optimization of microscope hardware parameters and image restoration by deconvolution. We compare two different deconvolution algorithms, evaluate the necessity of denoising and establish the optimal image restoration procedure. We validate our workflow by imaging sub resolution fluorescent beads and measuring the maximum lateral and axial resolution of the confocal system. Subsequently, we apply the parameters to the imaging and data restoration of fluorescently labelled meiotic spindles of mouse oocytes. We measure a resolution increase of approximately 2-fold in the lateral and 3-fold in the axial direction throughout a depth of 60μm. This demonstrates that with our optimized workflow we reach a resolution that is comparable to 3D-SIM-imaging, but with better depth penetration for confocal images of beads and the biological sample. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

    Science.gov (United States)

    Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

    2016-07-01

    We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

  12. Tissue clearing for confocal imaging of native and bio-artificial skeletal muscle.

    Science.gov (United States)

    Decroix, L; Van Muylder, V; Desender, L; Sampaolesi, M; Thorrez, L

    2015-01-01

    Novel clearing techniques have revolutionized three-dimensional confocal imaging of the brain without the need for physical tissue sectioning. We evaluated three clearing methods, ScaleA2, Clear(T2), and 3DISCO for visualizing native and tissue engineered muscle by confocal microscopy. We found that Clear(T2) treatment improved the depth of visualization of immunohistochemical staining slightly, but did not improve depth of visualization of endogenous green fluorescent protein (GFP). ScaleA2 preserved endogenous GFP signal better and permitted significantly deeper GFP imaging, but it was incompatible with tropomyosin immunohistochemical staining. 3DISCO treatment preserved both endogenous GFP and immunohistochemical staining, and permitted significantly deeper imaging. Clearing time for the 3DISCO procedure is short compared to ScaleA2 and Clear(T2). We suggest that 3DISCO is the preferable clearing method for native and tissue engineered skeletal muscle tissue.

  13. Confocal pore size measurement based on super-resolution image restoration.

    Science.gov (United States)

    Liu, Dali; Wang, Yun; Qiu, Lirong; Mao, Xinyue; Zhao, Weiqian

    2014-09-01

    A confocal pore size measurement based on super-resolution image restoration is proposed to obtain a fast and accurate measurement for submicrometer pore size of nuclear track-etched membranes (NTEMs). This method facilitates the online inspection of the pore size evolution during etching. Combining confocal microscopy with super-resolution image restoration significantly improves the lateral resolution of the NTEM image, yields a reasonable circle edge-setting criterion of 0.2408, and achieves precise pore edge detection. Theoretical analysis shows that the minimum measuring diameter can reach 0.19 μm, and the root mean square of the residuals is only 1.4 nm. Edge response simulation and experiment reveal that the edge response of the proposed method is better than 80 nm. The NTEM pore size measurement results obtained by the proposed method agree well with that obtained by scanning electron microscopy.

  14. 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.

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

    Science.gov (United States)

    Todorova, Biliana; Salabert, Nina; Tricot, Sabine; Boisgard, Raphaël; Rathaux, Mélanie; Le Grand, Roger; Chapon, Catherine

    2017-01-01

    We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. 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). 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. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

  16. Confocal non-line-of-sight imaging based on the light-cone transform

    Science.gov (United States)

    O’Toole, Matthew; Lindell, David B.; Wetzstein, Gordon

    2018-03-01

    How to image objects that are hidden from a camera’s view is a problem of fundamental importance to many fields of research, with applications in robotic vision, defence, remote sensing, medical imaging and autonomous vehicles. Non-line-of-sight (NLOS) imaging at macroscopic scales has been demonstrated by scanning a visible surface with a pulsed laser and a time-resolved detector. Whereas light detection and ranging (LIDAR) systems use such measurements to recover the shape of visible objects from direct reflections, NLOS imaging reconstructs the shape and albedo of hidden objects from multiply scattered light. Despite recent advances, NLOS imaging has remained impractical owing to the prohibitive memory and processing requirements of existing reconstruction algorithms, and the extremely weak signal of multiply scattered light. Here we show that a confocal scanning procedure can address these challenges by facilitating the derivation of the light-cone transform to solve the NLOS reconstruction problem. This method requires much smaller computational and memory resources than previous reconstruction methods do and images hidden objects at unprecedented resolution. Confocal scanning also provides a sizeable increase in signal and range when imaging retroreflective objects. We quantify the resolution bounds of NLOS imaging, demonstrate its potential for real-time tracking and derive efficient algorithms that incorporate image priors and a physically accurate noise model. Additionally, we describe successful outdoor experiments of NLOS imaging under indirect sunlight.

  17. Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

    Science.gov (United States)

    Nyström, Sofie; Bäck, Marcus; Nilsson, K Peter R; Hammarström, Per

    2017-10-20

    Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

  18. Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

    Science.gov (United States)

    Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

    2015-01-27

    Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.

  19. Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

    Science.gov (United States)

    Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

    2012-10-17

    There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. 3-D reconstruction of neurons from multichannel confocal laser scanning image series.

    Science.gov (United States)

    Wouterlood, Floris G

    2014-04-10

    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 to 3-D reconstruct structures of interest. The operator first configures separate channels (e.g., laser, filters, and detector settings) for each applied fluorochrome and then acquires Z-series of confocal images: one series per channel. Channel signal separation is extremely important. Measures to avoid bleaching are vital. Post-acquisition deconvolution of the image series is often performed to increase resolution before 3-D reconstruction takes place. In the 3-D reconstruction programs described in this unit, reconstructions can be inspected in real time from any viewing angle. By altering viewing angles and by switching channels off and on, the spatial relationships of 3-D-reconstructed structures with respect to structures visualized in other channels can be studied. Since each brand of CLSM, computer program, and 3-D reconstruction package has its own proprietary set of procedures, a general approach is provided in this protocol wherever possible. Copyright © 2014 John Wiley & Sons, Inc.

  1. A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy.

    Science.gov (United States)

    Verveer, P. J; Gemkow, M. J; Jovin, T. M

    1999-01-01

    We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide-field data. In general, restored confocal data are superior to restored wide-field data, but given sufficient higher signal level for the wide-field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide-field data is presented.

  2. Chromatic confocal microscopy for multi-depth imaging of epithelial tissue

    Science.gov (United States)

    Olsovsky, Cory; Shelton, Ryan; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

    2013-01-01

    We present a novel chromatic confocal microscope capable of volumetric reflectance imaging of microstructure in non-transparent tissue. Our design takes advantage of the chromatic aberration of aspheric lenses that are otherwise well corrected. Strong chromatic aberration, generated by multiple aspheres, longitudinally disperses supercontinuum light onto the sample. The backscattered light detected with a spectrometer is therefore wavelength encoded and each spectrum corresponds to a line image. This approach obviates the need for traditional axial mechanical scanning techniques that are difficult to implement for endoscopy and susceptible to motion artifact. A wavelength range of 590-775 nm yielded a >150 µm imaging depth with ~3 µm axial resolution. The system was further demonstrated by capturing volumetric images of buccal mucosa. We believe these represent the first microstructural images in non-transparent biological tissue using chromatic confocal microscopy that exhibit long imaging depth while maintaining acceptable resolution for resolving cell morphology. Miniaturization of this optical system could bring enhanced speed and accuracy to endomicroscopic in vivo volumetric imaging of epithelial tissue. PMID:23667789

  3. Spectral imaging technique for retinal perfusion detection using confocal scanning laser ophthalmoscopy

    Science.gov (United States)

    Rasta, Seyed Hossein; Manivannan, Ayyakkannu; Sharp, Peter F.

    2012-11-01

    To evaluate retinal perfusion in the human eye, a dual-wavelength confocal scanning laser ophthalmoscope (cSLO) was developed that provides spectral imaging of the fundus using a combination of red (670 nm) and near-infrared (810 nm) wavelengths. The image of the ocular fundus was analyzed to find out if quantitative measurements of the reflectivity of tissue permit assessment of the oxygen perfusion of tissue. We explored problems that affect the reproducibility of patient measurements such as non-uniformity errors on the image. For the first time, an image processing technique was designed and used to minimize the errors of oxygen saturation measurements by illumination correction in retina wide field by increasing SNR. Retinal images were taken from healthy and diabetic retinopathy eyes using the cSLO with a confocal aperture of 100 μm. The ratio image (RI) of red/IR, as oxygen saturation (SO2) index, was calculated for normal eyes. The image correction technique improved the reproducibility of the measurements. Average RI intensity variation of healthy retina tissue was determined within a range of about 5.5%. The capability of the new technique to discriminate oxygenation levels of retinal artery and vein was successfully demonstrated and showed good promise in the diagnosis of the perfused retina.

  4. 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.

  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...... scanning microscopy images can be used to provide information on the protein microstructure in yogurt products. For large numbers of microscopy images, subjective evaluation becomes a difficult or even impossible approach, if the images should be incorporated in any form of statistical analysis alongside...

  6. Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens.

    Science.gov (United States)

    Minker, Katharine R; Biedrzycki, Meredith L; Kolagunda, Abhishek; Rhein, Stephen; Perina, Fabiano J; Jacobs, Samuel S; Moore, Michael; Jamann, Tiffany M; Yang, Qin; Nelson, Rebecca; Balint-Kurti, Peter; Kambhamettu, Chandra; Wisser, Randall J; Caplan, Jeffrey L

    2018-02-01

    The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. High Definition Confocal Imaging Modalities for the Characterization of Tissue-Engineered Substitutes.

    Science.gov (United States)

    Mayrand, Dominique; Fradette, Julie

    2018-01-01

    Optimal imaging methods are necessary in order to perform a detailed characterization of thick tissue samples from either native or engineered tissues. Tissue-engineered substitutes are featuring increasing complexity including multiple cell types and capillary-like networks. Therefore, technical approaches allowing the visualization of the inner structural organization and cellular composition of tissues are needed. This chapter describes an optical clearing technique which facilitates the detailed characterization of whole-mount samples from skin and adipose tissues (ex vivo tissues and in vitro tissue-engineered substitutes) when combined with spectral confocal microscopy and quantitative analysis on image renderings.

  8. Imaging rat esophagus using combination of reflectance confocal and multiphoton microscopy

    International Nuclear Information System (INIS)

    Zhuo, S M; Chen, J X; Jiang, X S; Lu, K C; Xie, S S

    2008-01-01

    We combine reflectance confocal microscopy (RCM) with multiphoton microscopy (MPM) to image rat esophagus. The two imaging modalities allow detection of layered–resolved complementary information from esophagus. In the keratinizing layer, the keratinocytes boundaries can be characterized by RCM, while the keratinocytes cytoplasm (keratin) can be further imaged by multiphoton autofluorescence signal. In the epithelium, the epithelial cellular boundaries and nucleus can be detected by RCM, and MPM can be used for imaging epithelial cell cytoplasm and monitoring metabolic state of epithelium. In the stroma, multiphoton autofluorescence signal is used to image elastin and second harmonic generation signal is utilized to detect collagen, while RCM is used to determine the optical property of stroma. Overall, these results suggest that the combination of RCM and MPM has potential to provide more important and comprehensive information for early diagnosis of esophageal cancer

  9. A principal skeleton algorithm for standardizing confocal images of fruit fly nervous systems

    Science.gov (United States)

    Qu, Lei; Peng, Hanchuan

    2010-01-01

    Motivation: The fruit fly (Drosophila melanogaster) is a commonly used model organism in biology. We are currently building a 3D digital atlas of the fruit fly larval nervous system (LNS) based on a large collection of fly larva GAL4 lines, each of which targets a subset of neurons. To achieve such a goal, we need to automatically align a number of high-resolution confocal image stacks of these GAL4 lines. One commonly employed strategy in image pattern registration is to first globally align images using an affine transform, followed by local non-linear warping. Unfortunately, the spatially articulated and often twisted LNS makes it difficult to globally align the images directly using the affine method. In a parallel project to build a 3D digital map of the adult fly ventral nerve cord (VNC), we are confronted with a similar problem. Results: We proposed to standardize a larval image by best aligning its principal skeleton (PS), and thus used this method as an alternative of the usually considered affine alignment. The PS of a shape was defined as a series of connected polylines that spans the entire shape as broadly as possible, but with the shortest overall length. We developed an automatic PS detection algorithm to robustly detect the PS from an image. Then for a pair of larval images, we designed an automatic image registration method to align their PSs and the entire images simultaneously. Our experimental results on both simulated images and real datasets showed that our method does not only produce satisfactory results for real confocal larval images, but also perform robustly and consistently when there is a lot of noise in the data. We also applied this method successfully to confocal images of some other patterns such as the adult fruit fly VNC and center brain, which have more complicated PS. This demonstrates the flexibility and extensibility of our method. Availability: The supplementary movies, full size figures, test data, software, and tutorial on

  10. Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications.

    Science.gov (United States)

    Sfakis, Lauren; Kamaldinov, Tim; Larsen, Melinda; Castracane, James; Khmaladze, Alexander

    2016-11-01

    Quantifying confocal images to enable location of specific proteins of interest in three-dimensional (3D) is important for many tissue engineering (TE) applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in TE and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often the key to evaluating scaffold efficacy. Functional epithelial cell monolayers must be organized with apicobasal polarity with proteins specifically localized to the apical or basolateral regions of cells in many organs. In this work, a customized program was developed using the LabVIEW platform to quantify protein positions in Z-stacks of confocal images of epithelial cell monolayers. The program's functionality is demonstrated through salivary gland TE, since functional salivary epithelial cells must correctly orient many proteins on the apical and basolateral membranes. Bio-LabVIEW Image Matrix Evaluation (Bio-LIME) takes 3D information collected from confocal Z-stack images and processes the fluorescence at each pixel to determine cell heights, nuclei heights, nuclei widths, protein localization, and cell count. As a demonstration of its utility, Bio-LIME was used to quantify the 3D location of the Zonula occludens-1 protein contained within tight junctions and its change in 3D position in response to chemical modification of the scaffold with laminin. Additionally, Bio-LIME was used to demonstrate that there is no advantage of sub-100 nm poly lactic-co-glycolic acid nanofibers over 250 nm fibers for epithelial apicobasal polarization. Bio-LIME will be broadly applicable for quantification of proteins in 3D that are grown in many different contexts.

  11. High resolution 3D confocal microscope imaging of volcanic ash particles.

    Science.gov (United States)

    Wertheim, David; Gillmore, Gavin; Gill, Ian; Petford, Nick

    2017-07-15

    We present initial results from a novel high resolution confocal microscopy study of the 3D surface structure of volcanic ash particles from two recent explosive basaltic eruptions, Eyjafjallajökull (2010) and Grimsvötn (2011), in Iceland. The majority of particles imaged are less than 100μm in size and include PM 10 s, known to be harmful to humans if inhaled. Previous studies have mainly used 2D microscopy to examine volcanic particles. The aim of this study was to test the potential of 3D laser scanning confocal microscopy as a reliable analysis tool for these materials and if so to what degree high resolution surface and volume data could be obtained that would further aid in their classification. First results obtained using an Olympus LEXT scanning confocal microscope with a ×50 and ×100 objective lens are highly encouraging. They reveal a range of discrete particle types characterised by sharp or concave edges consistent with explosive formation and sudden rupture of magma. Initial surface area/volume ratios are given that may prove useful in subsequent modelling of damage to aircraft engines and human tissue where inhalation has occurred. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Characterization of LiF-based soft X-ray imaging detectors by confocal fluorescence microscopy

    International Nuclear Information System (INIS)

    Bonfigli, F; Gaudio, P; Lupelli, I; Nichelatti, E; Richetta, M; Vincenti, M A; Montereali, R M

    2010-01-01

    X-ray microscopy represents a powerful tool to obtain images of samples with very high spatial resolution. The main limitation of this technique is represented by the poor spatial resolution of standard imaging detectors. We proposed an innovative high-performance X-ray imaging detector based on the visible photoluminescence of colour centres in lithium fluoride. In this work, a confocal microscope in fluorescence mode was used to characterize LiF-based imaging detectors measuring CC integrated visible fluorescence signals of LiF crystals and films (grown on several kinds of substrates) irradiated by soft X-rays produced by a laser plasma source in different exposure conditions. The results are compared with the CC photoluminescence spectra measured on the same samples and discussed.

  13. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy.

    Science.gov (United States)

    Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H; Wouters, Fred S; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

    2013-12-24

    We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

  14. 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®

  15. Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy

    Czech Academy of Sciences Publication Activity Database

    Michálek, Jan; Čapek, Martin; Kubínová, Lucie

    2011-01-01

    Roč. 74, č. 9 (2011), s. 831-838 ISSN 1059-910X R&D Projects: GA ČR(CZ) GA102/08/0691; GA ČR(CZ) GA304/09/0733; GA MŠk(CZ) LC06063; GA MŠk(CZ) ME09010 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal laser scanning microscopy * image enhancement * morphology filters Subject RIV: JC - Computer Hardware ; Software Impact factor: 1.792, year: 2011

  16. Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue.

    Science.gov (United States)

    Longo, C; Rajadhyaksha, M; Ragazzi, M; Nehal, K; Gardini, S; Moscarella, E; Lallas, A; Zalaudek, I; Piana, S; Argenziano, G; Pellacani, G

    2014-09-01

    Fluorescence confocal microscopy (FCM) is an emerging technology for rapid imaging of excised tissue, without the need for frozen- or fixed-section processing. Basal cell carcinomas (BCCs) can be detected in Mohs excisions although few studies have described the major BCC findings as seen on FCM. To describe the major BCC findings of excised tissue during Mohs surgery and to correlate them with histopathology. Freshly excised tumours and frozen-thawed discarded tissue of BCC during Mohs surgery were analysed by means of FCM. A side-by-side correlation between FCM images and histological sections was performed. The FCM features of overlying skin and adnexal structures were also described. Sixty-four BCC cases were analysed. Distinct BCC types appeared unique in terms of shape and size of tumour islands [bigger in nodular (18/25), smaller and rounded in micronodular (7/7) and tiny cords for infiltrative ones (24/30)] and for the presence of clefting, palisading and increased nucleus/cytoplasm ratio. An excellent correlation was found between FCM and histological findings (Cohen's κ statistics = 0·9). In six cases, the presence of sebaceous glands and intense stroma reaction represented possible confounders. Fluorescence confocal microscopy is a fast and new imaging technique that allows an excellent visualization of skin structures and BCC findings during Mohs surgery. © 2014 British Association of Dermatologists.

  17. 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.

  18. Confocal microscopy and imaging profilometry: A new tool aimed to evaluate aesthetic procedures.

    Science.gov (United States)

    Fabbrocini, Gabriella; Mazzella, Caterina; Montagnaro, Fabio; De Padova, Maria Pia; Lorenzi, Sandra; Tedeschi, Aurora; Forgione, Patrizia; Capasso, Claudia; Sivero, Luigi; Velotti, Carla; Russo, Daniela; Vitiello, Rosa; Ilardi, Gennaro

    2017-02-01

    According to the American Academy of Aesthetic Plastic Surgeons, more than 11 million cosmetic surgical and nonsurgical procedures were performed by board-certified plastic surgeons, dermatologists and otolaryngologists in the United States, totaling more than 12 billion dollars. We performed a retrospective observational multi-centric study on patients treated with a non-animal origin cross-linked hyaluronic acid with different molecular weights for nasolabial folds, evaluating through a new imaging system, profilometric techniques with the confocal microscopy, the durability, the efficacy and the safety of this product. From 25 patients, 150 silicone casts were obtained: 75 casts of the right nasolabial fold and 75 casts of the left nasolabial fold. Roughness arithmetical average of the right fold at T2 decreased by 50% versus T0 and by 40% compared to T1; at T2, it decreased by the 45% versus T0 and by 35% compared to T1. No side effects were reported. Results proved that the analysis of the skin microreliefs through confocal microscopy is a new imaging system that allows to evaluate with precision and safety the results of aesthetic treatments such as fillers objectively.

  19. Automatic segmentation of cell nuclei from confocal laser scanning microscopy images

    International Nuclear Information System (INIS)

    Kelemen, A.; Reist, H.W.

    1997-01-01

    A newly developed experimental method combines the possibility of irradiating more than a thousand cells simultaneous with an efficient colony-forming ability and with the capability of localizing a particle track through a cell nucleus together with the assessment of the energy transfer by digital superposition of the image containing the track with that of the cells. To assess the amount of energy deposition by particles traversing the cell nucleus the intersection lengths of the particle tracks have to be known. Intersection lengths can be obtained by determining the 3D surface contours of the irradiated cell nuclei. Confocal laser scanning microscopy using specific DNA fluorescent dye offers a possible way for the determination of the 3D shape of individual nuclei. Unfortunately, such experiments cannot be performed on living cells. One solution to this problem can be provided by building a statistical model of the shape of the nuclei of the exposed cells. In order to build such a statistical model, a large number of cell nuclei have to be identified and segmented from confocal laser scanning microscopy images. The present paper describes a method to perform this 3D segmentation in an automatic manner in order to create a solid basis for the statistical model. (author) 2 figs., 4 refs

  20. 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

  1. 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.

  2. Dual-model automatic detection of nerve-fibres in corneal confocal microscopy images.

    Science.gov (United States)

    Dabbah, M A; Graham, J; Petropoulos, I; Tavakoli, M; Malik, R A

    2010-01-01

    Corneal Confocal Microscopy (CCM) imaging is a non-invasive surrogate of detecting, quantifying and monitoring diabetic peripheral neuropathy. This paper presents an automated method for detecting nerve-fibres from CCM images using a dual-model detection algorithm and compares the performance to well-established texture and feature detection methods. The algorithm comprises two separate models, one for the background and another for the foreground (nerve-fibres), which work interactively. Our evaluation shows significant improvement (p approximately 0) in both error rate and signal-to-noise ratio of this model over the competitor methods. The automatic method is also evaluated in comparison with manual ground truth analysis in assessing diabetic neuropathy on the basis of nerve-fibre length, and shows a strong correlation (r = 0.92). Both analyses significantly separate diabetic patients from control subjects (p approximately 0).

  3. Imaging phospholipid conformational disorder and packing in giant multilamellar liposome by confocal Raman microspectroscopy

    Science.gov (United States)

    Noothalapati, Hemanth; Iwasaki, Keita; Yoshimoto, Chikako; Yoshikiyo, Keisuke; Nishikawa, Tomoe; Ando, Masahiro; Hamaguchi, Hiro-o.; Yamamoto, Tatsuyuki

    2017-12-01

    Liposomes are closed phospholipid bilayer systems that have profound applications in fundamental cell biology, pharmaceutics and medicine. Depending on the composition (pure or mixture of phospholipids, presence of cholesterol) and preparation protocol, intra- and inter-chain molecular interactions vary leading to changes in the quality (order and packing) of liposomes. So far it is not possible to image conformational disorders and packing densities within a liposome in a straightforward manner. In this study, we utilized confocal Raman microspectroscopy to visualize structural disorders and packing efficiency within a giant multilamellar liposome model by focusing mainly on three regions in the vibrational spectrum (Csbnd C stretching, Csbnd H deformation and Csbnd H stretching). We estimated properties such as trans/gauche isomers and lateral packing probability. Interestingly, our Raman imaging studies revealed gel phase rich domains and heterogeneous lateral packing within the giant multilamellar liposome.

  4. Statistical region based active contour using a fractional entropy descriptor: Application to nuclei cell segmentation in confocal \\ud microscopy images

    OpenAIRE

    Histace, A; Meziou, B J; Matuszewski, Bogdan; Precioso, F; Murphy, M F; Carreiras, F

    2013-01-01

    We propose an unsupervised statistical region based active contour approach integrating an original fractional entropy measure for image segmentation with a particular application to single channel actin tagged fluorescence confocal microscopy image segmentation. Following description of statistical based active contour segmentation and the mathematical definition of the proposed fractional entropy descriptor, we demonstrate comparative segmentation results between the proposed approach and s...

  5. Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system

    Science.gov (United States)

    Kelner, Roy; Katz, Barak; Rosen, Joseph

    2015-01-01

    We propose a new type of confocal microscope using Fresnel incoherent correlation holography (FINCH). Presented here is a confocal configuration of FINCH using a phase pinhole and point illumination that is able to suppress out-of-focus information from the recorded hologram and hence combine the super-resolution capabilities of FINCH with the sectioning capabilities of confocal microscopy. PMID:26413560

  6. Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system

    OpenAIRE

    Kelner, Roy; Katz, Barak; Rosen, Joseph

    2014-01-01

    We propose a new type of confocal microscope using Fresnel incoherent correlation holography (FINCH). Presented here is a confocal configuration of FINCH using a phase pinhole and point illumination that is able to suppress out-of-focus information from the recorded hologram and hence combine the super-resolution capabilities of FINCH with the sectioning capabilities of confocal microscopy.

  7. Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke.

    Science.gov (United States)

    Talley Watts, Lora; Zheng, Wei; Garling, R Justin; Frohlich, Victoria C; Lechleiter, James Donald

    2015-06-23

    In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

  8. Virtual pinhole confocal microscope

    Energy Technology Data Exchange (ETDEWEB)

    George, J.S.; Rector, D.M.; Ranken, D.M. [Los Alamos National Lab., NM (United States). Biophysics Group; Peterson, B. [SciLearn Inc. (United States); Kesteron, J. [VayTech Inc. (United States)

    1999-06-01

    Scanned confocal microscopes enhance imaging capabilities, providing improved contrast and image resolution in 3-D, but existing systems have significant technical shortcomings and are expensive. Researchers at Los Alamos National Laboratory have developed a novel approach--virtual pinhole confocal microscopy--that uses state of the art illumination, detection, and data processing technologies to produce an imager with a number of advantages: reduced cost, faster imaging, improved efficiency and sensitivity, improved reliability and much greater flexibility. Work at Los Alamos demonstrated proof of principle; prototype hardware and software have been used to demonstrate technical feasibility of several implementation strategies. The system uses high performance illumination, patterned in time and space. The authors have built functional confocal imagers using video display technologies (LCD or DLP) and novel scanner based on a micro-lens array. They have developed a prototype system for high performance data acquisition and processing, designed to support realtime confocal imaging. They have developed algorithms to reconstruct confocal images from a time series of spatially sub-sampled images; software development remains an area of active development. These advances allow the collection of high quality confocal images (in fluorescence, reflectance and transmission modes) with equipment that can inexpensively retrofit to existing microscopes. Planned future extensions to these technologies will significantly enhance capabilities for microscopic imaging in a variety of applications, including confocal endoscopy, and confocal spectral imaging.

  9. Glycosylation Engineering

    DEFF Research Database (Denmark)

    Clausen, Henrik; Wandall, Hans H.; Steentoft, Catharina

    2017-01-01

    Knowledge of the cellular pathways of glycosylation across phylogeny provides opportunities for designing glycans via genetic engineering in a wide variety of cell types including bacteria, fungi, plant cells, and mammalian cells. The commercial demand for glycosylation engineering is broad......, including production of biological therapeutics with defined glycosylation (Chapter 57). This chapter describes how knowledge of glycan structures and their metabolism (Parts I–III of this book) has led to the current state of glycosylation engineering in different cell types. Perspectives for rapid...

  10. Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

    Science.gov (United States)

    Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

    2012-12-01

    Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.

  11. Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

    Science.gov (United States)

    Diaspro, A; Corosu, M; Ramoino, P; Robello, M

    1999-11-01

    Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode. Copyright 1999 Wiley-Liss, Inc.

  12. Electrophoretic Detection and Confocal Microscopic Imaging of Tyrosine Nitrated Proteins in Plant Tissue.

    Science.gov (United States)

    Arora, Dhara; Singh, Neha; Bhatla, Satish C

    2018-01-01

    Tyrosine nitrated proteins can be detected in plant cells electrophoretically and their distribution can be monitored by confocal laser scanning microscopy (CLSM) imaging. One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by Western blotting using polyclonal antibody against 3-nitrotyrosine residues enables detection of tyrosine nitrated proteins in plant cells. Here we describe detection of tyrosine nitrated proteins in the homogenates derived from sunflower (Helianthus annuus L.) seedling cotyledons. Total soluble proteins obtained from tissue homogenates are resolved using vertical gel electrophoresis followed by their electrophoretic transfer on to a microporous membrane support for immunodetection. Spatial distribution of tyrosine nitrated proteins can be visualized using an antibody against 3-nitrotyrosine residues. Immunofluorescent localization is performed by cutting 7 μm thick wax sections of tissue followed by incubation in primary anti-nitrotyrosine antibody (dilution 1:200) and secondary Cy-3 labeled anti-rabbit IgG antibody (dilution 1:1500). Confocal laser scanning microscopy analysis is undertaken using argon lasers (ex: 530-550 nm and em: 570 nm) at pinhole 1. Modulation in the abundance and spatial localization of tyrosine nitrated proteins in plant tissues can be monitored using these techniques.

  13. Confocal laser endomicroscopy for diagnosis and histomorphologic imaging of brain tumors in vivo.

    Directory of Open Access Journals (Sweden)

    Sebastian Foersch

    Full Text Available Early detection and evaluation of brain tumors during surgery is crucial for accurate resection. Currently cryosections during surgery are regularly performed. Confocal laser endomicroscopy (CLE is a novel technique permitting in vivo histologic imaging with miniaturized endoscopic probes at excellent resolution. Aim of the current study was to evaluate CLE for in vivo diagnosis in different types and models of intracranial neoplasia. In vivo histomorphology of healthy brains and two different C6 glioma cell line allografts was evaluated in rats. One cell line expressed EYFP, the other cell line was used for staining with fluorescent dyes (fluorescein, acriflavine, FITC-dextran and Indocyanine green. To evaluate future application in patients, fresh surgical resection specimen of human intracranial tumors (n = 15 were examined (glioblastoma multiforme, meningioma, craniopharyngioma, acoustic neurinoma, brain metastasis, medulloblastoma, epidermoid tumor. Healthy brain tissue adjacent to the samples served as control. CLE yielded high-quality histomorphology of normal brain tissue and tumors. Different fluorescent agents revealed distinct aspects of tissue and cell structure (nuclear pattern, axonal pathways, hemorrhages. CLE discrimination of neoplastic from healthy brain tissue was easy to perform based on tissue and cellular architecture and resemblance with histopathology was excellent. Confocal laser endomicroscopy allows immediate in vivo imaging of normal and neoplastic brain tissue at high resolution. The technology might be transferred to scientific and clinical application in neurosurgery and neuropathology. It may become helpful to screen for tumor free margins and to improve the surgical resection of malignant brain tumors, and opens the door to in vivo molecular imaging of tumors and other neurologic disorders.

  14. A statistical pixel intensity model for segmentation of confocal laser scanning microscopy images.

    Science.gov (United States)

    Calapez, Alexandre; Rosa, Agostinho

    2010-09-01

    Confocal laser scanning microscopy (CLSM) has been widely used in the life sciences for the characterization of cell processes because it allows the recording of the distribution of fluorescence-tagged macromolecules on a section of the living cell. It is in fact the cornerstone of many molecular transport and interaction quantification techniques where the identification of regions of interest through image segmentation is usually a required step. In many situations, because of the complexity of the recorded cellular structures or because of the amounts of data involved, image segmentation either is too difficult or inefficient to be done by hand and automated segmentation procedures have to be considered. Given the nature of CLSM images, statistical segmentation methodologies appear as natural candidates. In this work we propose a model to be used for statistical unsupervised CLSM image segmentation. The model is derived from the CLSM image formation mechanics and its performance is compared to the existing alternatives. Results show that it provides a much better description of the data on classes characterized by their mean intensity, making it suitable not only for segmentation methodologies with known number of classes but also for use with schemes aiming at the estimation of the number of classes through the application of cluster selection criteria.

  15. PIV as a method for quantifying root cell growth and particle displacement in confocal images.

    Science.gov (United States)

    Bengough, A Glyn; Hans, Joachim; Bransby, M Fraser; Valentine, Tracy A

    2010-01-01

    Particle image velocimetry (PIV) quantifies displacement of patches of pixels between successive images. We evaluated PIV as a tool for microscopists by measuring displacements of cells and of a surrounding granular medium in confocal laser scanning microscopy images of Arabidopsis thaliana roots labeled with cell-membrane targeted green fluorescent protein. Excellent accuracy (e.g., displacement standard deviation PIV-predicted and actual displacements (r(2) > 0.83). Root mean squared error for these distorted images was 0.4-1.1 pixels, increasing at higher magnification factors. Cell growth and rhizosphere deformation were tracked with good temporal (e.g., 1-min interval) and spatial resolution, with PIV patches located on recognizable cell features being tracked more successfully. Appropriate choice of GFP-label was important to decrease small-scale biological noise due to intracellular motion. PIV of roots grown in stiff 2% versus 0.7% agar showed patterns of cell expansion consistent with physically impeded roots of other species. Roots in glass ballotini underwent rapid changes in growth direction on a timescale of minutes, associated with localized arching of ballotini. By tracking cell vertices, we monitored automatically cell length, width, and area every minute for 0.5 h for cells in different stages of development. In conclusion, PIV measured displacements successfully in images of living root cells and the external granular medium, revealing much potential for use by microscopists. (c) 2009 Wiley-Liss, Inc.

  16. Scattering features for lung cancer detection in fibered confocal fluorescence microscopy images.

    Science.gov (United States)

    Rakotomamonjy, Alain; Petitjean, Caroline; Salaün, Mathieu; Thiberville, Luc

    2014-06-01

    To assess the feasibility of lung cancer diagnosis using fibered confocal fluorescence microscopy (FCFM) imaging technique and scattering features for pattern recognition. FCFM imaging technique is a new medical imaging technique for which interest has yet to be established for diagnosis. This paper addresses the problem of lung cancer detection using FCFM images and, as a first contribution, assesses the feasibility of computer-aided diagnosis through these images. Towards this aim, we have built a pattern recognition scheme which involves a feature extraction stage and a classification stage. The second contribution relies on the features used for discrimination. Indeed, we have employed the so-called scattering transform for extracting discriminative features, which are robust to small deformations in the images. We have also compared and combined these features with classical yet powerful features like local binary patterns (LBP) and their variants denoted as local quinary patterns (LQP). We show that scattering features yielded to better recognition performances than classical features like LBP and their LQP variants for the FCFM image classification problems. Another finding is that LBP-based and scattering-based features provide complementary discriminative information and, in some situations, we empirically establish that performance can be improved when jointly using LBP, LQP and scattering features. In this work we analyze the joint capability of FCFM images and scattering features for lung cancer diagnosis. The proposed method achieves a good recognition rate for such a diagnosis problem. It also performs well when used in conjunction with other features for other classical medical imaging classification problems. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. 3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

    Energy Technology Data Exchange (ETDEWEB)

    Fredrich, J.T.

    1999-02-10

    We present advances in the application of laser scanning confocal microscopy (LSCM) to image, reconstruct, and characterize statistically the microgeometry of porous geologic and engineering materials. We discuss technical and practical aspects of this imaging technique, including both its advantages and limitations. Confocal imaging can be used to optically section a material, with sub-micron resolution possible in the lateral and axial planes. The resultant volumetric image data, consisting of fluorescence intensities for typically {approximately}50 million voxels in XYZ space, can be used to reconstruct the three-dimensional structure of the two-phase medium. We present several examples of this application, including studying pore geometry in sandstone, characterizing brittle failure processes in low-porosity rock deformed under triaxial loading conditions in the laboratory, and analyzing the microstructure of porous ceramic insulations. We then describe approaches to extract statistical microgeometric descriptions from volumetric image data, and present results derived from confocal volumetric data sets. Finally, we develop the use of confocal image data to automatically generate a three-dimensional mesh for numerical pore-scale flow simulations.

  18. Measurement of capillary lenght from 3D images acquired by confocal microscopy using image analysis and stereology

    Czech Academy of Sciences Publication Activity Database

    Kubínová, Lucie; Janáček, Jiří; Eržen, I.; Mao, X. W.

    2010-01-01

    Roč. 16, Suppl.2 (2010), s. 736-737 ISSN 1431-9276. [Microscopy and Microanalysis 2010. Portland, 01.08.2010-05.08.2010] R&D Projects: GA MŠk(CZ) LC06063; GA MŠk(CZ) ME09010; GA MŠk(CZ) MEB090910; GA ČR(CZ) GA304/09/0733 Institutional research plan: CEZ:AV0Z50110509 Keywords : capillary length * confocal microscopy * image analysis Subject RIV: EA - Cell Biology Impact factor: 2.179, year: 2010

  19. 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

  20. 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.

  1. Segmentation of confocal Raman microspectroscopic imaging data using edge-preserving denoising and clustering.

    Science.gov (United States)

    Alexandrov, Theodore; Lasch, Peter

    2013-06-18

    Over the past decade, confocal Raman microspectroscopic (CRM) imaging has matured into a useful analytical tool to obtain spatially resolved chemical information on the molecular composition of biological samples and has found its way into histopathology, cytology, and microbiology. A CRM imaging data set is a hyperspectral image in which Raman intensities are represented as a function of three coordinates: a spectral coordinate λ encoding the wavelength and two spatial coordinates x and y. Understanding CRM imaging data is challenging because of its complexity, size, and moderate signal-to-noise ratio. Spatial segmentation of CRM imaging data is a way to reveal regions of interest and is traditionally performed using nonsupervised clustering which relies on spectral domain-only information with the main drawback being the high sensitivity to noise. We present a new pipeline for spatial segmentation of CRM imaging data which combines preprocessing in the spectral and spatial domains with k-means clustering. Its core is the preprocessing routine in the spatial domain, edge-preserving denoising (EPD), which exploits the spatial relationships between Raman intensities acquired at neighboring pixels. Additionally, we propose to use both spatial correlation to identify Raman spectral features colocalized with defined spatial regions and confidence maps to assess the quality of spatial segmentation. For CRM data acquired from midsagittal Syrian hamster ( Mesocricetus auratus ) brain cryosections, we show how our pipeline benefits from the complex spatial-spectral relationships inherent in the CRM imaging data. EPD significantly improves the quality of spatial segmentation that allows us to extract the underlying structural and compositional information contained in the Raman microspectra.

  2. 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.

  3. The effect of compression on clinical diagnosis of glaucoma based on non-analyzed confocal scanning laser ophthalmoscopy images

    NARCIS (Netherlands)

    Abramoff, M.D.

    2006-01-01

    Knowledge of the effect of compression of ophthalmic images on diagnostic reading is essential for effective tele-ophthalmology applications. It was therefore with great anticipation that I read the article “The Effect of Compression on Clinical Diagnosis of Glaucoma Based on Non-analyzed Confocal

  4. Inter-rater and intra-rater agreement of confocal microscopy imaging in diagnosing and subtyping basal cell carcinoma

    NARCIS (Netherlands)

    Kadouch, D. J.; van Haersma de With, A.; Elshot, Y. S.; Peppelman, M.; Bekkenk, M. W.; Wolkerstorfer, A.; Eekhout, I.; Prinsen, C. A. C.; de Rie, M. A.

    2017-01-01

    Reflectance confocal microscopy (RCM) imaging can be used to diagnose and subtype basal cell carcinoma (BCC) but relies on individual morphologic pattern recognition that might vary among users. We assessed the inter-rater and intra-rater agreement of RCM in correctly diagnosing and subtyping BCC.

  5. Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

    Science.gov (United States)

    Mas, Abraham; Amenós, Montse; Lois, L Maria

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

  6. 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.

  7. Near-Infrared Confocal Laser Reflectance Cytoarchitectural Imaging of the Substantia Nigra and Cerebellum in the Fresh Human Cadaver.

    Science.gov (United States)

    Cheyuo, Cletus; Grand, Walter; Balos, Lucia L

    2017-01-01

    Cytoarchitectural neuroimaging remains critical for diagnosis of many brain diseases. Fluorescent dye-enhanced, near-infrared confocal in situ cellular imaging of the brain has been reported. However, impermeability of the blood-brain barrier to most fluorescent dyes limits clinical utility of this modality. The differential degree of reflectance from brain tissue with unenhanced near-infrared imaging may represent an alternative technique for in situ cytoarchitectural neuroimaging. We assessed the utility of unenhanced near-infrared confocal laser reflectance imaging of the cytoarchitecture of the cerebellum and substantia nigra in 2 fresh human cadaver brains using a confocal near-infrared laser probe. Cellular images based on near-infrared differential reflectance were captured at depths of 20-180 μm from the brain surface. Parts of the cerebellum and substantia nigra imaged using the probe were subsequently excised and stained with hematoxylin and eosin for histologic correlation. Near-infrared reflectance imaging revealed the 3-layered cytoarchitecture of the cerebellum, with Purkinje cells appearing hyperreflectant. In the substantia nigra, neurons appeared hyporeflectant with hyperreflectant neuromelanin cytoplasmic inclusions. Cytoarchitecture of the cerebellum and substantia nigra revealed on near-infrared imaging closely correlated with the histology on hematoxylin-eosin staining. We showed that unenhanced near-infrared reflectance imaging of fresh human cadaver brain can reliably identify and distinguish neurons and detailed cytoarchitecture of the cerebellum and substantia nigra. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. In vivo subsurface morphological and functional cellular and subcellular imaging of the gastrointestinal tract with confocal mini-microscopy

    Institute of Scientific and Technical Information of China (English)

    Martin Goetz; Beena Memadathil; Stefan Biesterfeld; Constantin Schneider; Sebastian Gregor; Peter R Galle; Markus F Neurath; Ralf Kiesslich

    2007-01-01

    AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents.METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation.Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths (surface to 250 μm)were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle,stable contact. Tissue specimens were sampled for histopathological correlation.RESULTS: The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice.Real time microscopic imaging with the confocal minimicroscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging.CONCLUSION: Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures.The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients.

  9. 3D image restoration for confocal microscopy: toward a wavelet deconvolution for the study of complex biological structures

    Science.gov (United States)

    Boutet de Monvel, Jacques; Le Calvez, Sophie; Ulfendahl, Mats

    2000-05-01

    Image restoration algorithms provide efficient tools for recovering part of the information lost in the imaging process of a microscope. We describe recent progress in the application of deconvolution to confocal microscopy. The point spread function of a Biorad-MRC1024 confocal microscope was measured under various imaging conditions, and used to process 3D-confocal images acquired in an intact preparation of the inner ear developed at Karolinska Institutet. Using these experiments we investigate the application of denoising methods based on wavelet analysis as a natural regularization of the deconvolution process. Within the Bayesian approach to image restoration, we compare wavelet denoising with the use of a maximum entropy constraint as another natural regularization method. Numerical experiments performed with test images show a clear advantage of the wavelet denoising approach, allowing to `cool down' the image with respect to the signal, while suppressing much of the fine-scale artifacts appearing during deconvolution due to the presence of noise, incomplete knowledge of the point spread function, or undersampling problems. We further describe a natural development of this approach, which consists of performing the Bayesian inference directly in the wavelet domain.

  10. Confocal Microscopy

    Science.gov (United States)

    Liu, Jian; Tan, Jiubin

    2016-12-01

    The confocal microscope is appropriate for imaging cells or the measurement of industrial artefacts. However, junior researchers and instrument users sometimes misuse imaging concepts and metrological characteristics, such as position resolution in industrial metrology and scale resolution in bio-imaging. And, metrological characteristics or influence factors in 3D measurement such as height assessment error caused by 3D coupling effect are so far not yet identified. In this book, the authors outline their practices by the working experiences on standardization and system design. This book assumes little previous knowledge of optics, but rich experience in engineering of industrial measurements, in particular with profile metrology or areal surface topography will be very helpful to understand the theoretical concerns and value of the technological advances. It should be useful for graduate students or researchers as extended reading material, as well as microscope users alongside their handbook.

  11. Efficacy of photodynamic therapy against larvae of Aedes aegypti: confocal microscopy and fluorescence-lifetime imaging

    Science.gov (United States)

    de Souza, L. M.; Pratavieira, S.; Inada, N. M.; Kurachi, C.; Corbi, J.; Guimarães, F. E. G.; Bagnato, V. S.

    2014-03-01

    Recently a few demonstration on the use of Photodynamic Reaction as possibility to eliminate larvae that transmit diseases for men has been successfully demonstrated. This promising tool cannot be vastly used due to many problems, including the lake of investigation concerning the mechanisms of larvae killing as well as security concerning the use of photosensitizers in open environment. In this study, we investigate some of the mechanisms in which porphyrin (Photogem) is incorporated on the Aedes aegypti larvae previously to illumination and killing. Larvae at second instar were exposed to the photosensitizer and after 30 minutes imaged by a confocal fluorescence microscope. It was observed the presence of photosensitizer in the gut and at the digestive tract of the larva. Fluorescence-Lifetime Imaging showed greater photosensitizer concentration in the intestinal wall of the samples, which produces a strong decrease of the Photogem fluorescence lifetime. For Photodynamic Therapy exposition to different light doses and concentrations of porphyrin were employed. Three different light sources (LED, Fluorescent lamp, Sun light) also were tested. Sun light and fluorescent lamp shows close to 100% of mortality after 24 hrs. of illumination. These results indicate the potential use of photodynamic effect against the LARVAE of Aedes aegypti.

  12. Characterization of hydrogel microstructure using laser tweezers particle tracking and confocal reflection imaging

    International Nuclear Information System (INIS)

    Kotlarchyk, M A; Botvinick, E L; Putnam, A J

    2010-01-01

    Hydrogels are commonly used as extracellular matrix mimetics for applications in tissue engineering and increasingly as cell culture platforms with which to study the influence of biophysical and biochemical cues on cell function in 3D. In recent years, a significant number of studies have focused on linking substrate mechanical properties to cell function using standard methodologies to characterize the bulk mechanical properties of the hydrogel substrates. However, current understanding of the correlations between the microstructural mechanical properties of hydrogels and cell function in 3D is poor, in part because of a lack of appropriate techniques. Here we have utilized a laser tracking system, based on passive optical microrheology instrumentation, to characterize the microstructure of viscoelastic fibrin clots. Trajectories and mean square displacements were observed as bioinert PEGylated (PEG: polyethylene glycol) microspheres (1, 2 or 4.7 μm in diameter) diffused within confined pores created by the protein phase of fibrin hydrogels. Complementary confocal reflection imaging revealed microstructures comprised of a highly heterogeneous fibrin network with a wide range of pore sizes. As the protein concentration of fibrin gels was increased, our quantitative laser tracking measurements showed a corresponding decrease in particle mean square displacements with greater resolution and sensitivity than conventional imaging techniques. This platform-independent method will enable a more complete understanding of how changes in substrate mechanical properties simultaneously influence other microenvironmental parameters in 3D cultures.

  13. A portable confocal hyperspectral microscope without any scan or tube lens and its application in fluorescence and Raman spectral imaging

    Science.gov (United States)

    Li, Jingwei; Cai, Fuhong; Dong, Yongjiang; Zhu, Zhenfeng; Sun, Xianhe; Zhang, Hequn; He, Sailing

    2017-06-01

    In this study, a portable confocal hyperspectral microscope is developed. In traditional confocal laser scanning microscopes, scan lens and tube lens are utilized to achieve a conjugate relationship between the galvanometer and the back focal plane of the objective, in order to achieve a better resolution. However, these lenses make it difficult to scale down the volume of the system. In our portable confocal hyperspectral microscope (PCHM), the objective is placed directly next to the galvomirror. Thus, scan lens and tube lens are not included in our system and the size of this system is greatly reduced. Furthermore, the resolution is also acceptable in many biomedical and food-safety applications. Through reducing the optical length of the system, the signal detection efficiency is enhanced. This is conducive to realizing both the fluorescence and Raman hyperspectral imaging. With a multimode fiber as a pinhole, an improved image contrast is also achieved. Fluorescent spectral images for HeLa cells/fingers and Raman spectral images of kumquat pericarp are present. The spectral resolution and spatial resolutions are about 0.4 nm and 2.19 μm, respectively. These results demonstrate that this portable hyperspectral microscope can be used in in-vivo fluorescence imaging and in situ Raman spectral imaging.

  14. Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents

    Science.gov (United States)

    Higgins, Laura M.; Zevon, Margot; Ganapathy, Vidya; Sheng, Yang; Tan, Mei Chee; Riman, Richard E.; Roth, Charles M.; Moghe, Prabhas V.; Pierce, Mark C.

    2015-01-01

    Abstract. Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4:Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic. PMID:26603495

  15. Ca(2+ release events in cardiac myocytes up close: insights from fast confocal imaging.

    Directory of Open Access Journals (Sweden)

    Vyacheslav M Shkryl

    Full Text Available The spatio-temporal properties of Ca(2+ transients during excitation-contraction coupling and elementary Ca(2+ release events (Ca(2+ sparks were studied in atrial and ventricular myocytes with ultra-fast confocal microscopy using a Zeiss LSM 5 LIVE system that allows sampling rates of up to 60 kHz. Ca(2+ sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca(2+ sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca(2+ spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca(2+]i. 2-D imaging of Ca(2+ transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca(2+ entry through surface membrane Ca(2+ channels and subsequent activation of Ca(2+-induced Ca(2+ release. With a latency of 2.5 ms after application of an electrical stimulus, Ca(2+ entry could be detected that was followed by SR Ca(2+ release after an additional 3 ms delay. Maximum Ca(2+ release was observed 4 ms after the beginning of release. The timing of Ca(2+ entry and release was confirmed by simultaneous [Ca(2+]i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca(2+ release events that fused into a peripheral ring of elevated [Ca(2+]i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca(2+ release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca(2+ transient. In summary, ultra-fast confocal imaging allows investigation of Ca(2+ signals with a time resolution similar to patch clamp technique, however in a less invasive fashion.

  16. 3D imaging of cement-based materials at submicron resolution by combining laser scanning confocal microscopy with serial sectioning.

    Science.gov (United States)

    Yio, M H N; Mac, M J; Wong, H S; Buenfeld, N R

    2015-05-01

    In this paper, we present a new method to reconstruct large volumes of nontransparent porous materials at submicron resolution. The proposed method combines fluorescence laser scanning confocal microscopy with serial sectioning to produce a series of overlapping confocal z-stacks, which are then aligned and stitched based on phase correlation. The method can be extended in the XY plane to further increase the overall image volume. Resolution of the reconstructed image volume does not degrade with increase in sample size. We have used the method to image cementitious materials, hardened cement paste and concrete and the results obtained show that the method is reliable. Possible applications of the method such as three-dimensional characterization of the pores and microcracks in hardened concrete, three-dimensional particle shape characterization of cementitious materials and three-dimensional characterization of other porous materials such as rocks and bioceramics are discussed. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  17. Confocal spectroscopic imaging measurements of depth dependent hydration dynamics in human skin in-vivo

    Science.gov (United States)

    Behm, P.; Hashemi, M.; Hoppe, S.; Wessel, S.; Hagens, R.; Jaspers, S.; Wenck, H.; Rübhausen, M.

    2017-11-01

    We present confocal spectroscopic imaging measurements applied to in-vivo studies to determine the depth dependent hydration profiles of human skin. The observed spectroscopic signal covers the spectral range from 810 nm to 2100 nm allowing to probe relevant absorption signals that can be associated with e.g. lipid and water-absorption bands. We employ a spectrally sensitive autofocus mechanism that allows an ultrafast focusing of the measurement spot on the skin and subsequently probes the evolution of the absorption bands as a function of depth. We determine the change of the water concentration in m%. The water concentration follows a sigmoidal behavior with an increase of the water content of about 70% within 5 μm in a depth of about 14 μm. We have applied our technique to study the hydration dynamics of skin before and after treatment with different concentrations of glycerol indicating that an increase of the glycerol concentration leads to an enhanced water concentration in the stratum corneum. Moreover, in contrast to traditional corneometry we have found that the application of Aluminium Chlorohydrate has no impact to the hydration of skin.

  18. Confocal spectroscopic imaging measurements of depth dependent hydration dynamics in human skin in-vivo

    Directory of Open Access Journals (Sweden)

    P. Behm

    2017-11-01

    Full Text Available We present confocal spectroscopic imaging measurements applied to in-vivo studies to determine the depth dependent hydration profiles of human skin. The observed spectroscopic signal covers the spectral range from 810 nm to 2100 nm allowing to probe relevant absorption signals that can be associated with e.g. lipid and water-absorption bands. We employ a spectrally sensitive autofocus mechanism that allows an ultrafast focusing of the measurement spot on the skin and subsequently probes the evolution of the absorption bands as a function of depth. We determine the change of the water concentration in m%. The water concentration follows a sigmoidal behavior with an increase of the water content of about 70% within 5 μm in a depth of about 14 μm. We have applied our technique to study the hydration dynamics of skin before and after treatment with different concentrations of glycerol indicating that an increase of the glycerol concentration leads to an enhanced water concentration in the stratum corneum. Moreover, in contrast to traditional corneometry we have found that the application of Aluminium Chlorohydrate has no impact to the hydration of skin.

  19. Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

    Energy Technology Data Exchange (ETDEWEB)

    Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz; Lukasik, Beata; Garner, Logan E.; Chworos, Arkadiusz

    2017-05-01

    Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining was determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.

  20. Monitoring pancreatic carcinogenesis by the molecular imaging of cathepsin E in vivo using confocal laser endomicroscopy.

    Directory of Open Access Journals (Sweden)

    Hui Li

    Full Text Available The monitoring of pancreatic ductal adenocarcinoma (PDAC in high-risk populations is essential. Cathepsin E (CTSE is specifically and highly expressed in PDAC and pancreatic intraepithelial neoplasias (PanINs, and its expression gradually increases along with disease progression. In this study, we first established an in situ 7,12-dimethyl-1,2-benzanthracene (DMBA-induced rat model for PanINs and PDAC and then confirmed that tumorigenesis properties in this model were consistent with those of human PDAC in that CTSE expression gradually increased with tumor development using histology and immunohistochemistry. Then, using in vivo imaging of heterotopically implanted tumors generated from CTSE- overexpressing cells (PANC-1-CTSE in nude mice and in vitro imaging of PanINs and PDAC in DMBA-induced rats, the specificity of the synthesized CTSE-activatable probe was verified. Quantitative determination identified that the fluorescence signal ratio of pancreatic tumor to normal pancreas gradually increased in association with progressive pathological grades, with the exception of no significant difference between PanIN-II and PanIN-III grades. Finally, we monitored pancreatic carcinogenesis in vivo using confocal laser endomicroscopy (CLE in combination with the CTSE-activatable probe. A prospective double-blind control study was performed to evaluate the accuracy of this method in diagnosing PDAC and PanINs of all grades (>82.7%. This allowed us to establish effective diagnostic criteria for CLE in PDAC and PanINs to facilitate the monitoring of PDAC in high-risk populations.

  1. Anatomical and metabolic small-animal whole-body imaging using ring-shaped confocal photoacoustic computed tomography

    Science.gov (United States)

    Xia, Jun; Chatni, Muhammad; Maslov, Konstantin; Wang, Lihong V.

    2013-03-01

    Due to the wide use of animals for human disease studies, small animal whole-body imaging plays an increasingly important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical and glucose metabolic information, leading to higher costs of building dual-modality systems. Even with image coregistration, the spatial resolution of the metabolic imaging modality is not improved. We present a ring-shaped confocal photoacoustic computed tomography (RC-PACT) system that can provide both assessments in a single modality. Utilizing the novel design of confocal full-ring light delivery and ultrasound transducer array detection, RC-PACT provides full-view cross-sectional imaging with high spatial resolution. Scanning along the orthogonal direction provides three-dimensional imaging. While the mouse anatomy was imaged with endogenous hemoglobin contrast, the glucose metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose. Through mouse tumor models, we demonstrate that RC-PACT may be a paradigm shifting imaging method for preclinical research.

  2. Probe-based confocal laser endomicroscopy (pCLE) - a new imaging technique for in situ localization of spermatozoa.

    Science.gov (United States)

    Trottmann, Matthias; Stepp, Herbert; Sroka, Ronald; Heide, Michael; Liedl, Bernhard; Reese, Sven; Becker, Armin J; Stief, Christian G; Kölle, Sabine

    2015-05-01

    In azoospermic patients, spermatozoa are routinely obtained by testicular sperm extraction (TESE). However, success rates of this technique are moderate, because the site of excision of testicular tissue is determined arbitrarily. Therefore the aim of this study was to establish probe-based laser endomicroscopy (pCLE) a noval biomedical imaging technique, which provides the opportunity of non-invasive, real-time visualisation of tissue at histological resolution. Using pCLE we clearly visualized longitudinal and horizontal views of the tubuli seminiferi contorti and localized vital spermatozoa. Obtained images and real-time videos were subsequently compared with confocal laser scanning microscopy (CLSM) of spermatozoa and tissues, respectively. Comparative visualization of single native Confocal laser scanning microscopy (CLSM, left) and probe-based laser endomicroscopy (pCLE, right) using Pro Flex(TM) UltraMini O after staining with acriflavine. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. 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

    2016-01-01

    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. PMID:28018724

  4. A linear programming approach to reconstructing subcellular structures from confocal images for automated generation of representative 3D cellular models.

    Science.gov (United States)

    Wood, Scott T; Dean, Brian C; Dean, Delphine

    2013-04-01

    This paper presents a novel computer vision algorithm to analyze 3D stacks of confocal images of fluorescently stained single cells. The goal of the algorithm is to create representative in silico model structures that can be imported into finite element analysis software for mechanical characterization. Segmentation of cell and nucleus boundaries is accomplished via standard thresholding methods. Using novel linear programming methods, a representative actin stress fiber network is generated by computing a linear superposition of fibers having minimum discrepancy compared with an experimental 3D confocal image. Qualitative validation is performed through analysis of seven 3D confocal image stacks of adherent vascular smooth muscle cells (VSMCs) grown in 2D culture. The presented method is able to automatically generate 3D geometries of the cell's boundary, nucleus, and representative F-actin network based on standard cell microscopy data. These geometries can be used for direct importation and implementation in structural finite element models for analysis of the mechanics of a single cell to potentially speed discoveries in the fields of regenerative medicine, mechanobiology, and drug discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Real time detection of antibody-antigen interaction using a laser scanning confocal imaging-surface plasmon resonance system

    International Nuclear Information System (INIS)

    Zhang Hong-Yan; Yang Li-Quan; Ning Ting-Yin; Liu Wei-Min; Sun Jia-Yu; Wang Peng-Fei; Meng Lan; Nie Jia-Cai

    2012-01-01

    A laser scanning confocal imaging-surface plasmon resonance (LSCI-SPR) instrument integrated with a wavelength-dependent surface plasmon resonance (SPR) sensor and a laser scanning confocal microscopy (LSCM) is built to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibodies in real time. The shifts of resonant wavelength at different reaction time stages are obtained by SPR, corresponding well with the changes of the fluorescence intensity collected by using LSCM. The instrument shows the merits of the combination and complementation of the SPR and LSCM, with such advantages as quantificational analysis, high spatial resolution and real time monitor, which are of great importance for practical applications in biosensor and life science. (general)

  6. Imaging subsurface damage of grinded fused silica optics by confocal fluorescence microscopy

    International Nuclear Information System (INIS)

    Neauport, J.; Cormont, P.; Destribats, J.; Legros, P.; Ambard, C.

    2009-01-01

    We report an experimental investigation of fluorescence confocal microscopy as a tool to measure subsurface damage on grinded fused silica optics. Confocal fluorescence microscopy was performed with an excitation at the wavelength of 405 nm on fixed abrasive diamond grinded fused silica samples. We detail the measured fluorescence spectrums and compare them to those of oil based coolants and grinding slurries. We evidence that oil based coolant used in diamond grinding induces a fluorescence that marks the subsurface damages and eases its observation. Such residual traces might also be involved in the laser damage process. (authors)

  7. Visualization and quantitation of abundant macroautophagy in virus-infected cells by confocal three-dimensional fluorescence imaging.

    Science.gov (United States)

    Jackson, Wallen; Yamada, Masaki; Moninger, Thomas; Grose, Charles

    2013-10-01

    Varicella-zoster virus (VZV) is a human herpesvirus. Primary infection causes varicella (chickenpox), a viremic illness typified by an exanthem consisting of several hundred vesicles. When VZV reactivates from latency in the spinal ganglia during late adulthood, the emerging virus causes a vesicular dermatomal rash (herpes zoster or shingles). To expand investigations of autophagy during varicella and zoster, newer 3D imaging technology was combined with laser scanning confocal microscopy to provide animations of autophagosomes in the vesicular rash. First, the cells were immunolabeled with antibodies against VZV proteins and the LC3 protein, an integral autophagosomal protein. Antibody reagents lacking activity against the human blood group A1 antigen were selected. After laser excitation of the samples, optimized emission detection bandwidths were configured by Zeiss Zen control software. Confocal Z-stacks comprising up to 40 optical slices were reconstructed into 3D animations with the aid of Imaris software. With this imaging technology, individual autophagosomes were clearly detectable as spheres within each vesicular cell. To enumerate the number of autophagosomes, data sets from 50 cells were reconstructed as 3D fluorescence images and analyzed with MeasurementPro software. The mean number of autophagosomes per infected vesicular cell was >100, although over 200 autophagosomes were seen in a few cells. In summary, macroautophagy was easily quantitated within VZV-infected cells after immunolabeling and imaging by 3D confocal animation technology. These same 3D imaging techniques will be applicable for investigations of autophagy in other virus-infected cells. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  8. Self-interference fluorescence microscopy with three-phase detection for depth-resolved confocal epi-fluorescence imaging.

    Science.gov (United States)

    Braaf, Boy; de Boer, Johannes F

    2017-03-20

    Three-dimensional confocal fluorescence imaging of in vivo tissues is challenging due to sample motion and limited imaging speeds. In this paper a novel method is therefore presented for scanning confocal epi-fluorescence microscopy with instantaneous depth-sensing based on self-interference fluorescence microscopy (SIFM). A tabletop epi-fluorescence SIFM setup was constructed with an annular phase plate in the emission path to create a spectral self-interference signal that is phase-dependent on the axial position of a fluorescent sample. A Mach-Zehnder interferometer based on a 3 × 3 fiber-coupler was developed for a sensitive phase analysis of the SIFM signal with three photon-counter detectors instead of a spectrometer. The Mach-Zehnder interferometer created three intensity signals that alternately oscillated as a function of the SIFM spectral phase and therefore encoded directly for the axial sample position. Controlled axial translation of fluorescent microsphere layers showed a linear dependence of the SIFM spectral phase with sample depth over axial image ranges of 500 µm and 80 µm (3.9 × Rayleigh range) for 4 × and 10 × microscope objectives respectively. In addition, SIFM was in good agreement with optical coherence tomography depth measurements on a sample with indocyanine green dye filled capillaries placed at multiple depths. High-resolution SIFM imaging applications are demonstrated for fluorescence angiography on a dye-filled capillary blood vessel phantom and for autofluorescence imaging on an ex vivo fly eye.

  9. Concurrent Reflectance Confocal Microscopy and Laser Doppler Flowmetry to Improve Skin Cancer Imaging: A Monte Carlo Model and Experimental Validation

    Directory of Open Access Journals (Sweden)

    Alireza Mowla

    2016-09-01

    Full Text Available Optical interrogation of suspicious skin lesions is standard care in the management of skin cancer worldwide. Morphological and functional markers of malignancy are often combined to improve expert human diagnostic power. We propose the evaluation of the combination of two independent optical biomarkers of skin tumours concurrently. The morphological modality of reflectance confocal microscopy (RCM is combined with the functional modality of laser Doppler flowmetry, which is capable of quantifying tissue perfusion. To realize the idea, we propose laser feedback interferometry as an implementation of RCM, which is able to detect the Doppler signal in addition to the confocal reflectance signal. Based on the proposed technique, we study numerical models of skin tissue incorporating two optical biomarkers of malignancy: (i abnormal red blood cell velocities and concentrations and (ii anomalous optical properties manifested through tissue confocal reflectance, using Monte Carlo simulation. We also conduct a laboratory experiment on a microfluidic channel containing a dynamic turbid medium, to validate the efficacy of the technique. We quantify the performance of the technique by examining a signal to background ratio (SBR in both the numerical and experimental models, and it is shown that both simulated and experimental SBRs improve consistently using this technique. This work indicates the feasibility of an optical instrument, which may have a role in enhanced imaging of skin malignancies.

  10. 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.

  11. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    International Nuclear Information System (INIS)

    Al-Gubory, Kais H.

    2005-01-01

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals

  12. SU-F-T-665: Confocal Microscopy Imaging of Cell Cycle Distribution in Cells Treated with Pegylated Gold Nanoshells

    Energy Technology Data Exchange (ETDEWEB)

    Sadetaporn, D [Rice University, Houston, TX (United States); The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Flint, D; McFadden, C; Sawakuchi, G [The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Asaithamby, A [UT Southwestern Medical Center, Dallas, TX (United States)

    2016-06-15

    Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

  13. SU-F-T-665: Confocal Microscopy Imaging of Cell Cycle Distribution in Cells Treated with Pegylated Gold Nanoshells

    International Nuclear Information System (INIS)

    Sadetaporn, D; Flint, D; McFadden, C; Sawakuchi, G; Asaithamby, A

    2016-01-01

    Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

  14. Confocal imaging of whole vertebrate embryos reveals novel insights into molecular and cellular mechanisms of organ development

    Science.gov (United States)

    Hadel, Diana M.; Keller, Bradley B.; Sandell, Lisa L.

    2014-03-01

    Confocal microscopy has been an invaluable tool for studying cellular or sub-cellular biological processes. The study of vertebrate embryology is based largely on examination of whole embryos and organs. The application of confocal microscopy to immunostained whole mount embryos, combined with three dimensional (3D) image reconstruction technologies, opens new avenues for synthesizing molecular, cellular and anatomical analysis of vertebrate development. Optical cropping of the region of interest enables visualization of structures that are morphologically complex or obscured, and solid surface rendering of fluorescent signal facilitates understanding of 3D structures. We have applied these technologies to whole mount immunostained mouse embryos to visualize developmental morphogenesis of the mammalian inner ear and heart. Using molecular markers of neuron development and transgenic reporters of neural crest cell lineage we have examined development of inner ear neurons that originate from the otic vesicle, along with the supporting glial cells that derive from the neural crest. The image analysis reveals a previously unrecognized coordinated spatial organization between migratory neural crest cells and neurons of the cochleovestibular nerve. The images also enable visualization of early cochlear spiral nerve morphogenesis relative to the developing cochlea, demonstrating a heretofore unknown association of neural crest cells with extending peripheral neurite projections. We performed similar analysis of embryonic hearts in mouse and chick, documenting the distribution of adhesion molecules during septation of the outflow tract and remodeling of aortic arches. Surface rendering of lumen space defines the morphology in a manner similar to resin injection casting and micro-CT.

  15. FluoRender: An application of 2D image space methods for 3D and 4D confocal microscopy data visualization in neurobiology research

    KAUST Repository

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

    2012-01-01

    2D image space methods are processing methods applied after the volumetric data are projected and rendered into the 2D image space, such as 2D filtering, tone mapping and compositing. In the application domain of volume visualization, most 2D image space methods can be carried out more efficiently than their 3D counterparts. Most importantly, 2D image space methods can be used to enhance volume visualization quality when applied together with volume rendering methods. In this paper, we present and discuss the applications of a series of 2D image space methods as enhancements to confocal microscopy visualizations, including 2D tone mapping, 2D compositing, and 2D color mapping. These methods are easily integrated with our existing confocal visualization tool, FluoRender, and the outcome is a full-featured visualization system that meets neurobiologists' demands for qualitative analysis of confocal microscopy data. © 2012 IEEE.

  16. FluoRender: An application of 2D image space methods for 3D and 4D confocal microscopy data visualization in neurobiology research

    KAUST Repository

    Wan, Yong

    2012-02-01

    2D image space methods are processing methods applied after the volumetric data are projected and rendered into the 2D image space, such as 2D filtering, tone mapping and compositing. In the application domain of volume visualization, most 2D image space methods can be carried out more efficiently than their 3D counterparts. Most importantly, 2D image space methods can be used to enhance volume visualization quality when applied together with volume rendering methods. In this paper, we present and discuss the applications of a series of 2D image space methods as enhancements to confocal microscopy visualizations, including 2D tone mapping, 2D compositing, and 2D color mapping. These methods are easily integrated with our existing confocal visualization tool, FluoRender, and the outcome is a full-featured visualization system that meets neurobiologists\\' demands for qualitative analysis of confocal microscopy data. © 2012 IEEE.

  17. Impact of immersion oils and mounting media on the confocal imaging of dendritic spines.

    Science.gov (United States)

    Peterson, Brittni M; Mermelstein, Paul G; Meisel, Robert L

    2015-03-15

    Structural plasticity, such as changes in dendritic spine morphology and density, reflect changes in synaptic connectivity and circuitry. Procedural variables used in different methods for labeling dendritic spines have been quantitatively evaluated for their impact on the ability to resolve individual spines in confocal microscopic analyses. In contrast, there have been discussions, though no quantitative analyses, of the potential effects of choosing specific mounting media and immersion oils on dendritic spine resolution. Here we provide quantitative data measuring the impact of these variables on resolving dendritic spines in 3D confocal analyses. Medium spiny neurons from the rat striatum and nucleus accumbens are used as examples. Both choice of mounting media and immersion oil affected the visualization of dendritic spines, with choosing the appropriate immersion oil as being more imperative. These biologic data are supported by quantitative measures of the 3D diffraction pattern (i.e. point spread function) of a point source of light under the same mounting medium and immersion oil combinations. Although not a new method, this manuscript provides quantitative data demonstrating that different mounting media and immersion oils can impact the ability to resolve dendritic spines. These findings highlight the importance of reporting which mounting medium and immersion oil are used in preparations for confocal analyses, especially when comparing published results from different laboratories. Collectively, these data suggest that choosing the appropriate immersion oil and mounting media is critical for obtaining the best resolution, and consequently more accurate measures of dendritic spine densities. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. 3D confocal Raman imaging of oil-rich emulsion from enzyme-assisted aqueous extraction of extruded soybean powder.

    Science.gov (United States)

    Wu, Longkun; Wang, Limin; Qi, Baokun; Zhang, Xiaonan; Chen, Fusheng; Li, Yang; Sui, Xiaonan; Jiang, Lianzhou

    2018-05-30

    The understanding of the structure morphology of oil-rich emulsion from enzyme-assisted extraction processing (EAEP) was a critical step to break the oil-rich emulsion structure in order to recover oil. Albeit EAEP method has been applied as an alternative way to conventional solvent extraction method, the structure morphology of oil-rich emulsion was still unclear. The current study aimed to investigate the structure morphology of oil-rich emulsion from EAEP using 3D confocal Raman imaging technique. With increasing the enzymatic hydrolysis duration from 1 to 3 h, the stability of oil-rich emulsion was decreased as visualized in the 3D confocal Raman images that the protein and oil were mixed together. The subsequent Raman spectrum analysis further revealed that the decreased stability of oil-rich emulsion was due to the protein aggregations via SS bonds or protein-lipid interactions. The conformational transfer in protein indicated the formation of a compact structure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Lateral Brightness Correction in Confocal Microscopy Images Using Mathematical Morphology Filters

    Czech Academy of Sciences Publication Activity Database

    Michálek, Jan; Čapek, M.; Mao, X. W.; Kubínová, Lucie

    2010-01-01

    Roč. 16, Suppl.2 (2010), s. 758-759 ISSN 1431-9276. [Microscopy and Microanalysis 2010. Portland, 01.08.2010-05.08.2010] R&D Projects: GA MŠk(CZ) LC06063; GA MŠk(CZ) ME09010; GA ČR(CZ) GA102/08/0691; GA ČR(CZ) GA304/09/0733 Institutional research plan: CEZ:AV0Z50110509 Keywords : Lipschitz cover * lateral intensity correction * confocal microscopy Subject RIV: JD - Computer Applications, Robotics Impact factor: 2.179, year: 2010

  20. 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

  1. Spatiotemporal closure of fractional laser-ablated channels imaged by optical coherence tomography and reflectance confocal microscopy

    DEFF Research Database (Denmark)

    Banzhaf, Christina A.; Wind, Bas S.; Mogensen, Mette

    2016-01-01

    Background and Objective 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 channels and dynamics in their spatiotemporal closure using in vivo OCT and RCM techniques. Study design/Materials and Methods The inner forearm of healthy subjects (n = 6) was exposed to 10,600 nm fractional CO2 laser using 5 and 25% densities, 120 μm beam diameter, 5, 15, and 25 m......J/microbeam. Treatment sites were scanned with OCT to evaluate closure of AFXL-channels and RCM to evaluate subsequent re-epithelialization. Results OCT and RCM identified laser channels in epidermis and upper dermis as black, ablated tissue defects surrounded by characteristic hyper-and hyporeflective zones. OCT imaged...

  2. WE-FG-BRA-04: A Portable Confocal Microscope to Image Live Cell Damage Response Induced by Therapeutic Radiation

    Energy Technology Data Exchange (ETDEWEB)

    McFadden, C; Flint, D; Grosshans, D; Sawakuchi, G [The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Sadetaporn, D [The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Rice University, Houston, TX (United States); Asaithamby, A [UT Southwestern Medical Center, Dallas, TX (United States)

    2016-06-15

    Purpose: To construct a custom and portable fluorescence confocal laser-scanning microscope (FCLSM) that can be placed in the path of therapeutic radiation beams to study real-time radiation-induced damage response in live cells. Methods: We designed and constructed a portable FCLSM with three laser diodes for excitation (405, 488, and 635 nm). An objective lens focuses the excitation light and collects fluorescence from the sample. A pair of galvanometer mirrors scans/collects the laser beam/fluorescence along the focal plane (x/y-directions). A stepper motor stage scans in the axial direction and positions the x/y of the image field. Barrier filters and dichroic mirrors are used to route the spectral emission bands to the appropriate photodetector. An avalanche photodiode collects near-infrared fluorescence; a photodiode collects back-reflected 635 nm light; and a photomultiplier tube collects green fluorescence in the range of eGFP/eYFP. A 200-µm diameter pinhole was used to implement the confocal geometry for near-infrared and red channels and a 150-µm diameter pinhole for the green channel. Data acquisition and system control were achieved using a high-throughput data acquisition card. In-house software developed in LabVIEW was used to control the hardware, collect data from the photodetectors and reconstruct the confocal images. Results: 6 frames/s can be acquired for a 25 µm{sup 2} (128×128 pixels) field of view, visualizing the entire volume of the cell nucleus (∼10 µm depth) in <10 s. To demonstrate the usefulness of our FCLSM, we imaged gold nanoshells in live cells, radiation-induced damage in fibrosarcoma cells expressing eGFP tagged to a DNA repair protein, and neurons expressing eGFP. The system can also image particle tracks in fluorescent nuclear track detectors. Conclusion: We developed a versatile and portable FCLSM that allows radiobiology studies in live cells exposed to therapeutic radiation. The FCLSM can be placed in any vertical beam

  3. WE-FG-BRA-04: A Portable Confocal Microscope to Image Live Cell Damage Response Induced by Therapeutic Radiation

    International Nuclear Information System (INIS)

    McFadden, C; Flint, D; Grosshans, D; Sawakuchi, G; Sadetaporn, D; Asaithamby, A

    2016-01-01

    Purpose: To construct a custom and portable fluorescence confocal laser-scanning microscope (FCLSM) that can be placed in the path of therapeutic radiation beams to study real-time radiation-induced damage response in live cells. Methods: We designed and constructed a portable FCLSM with three laser diodes for excitation (405, 488, and 635 nm). An objective lens focuses the excitation light and collects fluorescence from the sample. A pair of galvanometer mirrors scans/collects the laser beam/fluorescence along the focal plane (x/y-directions). A stepper motor stage scans in the axial direction and positions the x/y of the image field. Barrier filters and dichroic mirrors are used to route the spectral emission bands to the appropriate photodetector. An avalanche photodiode collects near-infrared fluorescence; a photodiode collects back-reflected 635 nm light; and a photomultiplier tube collects green fluorescence in the range of eGFP/eYFP. A 200-µm diameter pinhole was used to implement the confocal geometry for near-infrared and red channels and a 150-µm diameter pinhole for the green channel. Data acquisition and system control were achieved using a high-throughput data acquisition card. In-house software developed in LabVIEW was used to control the hardware, collect data from the photodetectors and reconstruct the confocal images. Results: 6 frames/s can be acquired for a 25 µm 2 (128×128 pixels) field of view, visualizing the entire volume of the cell nucleus (∼10 µm depth) in <10 s. To demonstrate the usefulness of our FCLSM, we imaged gold nanoshells in live cells, radiation-induced damage in fibrosarcoma cells expressing eGFP tagged to a DNA repair protein, and neurons expressing eGFP. The system can also image particle tracks in fluorescent nuclear track detectors. Conclusion: We developed a versatile and portable FCLSM that allows radiobiology studies in live cells exposed to therapeutic radiation. The FCLSM can be placed in any vertical beam line

  4. Nondestructive 3D confocal laser imaging with deconvolution of seven whole stardust tracks with complementary XRF and quantitative analysis

    International Nuclear Information System (INIS)

    Greenberg, M.; Ebel, D.S.

    2009-01-01

    We present a nondestructive 3D system for analysis of whole Stardust tracks, using a combination of Laser Confocal Scanning Microscopy and synchrotron XRF. 3D deconvolution is used for optical corrections, and results of quantitative analyses of several tracks are presented. The Stardust mission to comet Wild 2 trapped many cometary and ISM particles in aerogel, leaving behind 'tracks' of melted silica aerogel on both sides of the collector. Collected particles and their tracks range in size from submicron to millimeter scale. Interstellar dust collected on the obverse of the aerogel collector is thought to have an average track length of ∼15 (micro)m. It has been our goal to perform a total non-destructive 3D textural and XRF chemical analysis on both types of tracks. To that end, we use a combination of Laser Confocal Scanning Microscopy (LCSM) and X Ray Florescence (XRF) spectrometry. Utilized properly, the combination of 3D optical data and chemical data provides total nondestructive characterization of full tracks, prior to flattening or other destructive analysis methods. Our LCSM techniques allow imaging at 0.075 (micro)m/pixel, without the use of oil-based lenses. A full textural analysis on track No.82 is presented here as well as analysis of 6 additional tracks contained within 3 keystones (No.128, No.129 and No.140). We present a method of removing the axial distortion inherent in LCSM images, by means of a computational 3D Deconvolution algorithm, and present some preliminary experiments with computed point spread functions. The combination of 3D LCSM data and XRF data provides invaluable information, while preserving the integrity of the samples for further analysis. It is imperative that these samples, the first extraterrestrial solids returned since the Apollo era, be fully mapped nondestructively in 3D, to preserve the maximum amount of information prior to other, destructive analysis.

  5. Fibre optic confocal imaging (FOCI) of keratinocytes, blood vessels and nerves in hairless mouse skin in vivo

    Science.gov (United States)

    BUSSAU, L. J.; VO, L. T.; DELANEY, P. M.; PAPWORTH, G. D.; BARKLA, D. H.; KING, R. G.

    1998-01-01

    Fibre optic confocal imaging (FOCI) enabled subsurface fluorescence microscopy of the skin of hairless mice in vivo. Application of acridine orange enabled imaging of the layers of the epidermis. The corneocytes of the stratum corneum, the keratinocytes in the basal layers and redundant hair follicles were visualised at depths greater than 100 μm. Cellular and nuclear membranes of keratinocytes of the skin were visualised by the use of acridine orange and DIOC5(3). Imaging of the skin after injection of FITC-dextran revealed an extensive network of blood vessels with a size range up to 20 μm. Blood cells could be seen moving through dermal vessels and the blood circulation through the dermal vascular bed was video-taped. The fluorescent dye 4-di-2-ASP showed the presence of nerves fibres around the hair follicles and subsurface blood vessels. Comparison was made between images obtained in vivo using FOCI and in vitro scanning electron microscopy and conventional histology. FOCI offers the potential to study dynamic events in vivo, such as blood flow, skin growth, nerve regeneration and many pathological processes, in ways which have not previously been possible. PMID:9643419

  6. Confocal laser microscopic imaging of conspicuous facial pores in vivo: relation between the appearance and the internal structure of skin.

    Science.gov (United States)

    Sugata, Keiichi; Nishijima, Takafumi; Kitahara, Takashi; Takema, Yoshinori

    2008-05-01

    Conspicuous facial pores are one of the more serious esthetic defects of most concern to women. Previous microscopic observations of the skin surface around conspicuous pores have discovered large hollows and uneven skin tone. In this study, the observation area was extended from the skin surface to deeper skin to find the characteristic features of conspicuous pores in a wider spectrum. First, a magnified surface image of the cheek skin was obtained using a video microscope. Second, replicas were collected from the same area. Third, the horizontal cross-sectioned images of the epidermis and papillary dermis in different depths were non-invasively obtained using in vivo confocal laser scanning microscopy. These images were compared with each other to find a correlation between features of the skin surface and those of deeper layers. In cross-sectioned images of conspicuous pores, a strongly undulated epidermal-dermal junction was commonly observed around a pore's opening. Areas with this feature correlated well to the areas with larger hollows and an uneven skin tone. Our results indicate that there is a positive correlation between the incidence of the characteristic feature at the epidermal-dermal junction and the visual appearance of a pore.

  7. 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.

  8. Time-Lapse, in Situ Imaging of Ice Crystal Growth Using Confocal Microscopy.

    Science.gov (United States)

    Marcellini, Moreno; Noirjean, Cecile; Dedovets, Dmytro; Maria, Juliette; Deville, Sylvain

    2016-11-30

    Ice crystals nucleate and grow when a water solution is cooled below its freezing point. The growth velocities and morphologies of the ice crystals depend on many parameters, such as the temperature of ice growth, the melting temperature, and the interactions of solutes with the growing crystals. Three types of morphologies may appear: dendritic, cellular (or fingerlike), or the faceted equilibrium form. Understanding and controlling which type of morphology is formed is essential in several domains, from biology to geophysics and materials science. Obtaining, in situ, three dimensional observations without introducing artifacts due to the experimental technique is nevertheless challenging. Here we show how we can use laser scanning confocal microscopy to follow in real-time the growth of smoothed and faceted ice crystals in zirconium acetate solutions. Both qualitative and quantitative observations can be made. In particular, we can precisely measure the lateral growth velocity of the crystals, a measure otherwise difficult to obtain. Such observations should help us understand the influence of the parameters that control the growth of ice crystals in various systems.

  9. Multicolor probe-based confocal laser endomicroscopy: a new world for in vivo and real-time cellular imaging

    Science.gov (United States)

    Vercauteren, Tom; Doussoux, François; Cazaux, Matthieu; Schmid, Guillaume; Linard, Nicolas; Durin, Marie-Amélie; Gharbi, Hédi; Lacombe, François

    2013-03-01

    Since its inception in the field of in vivo imaging, endomicroscopy through optical fiber bundles, or probe-based Confocal Laser Endomicroscopy (pCLE), has extensively proven the benefit of in situ and real-time examination of living tissues at the microscopic scale. By continuously increasing image quality, reducing invasiveness and improving system ergonomics, Mauna Kea Technologies has turned pCLE not only into an irreplaceable research instrument for small animal imaging, but also into an accurate clinical decision making tool with applications as diverse as gastrointestinal endoscopy, pulmonology and urology. The current implementation of pCLE relies on a single fluorescence spectral band making different sources of in vivo information challenging to distinguish. Extending the pCLE approach to multi-color endomicroscopy therefore appears as a natural plan. Coupling simultaneous multi-laser excitation with minimally invasive, microscopic resolution, thin and flexible optics, allows the fusion of complementary and valuable biological information, thus paving the way to a combination of morphological and functional imaging. This paper will detail the architecture of a new system, Cellvizio Dual Band, capable of video rate in vivo and in situ multi-spectral fluorescence imaging with a microscopic resolution. In its standard configuration, the system simultaneously operates at 488 and 660 nm, where it automatically performs the necessary spectral, photometric and geometric calibrations to provide unambiguously co-registered images in real-time. The main hardware and software features, including calibration procedures and sub-micron registration algorithms, will be presented as well as a panorama of its current applications, illustrated with recent results in the field of pre-clinical imaging.

  10. 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.

  11. Bacterial and abiotic decay in waterlogged archaeological Picea abies (L.) Karst studied by confocal Raman imaging and ATR-FTIR spectroscopy

    DEFF Research Database (Denmark)

    Pedersen, Nanna Bjerregaard; Gierlinger, Notburga; Thygesen, Lisbeth Garbrecht

    2015-01-01

    Waterlogged archaeological Norway spruce [Picea abies (L.) Karst] poles were studied by means of confocal Raman imaging (CRI) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) analysis to determine lignin and polysaccharide composition and distribution in the cell......, and minor oxidation of the lignin polymer compared to recent reference material. This is evidence for abiotic decay in the course of waterlogging....

  12. Image-guided intraocular injection using multimodality optical coherence tomography and fluorescence confocal scanning laser ophthalmoscopy in rodent ophthalmological models

    Science.gov (United States)

    Terrones, Benjamin D.; Benavides, Oscar R.; Leeburg, Kelsey C.; Mehanathan, Sankarathi B.; Levine, Edward M.; Tao, Yuankai K.

    2018-02-01

    Intraocular injections are routinely performed for delivery of anti-VEGF and anti-inflammatory therapies in humans. While these injections are also performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the injection location and volume are not well-controlled and reproducible. We overcome limitations of conventional injections methods by developing a multimodality, long working distance, non-contact optical coherence tomography (OCT) and fluorescence confocal scanning laser ophthalmoscopy (cSLO) system for retinal imaging before and after injections. Our OCT+cSLO system combines a custom-built spectraldomain OCT engine (875+/-85 nm) with 125 kHz line-rate with a modified commercial cSLO with a maximum frame-rate of 30 fps (512 x 512 pix.). The system was designed for an overlapping OCT+cSLO field-of-view of 1.1 mm with a 7.76 mm working distance to the pupil. cSLO excitation light sources and filters were optimized for simultaneous GFP and tdTomato imaging. Lateral resolution was 3.02 µm for OCT and 2.74 μm for cSLO. Intravitreal injections of 5%, 10%, and 20% intralipid with Alex Fluor 488 were manually injected intraocularly in C57BL/6 mice. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. OCT enables quantitative analysis of injection location and volumes whereas complementary cSLO improves specificity for identifying fluorescently labeled injected compounds and transgenic cells. The long working distance of our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections and may be applied for imaging of ophthalmic surgical dynamics and real-time image-guided injections.

  13. Confocal multispot microscope for fast and deep imaging in semicleared tissues

    Science.gov (United States)

    Adam, Marie-Pierre; Müllenbroich, Marie Caroline; Di Giovanna, Antonino Paolo; Alfieri, Domenico; Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco Saverio

    2018-02-01

    Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.

  14. 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

  15. In vivo imaging of the airway wall in asthma: fibered confocal fluorescence microscopy in relation to histology and lung function

    Directory of Open Access Journals (Sweden)

    Bel Elisabeth H

    2011-06-01

    Full Text Available Abstract Background Airway remodelling is a feature of asthma including fragmentation of elastic fibres observed in the superficial elastin network of the airway wall. Fibered confocal fluorescence microscopy (FCFM is a new and non-invasive imaging technique performed during bronchoscopy that may visualize elastic fibres, as shown by in vitro spectral analysis of elastin powder. We hypothesized that FCFM images capture in vivo elastic fibre patterns within the airway wall and that such patterns correspond with airway histology. We aimed to establish the concordance between the bronchial elastic fibre pattern in histology and FCFM. Second, we examined whether elastic fibre patterns in histology and FCFM were different between asthmatic subjects and healthy controls. Finally, the association between these patterns and lung function parameters was investigated. Methods In a cross-sectional study comprising 16 subjects (8 atopic asthmatic patients with controlled disease and 8 healthy controls spirometry and bronchoscopy were performed, with recording of FCFM images followed by endobronchial biopsy at the airway main carina. Elastic fibre patterns in histological sections and FCFM images were scored semi-quantitatively. Agreement between histology and FCFM was analysed using linearly weighted kappa κw. Results The patterns observed in histological sections and FCFM images could be divided into 3 distinct groups. There was good agreement between elastic fibre patterns in histology and FCFM patterns (κw 0.744. The semi-quantitative pattern scores were not different between asthmatic patients and controls. Notably, there was a significant difference in post-bronchodilator FEV1 %predicted between the different patterns by histology (p = 0.001 and FCFM (p = 0.048, regardless of asthma or atopy. Conclusion FCFM captures the elastic fibre pattern within the airway wall in humans in vivo. The association between post-bronchodilator FEV1 %predicted and

  16. Smart imaging of acute lung injury: exploration of myeloperoxidase activity using in vivo endoscopic confocal fluorescence microscopy.

    Science.gov (United States)

    Chagnon, Frédéric; Bourgouin, Alexandra; Lebel, Réjean; Bonin, Marc-André; Marsault, Eric; Lepage, Martin; Lesur, Olivier

    2015-09-15

    The pathophysiology of acute lung injury (ALI) is well characterized, but its real-time assessment at bedside remains a challenge. When patients do not improve after 1 wk despite supportive therapies, physicians have to consider open lung biopsy (OLB) to identify the process(es) at play. Sustained inflammation and inadequate repair are often observed in this context. OLB is neither easy to perform in a critical setting nor exempt from complications. Herein, we explore intravital endoscopic confocal fluorescence microscopy (ECFM) of the lung in vivo combined with the use of fluorescent smart probe(s) activated by myeloperoxidase (MPO). MPO is a granular enzyme expressed by polymorphonuclear neutrophils (PMNs) and alveolar macrophages (AMs), catalyzing the synthesis of hypoclorous acid, a by-product of hydrogen peroxide. Activation of these probes was first validated in vitro in relevant cells (i.e., AMs and PMNs) and on MPO-non-expressing cells (as negative controls) and then tested in vivo using three rat models of ALI and real-time intravital imaging with ECFM. Semiquantitative image analyses revealed that in vivo probe-related cellular/background fluorescence was associated with corresponding enhanced lung enzymatic activity and was partly prevented by specific MPO inhibition. Additional ex vivo phenotyping was performed, confirming that fluorescent cells were neutrophil elastase(+) (PMNs) or CD68(+) (AMs). This work is a first step toward "virtual biopsy" of ALI without OLB. Copyright © 2015 the American Physiological Society.

  17. Potential Role of In Vivo Confocal Microscopy for Imaging Corneal Nerves in Transthyretin Familial Amyloid Polyneuropathy.

    Science.gov (United States)

    Rousseau, Antoine; Cauquil, Cecile; Dupas, Benedicte; Labbé, Antoine; Baudouin, Christophe; Barreau, Emmanuel; Théaudin, Marie; Lacroix, Catherine; Guiochon-Mantel, Anne; Benmalek, Anouar; Labetoulle, Marc; Adams, David

    2016-09-01

    Small fiber neuropathy (SFN) is an important feature of transthyretin familial amyloid polyneuropathy (TTR-FAP). A practical and objective method for the clinical evaluation of SFN is needed to improve the management of this disease. In vivo confocal microscopy (IVCM) of the corneal nerves, a rapid noninvasive technique, may be used as a surrogate marker of SFN. To determine the correlation of SFN with IVCM in patients with TTR-FAP. A prospective, single-center, cross-sectional controlled study was conducted at the French National Reference Center for TTR-FAP from June 1, 2013, to June 30, 2014. Fifteen patients with TTR-FAP underwent a complete neurologic examination, including Neuropathy Impairment Score of the Lower Limbs, hand grip strength, and evaluation of vegetative dysfunction, as well as electrophysiologic studies (nerve conduction and electrochemical skin conductance) and intraepidermal nerve fiber density quantification. Patients and 15 controls (matched for age and sex) underwent ophthalmologic assessments, including corneal esthesiometry and IVCM. Correlation of corneal nerve fiber length (CNFL) with the severity of SFN. Of the 15 patients enrolled in the study, 6 were women (40%); mean (SD) age was 54.4 [13.7] years. The CNFL was shorter in the patients than in controls (13.08 vs 17.57 mm/mm2; difference of 4.49 [95% CI, 0.72 to 8.27]; P = .02). The patients' CNFL correlated with the severity of both autonomic neuropathy assessed by the Compound Autonomic Dysfunction Test (rs = 0.66 [95% CI, 0.22 to 0.87]; P = .008) or electrochemical skin conductance (rs = 0.80 [95% CI, 0.50 to 0.93]; P < .001) and sensorimotor neuropathy assessed using the Neuropathy Impairment Score of the Lower Limbs (rs = -0.58 [95% CI, -0.84 to -0.11]; P = .02). Patients with altered sensory nerve action potentials and intraepidermal nerve fiber density had a shorter CNFL (P = .04 and P = .02, respectively). The CNFL could be measured in all

  18. Thermally activated delayed fluorescence of fluorescein derivative for time-resolved and confocal fluorescence imaging.

    Science.gov (United States)

    Xiong, Xiaoqing; Song, Fengling; Wang, Jingyun; Zhang, Yukang; Xue, Yingying; Sun, Liangliang; Jiang, Na; Gao, Pan; Tian, Lu; Peng, Xiaojun

    2014-07-09

    Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

  19. Flexible Spectral Imaging Color Enhancement and Probe-based Confocal Laser Endomicroscopy in Minimal Change Esophageal Reflux Disease.

    Science.gov (United States)

    Pittayanon, Rapat; Aumkaew, Surasak; Rerknimitr, Rungsun; Wisedopas, Naruemon; Kullavanijaya, Pinit

    2016-07-25

    Although flexible spectral imaging color enhancement (FICE) can facilitate the diagnosis of minimal change esophageal reflux disease (MERD), the complicated diagnostic criteria cause suboptimal inter-observer agreement. Confocal laser endomicroscopy (CLE) yields good diagnostic results but its inter-observer agreement has never been explored. This study compares the diagnostic value of magnifying FICE and probe-based CLE (pCLE) for MERD and evaluates the inter-observer agreement of both techniques. Thirty-six patients with suspected MERD and 18 asymptomatic controls were recruited. Magnifying FICE was used for evaluation of distal esophagus. pCLE counted the number of intrapapillary capillary loops (IPCLs) using more than five IPCLs in 500×500 micron area as a criterion for MERD diagnosis. The validity scores and interobserever agreement of both FICE and pCLE were assessed. For FICE vs. pCLE, the accuracy was 79% vs. 87%, sensitivity 94% vs. 97%, specificity 50% vs. 66%, positive predictive value 79% vs. 85%, and negative predictive value 82% vs. 92%. Interobserver agreement of FICE was fair to substantial, whereas pCLE had substantial to almost perfect agreement. Both FICE and pCLE have good operating characteristics and can facilitate the MERD diagnosis. However, among different observers, pCLE is more consistent on MERD diagnosis.

  20. 3D Segmentations of Neuronal Nuclei from Confocal Microscope Image Stacks

    Directory of Open Access Journals (Sweden)

    Antonio eLaTorre

    2013-12-01

    Full Text Available In this paper, we present an algorithm to create 3D segmentations of neuronal cells from stacks of previously segmented 2D images. The idea behind this proposal is to provide a general method to reconstruct 3D structures from 2D stacks, regardless of how these 2D stacks have been obtained. The algorithm not only reuses the information obtained in the 2D segmentation, but also attempts to correct some typical mistakes made by the 2D segmentation algorithms (for example, under segmentation of tightly-coupled clusters of cells. We have tested our algorithm in a real scenario --- the segmentation of the neuronal nuclei in different layers of the rat cerebral cortex. Several representative images from different layers of the cerebral cortex have been considered and several 2D segmentation algorithms have been compared. Furthermore, the algorithm has also been compared with the traditional 3D Watershed algorithm and the results obtained here show better performance in terms of correctly identified neuronal nuclei.

  1. 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.

  2. In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice.

    Science.gov (United States)

    de la Rosa, Xavier; Santalucía, Tomàs; Fortin, Pierre-Yves; Purroy, Jesús; Calvo, Maria; Salas-Perdomo, Angélica; Justicia, Carles; Couillaud, Franck; Planas, Anna M

    2013-02-01

    Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.

  3. The Use of Intravital Two-Photon and Thick Section Confocal Imaging to Analyze B Lymphocyte Trafficking in Lymph Nodes and Spleen.

    Science.gov (United States)

    Park, Chung; Hwang, Il-Young; Kehrl, John H

    2018-01-01

    Intravital two-photon laser scanning microscopy (TP-LSM) has allowed the direct observation of immune cells in intact organs of living animals. In the B cell biology field TP-LSM has detailed the movement of B cells in high endothelial venules and during their transmigration into lymph organs; described the movement and positioning of B cells within lymphoid organs; outlined the mechanisms by which antigen is delivered to B cells; observed B cell interacting with T cells, other cell types, and even with pathogens; and delineated the egress of B cells from the lymph node (LN) parenchyma into the efferent lymphatics. As the quality of TP-LSM improves and as new fluorescent probes become available additional insights into B cell behavior and function await new investigations. Yet intravital TP-LSM has some disadvantages including a lower resolution than standard confocal microscopy, a narrow imaging window, and a shallow depth of imaging. We have found that supplementing intravital TP-LSM with conventional confocal microscopy using thick LN sections helps to overcome some of these shortcomings. Here, we describe procedures for visualizing the behavior and trafficking of fluorescently labeled, adoptively transferred antigen-activated B cells within the inguinal LN of live mice using two-photon microscopy. Also, we introduce procedures for fixed thick section imaging using standard confocal microscopy, which allows imaging of fluorescently labeled cells deep in the LN cortex and in the spleen with high resolution.

  4. Semi-automated algorithm for localization of dermal/epidermal junction in reflectance confocal microscopy images of human skin

    Science.gov (United States)

    Kurugol, Sila; Dy, Jennifer G.; Rajadhyaksha, Milind; Gossage, Kirk W.; Weissmann, Jesse; Brooks, Dana H.

    2011-03-01

    The examination of the dermis/epidermis junction (DEJ) is clinically important for skin cancer diagnosis. Reflectance confocal microscopy (RCM) is an emerging tool for detection of skin cancers in vivo. However, visual localization of the DEJ in RCM images, with high accuracy and repeatability, is challenging, especially in fair skin, due to low contrast, heterogeneous structure and high inter- and intra-subject variability. We recently proposed a semi-automated algorithm to localize the DEJ in z-stacks of RCM images of fair skin, based on feature segmentation and classification. Here we extend the algorithm to dark skin. The extended algorithm first decides the skin type and then applies the appropriate DEJ localization method. In dark skin, strong backscatter from the pigment melanin causes the basal cells above the DEJ to appear with high contrast. To locate those high contrast regions, the algorithm operates on small tiles (regions) and finds the peaks of the smoothed average intensity depth profile of each tile. However, for some tiles, due to heterogeneity, multiple peaks in the depth profile exist and the strongest peak might not be the basal layer peak. To select the correct peak, basal cells are represented with a vector of texture features. The peak with most similar features to this feature vector is selected. The results show that the algorithm detected the skin types correctly for all 17 stacks tested (8 fair, 9 dark). The DEJ detection algorithm achieved an average distance from the ground truth DEJ surface of around 4.7μm for dark skin and around 7-14μm for fair skin.

  5. Three-dimensional computer reconstruction of large tissue volumes based on composing series of high-resolution confocal images by GlueMRC and LinkMRC software

    Czech Academy of Sciences Publication Activity Database

    Karen, Petr; Jirkovská, M.; Tomori, Z.; Demjénová, E.; Janáček, Jiří; Kubínová, Lucie

    2003-01-01

    Roč. 62, č. 5 (2003), s. 415-422 ISSN 1059-910X R&D Projects: GA ČR GA304/01/0257 Grant - others:VEGA(SK) 2/1146/21; CZ-SK GA MŠk(CZ) KONTAKT 126/184 Institutional research plan: CEZ:AV0Z5011922 Keywords : 3D reconstruction * confocal microscopy * image processing Subject RIV: JC - Computer Hardware ; Software Impact factor: 2.307, year: 2003

  6. Multiphotonic Confocal Microscopy 3D imaging: Application to mantle sulfides in sub-arc environment (Avacha Volcano, Kamchatka)

    Science.gov (United States)

    Antoine, Bénard; Luc-Serge, Doucet; Sabine, Palle; Dmitri A., Ionov

    2010-05-01

    Petrogenetic relations in igneous rocks are usually studied in natural samples using classical optical microscopy and subsequent geochemical data acquisition. Multiphotonic Laser Scanning Confocal Microscopy (MLSCM) can be a powerful tool to section geological materials optically with sub-micrometric resolution and then generate a three-dimensional (3D) reconstruction (ca. 106 μm3 stack). MLSCM is used here to investigate textural relations of Monosulfide Solid Solution (MSS) with silicate phases in fresh spinel harzburgite xenoliths from the andesitic Avacha volcano (Kamchatka, Russia). The xenoliths contain MSS disseminated in olivine and orthopyroxene (opx) neoblasts as well as MSS-rich quenched magmatic opx veins [1]. First, Reflection Mode (RM) was tested on vein sulfides in resin-impregnated thick (120 μm) polished rock sections. Then we used a combination of Differential Interference Contrast (DIC) with a transmitted light detector, two photons-excited fluorescence (2PEF) and Second Harmonic Generation (SHG). Sequential imaging feature of the Leica TCS-SP2 software was applied. The excitation laser used for 2PEF was a COHERENT MIRA 900 with a 76Hz repetition rate and 800nm wavelength. Image stacks were analysed using ImageJ software [2]. The aim of the tests was to try to discriminate sulfides in silicate matrix as a tool for a better assessment of equilibrium conditions between the two phases. Preliminary results show that Fe-Ni rich MSS from vein and host rock have a strong auto-fluorescence in the Near UV-VIS domain (392-715 nm) whereas silicate matrix is only revealed through DIC. SHG is obtained only from dense nanocentrosymmetrical structures such as embedded medium (organic matter like glue and resin). The three images were recorded sequentially enabling efficient discrimination between the different components of the rock slices. RM permits reconstruction of the complete 3D structure of the rock slice. High resolution (ca. 0.2 μm along X-Y axis vs

  7. Tumor-specific antivascular effect of TZT-1027 (Soblidotin) elucidated by magnetic resonance imaging and confocal laser scanning microscopy

    International Nuclear Information System (INIS)

    Natsume, Tsugitaka; Watanabe, Junichi; Kobayashi, Motohiro; Ogawa, Kenji; Yasumura, Kazuhiko

    2007-01-01

    TZT-1027 (soblidotin), an antimicrotubule agent, has previously been evaluated in terms of its antivascular effects. In this study, Evans blue perfusion, magnetic resonance imaging (MRI), and confocal laser scanning microscopy (CLSM) were utilized to further elucidate the antivascular effect of TZT-1027 in female nude mice and rats bearing human breast tumor MX-1, as well as in female Sprague-Dawley rats that developed breast tumors induced by dimethylbenz(a)anthracene (DMBA). Therapeutic doses of TZT-1027 caused nearly complete regression of implanted MX-1 tumors in nude mice and rats as well as DMBA-induced tumors in rats. The perfusion in MX-1 tumor implanted in nude mice was drastically reduced within 30 min after TZT-1027 administration and was completely inhibited after 6 h or more, although not reduced in normal tissue of kidney. The study using MRI demonstrated that rich blood flow within tumors was remarkably reduced 1-3 h after TZT-1027 administration both in nude rats bearing MX-1 tumors and in rats with DMBA-induced tumors. Furthermore, the study with CLSM in nude mice bearing MX-1 tumors revealed a disruption of tumor microvessels at 1 h and a destruction of tumor microvessel network at 3 h after TZT-1027 administration. In contrast, these types of vascular disorders were not observed in heart and kidney. These results suggest that TZT-1027 specifically damages tumor vasculatures, leading to extensive tumor necrosis within tolerable dose range, and confirms earlier observations that TZT-1027 exerts a considerable antivascular effect in addition to an excellent cytotoxic effect. (author)

  8. High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy.

    Science.gov (United States)

    Manfredini, Marco; Arginelli, Federica; Dunsby, Christopher; French, Paul; Talbot, Clifford; König, Karsten; Pellacani, Giovanni; Ponti, Giovanni; Seidenari, Stefania

    2013-02-01

    The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view. © 2012 John Wiley & Sons A/S.

  9. Confocal absorption spectral imaging of MoS2: optical transitions depending on the atomic thickness of intrinsic and chemically doped MoS2.

    Science.gov (United States)

    Dhakal, Krishna P; Duong, Dinh Loc; Lee, Jubok; Nam, Honggi; Kim, Minsu; Kan, Min; Lee, Young Hee; Kim, Jeongyong

    2014-11-07

    We performed a nanoscale confocal absorption spectral imaging to obtain the full absorption spectra (over the range 1.5-3.2 eV) within regions having different numbers of layers and studied the variation of optical transition depending on the atomic thickness of the MoS2 film. Three distinct absorption bands corresponding to A and B excitons and a high-energy background (BG) peak at 2.84 eV displayed a gradual redshift as the MoS2 film thickness increased from the monolayer, to the bilayer, to the bulk MoS2 and this shift was attributed to the reduction of the gap energy in the Brillouin zone at the K-point as the atomic thickness increased. We also performed n-type chemical doping of MoS2 films using reduced benzyl viologen (BV) and the confocal absorption spectra modified by the doping showed a strong dependence on the atomic thickness: A and B exciton peaks were greatly quenched in the monolayer MoS2 while much less effect was shown in larger thickness and the BG peak either showed very small quenching for 1 L MoS2 or remained constant for larger thicknesses. Our results indicate that confocal absorption spectral imaging can provide comprehensive information on optical transitions of microscopic size intrinsic and doped two-dimensional layered materials.

  10. Simultaneous measurement of internal and surrounding flows of a moving droplet using multicolour confocal micro-particle image velocimetry (micro-PIV)

    International Nuclear Information System (INIS)

    Oishi, M; Kinoshita, H; Fujii, T; Oshima, M

    2011-01-01

    This paper presents a micro-multiphase flow measurement technique, 'multicolour confocal micro-particle image velocimetry (PIV), and its application to the internal and surrounding flow measurement of a droplet moving through a microchannel. The present system measures the dynamic interaction between flows in two different phases, such as solid–liquid or liquid–liquid, simultaneously and separately. Unlike conventional confocal micro-PIV, this system features a wavelength separation optical device. The optical components (e.g., filters and dichroic mirror) are designed to separate fluorescent lights of tracer particles and to eliminate unnecessary scattered light depending on the characteristic wavelengths. The system can record a sequence of images at up to 2000 frames per second. It also has an in-plane spatial resolution of 0.284 µm/pixel in a field of 227.2 µm × 170.4 µm and a confocal depth of 3.43 µm using 1.0 µm particles and a 40× objective lens. This paper examines the performance of the present system, such as its ability to separate wavelengths. Furthermore, this system is applied to liquid–liquid two-phase flow, which consists of a water droplet and surrounding oil flow, in a microchannel. We succeeded in measuring each phase movement separately and simultaneously. As a result of the estimation of the out-of-plane velocity component, a three-dimensional flow structure is obtained and the interaction between each phase is investigated

  11. Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique

    International Nuclear Information System (INIS)

    Lamprecht, C; Unterauer, B; Plochberger, B; Brameshuber, M; Hinterdorfer, P; Ebner, A; Gierlinger, N; Hild, S; Heister, E

    2012-01-01

    The uptake of carbon nanotubes (CNTs) by mammalian cells and their distribution within cells is being widely studied in recent years due to their increasing use for biomedical purposes. The two main imaging techniques used are confocal fluorescence microscopy and transmission electron microscopy (TEM). The former, however, requires labeling of the CNTs with fluorescent dyes, while the latter is a work-intensive technique that is unsuitable for in situ bio-imaging. Raman spectroscopy, on the other hand, presents a direct, straightforward and label-free alternative. Confocal Raman microscopy can be used to image the CNTs inside cells, exploiting the strong Raman signal connected to different vibrational modes of the nanotubes. In addition, cellular components, such as the endoplasmic reticulum and the nucleus, can be mapped. We first validate our method by showing that only when using the CNTs’ G band for intracellular mapping accurate results can be obtained, as mapping of the radial breathing mode (RBM) only shows a small fraction of CNTs. We then take a closer look at the exact localization of the nanotubes inside cells after folate receptor-mediated endocytosis and show that, after 8-10 h incubation, the majority of CNTs are localized around the nucleus. In summary, Raman imaging has enormous potential for imaging CNTs inside cells, which is yet to be fully realized. (paper)

  12. The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images

    DEFF Research Database (Denmark)

    Hidayat, Budi J.; Weisskopf, Carmen; Felby, Claus

    2015-01-01

    or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method...

  13. Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots.

    Directory of Open Access Journals (Sweden)

    Anje Sporbert

    Full Text Available This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.

  14. Confocal Microscopy and Image Analysis Indicates a Region-Specific Relation between Active Caspases and Cytoplasm in Ejaculated and Epididymal Sperm

    Science.gov (United States)

    García Vazquez, Susana; Aragón Martínez, Andrés; Flores-Alonso, Juan Carlos

    2012-01-01

    Previously, it was suggested a relation between the presence of apoptosis markers with cytoplasm in mammalian sperm. In this work, flow cytometry, confocal microscopy and image analysis were used to analyze the relationship between active caspase-3 and -7 and intracellular esterases expression in ejaculated or epididymal ram sperm. Sperm obtained from ejaculates from the caput, corpus, or cauda of the epididymis were treated with an inhibitor of active caspase-3 and -7 and a marker of cytoplasmic esterases. Additionally, ejaculated sperm were incubated for one, two, or three hours before evaluation for active caspases. Sperm subpopulations positive for active caspases and/or intracellular esterases were detected by flow cytometry and confocal microscopy; however, image analysis of confocal images showed that the correlation between active caspases and cytoplasmic esterases in sperm is region-specific. Lower values of Spearman correlation coefficients were found when whole sperm or head sperm was analyzed; however, a high correlation was observed for midpiece sperm. Incubation of sperm for two or three hours promoted the autoactivation of caspases. It has been suggested that the presence of apoptotic markers in sperm are related with a process of abortive apoptosis and with errors during spermiogenesis. Our results permit us suggest that the origin of the relationship between active caspases and cytoplasmic esterases is due to differentiation errors occurring during spermiogenesis because the percentages of sperm with active caspases are not different in the caput, corpus, or cauda of the epididymis. In this study we demonstrate that existing sperm subpopulations can express active caspases and intracellular esterases and that the correlation between these molecules is high in midpiece sperm. PMID:22530029

  15. Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

    Science.gov (United States)

    Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

    2017-03-27

    Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

  16. Synthesis and Evaluation of Novel Imaging Probes for the Study of Glycosylation and Fatty Acid Uptake In Vivo

    OpenAIRE

    Cohen, Allison Stacey

    2011-01-01

    Imaging represents a powerful method for advancing our understanding of biology. In particular, it has been used as a tool for the diagnosis and monitoring of diseases in vivo. Bioluminescence imaging (BLI) represents one of the molecular imaging modalities and has been applied to the study of numerous processes in cells and in animals. However, there is a need for the design of new bioluminescence imaging probes for the study of several key metabolic processes. Activatable bioluminescenc...

  17. 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.

  18. New algorithm to determine true colocalization in combination with image restoration and time-lapse confocal microscopy to MAP kinases in mitochondria.

    Directory of Open Access Journals (Sweden)

    Jorge Ignacio Villalta

    Full Text Available The subcellular localization and physiological functions of biomolecules are closely related and thus it is crucial to precisely determine the distribution of different molecules inside the intracellular structures. This is frequently accomplished by fluorescence microscopy with well-characterized markers and posterior evaluation of the signal colocalization. Rigorous study of colocalization requires statistical analysis of the data, albeit yet no single technique has been established as a standard method. Indeed, the few methods currently available are only accurate in images with particular characteristics. Here, we introduce a new algorithm to automatically obtain the true colocalization between images that is suitable for a wide variety of biological situations. To proceed, the algorithm contemplates the individual contribution of each pixel's fluorescence intensity in a pair of images to the overall Pearsońs correlation and Manders' overlap coefficients. The accuracy and reliability of the algorithm was validated on both simulated and real images that reflected the characteristics of a range of biological samples. We used this algorithm in combination with image restoration by deconvolution and time-lapse confocal microscopy to address the localization of MEK1 in the mitochondria of different cell lines. Appraising the previously described behavior of Akt1 corroborated the reliability of the combined use of these techniques. Together, the present work provides a novel statistical approach to accurately and reliably determine the colocalization in a variety of biological images.

  19. Multimodality optical coherence tomography and fluorescence confocal scanning laser ophthalmoscopy for image-guided feedback of intraocular injections in mouse models

    Science.gov (United States)

    Benavides, Oscar R.; Terrones, Benjamin D.; Leeburg, Kelsey C.; Mehanathan, Sankarathi B.; Levine, Edward M.; Tao, Yuankai K.

    2018-02-01

    Rodent models are robust tools for understanding human retinal disease and function because of their similarities with human physiology and anatomy and availability of genetic mutants. Optical coherence tomography (OCT) has been well-established for ophthalmic imaging in rodents and enables depth-resolved visualization of structures and image-based surrogate biomarkers of disease. Similarly, fluorescence confocal scanning laser ophthalmoscopy (cSLO) has demonstrated utility for imaging endogenous and exogenous fluorescence and scattering contrast in the mouse retina. Complementary volumetric scattering and en face fluorescence contrast from OCT and cSLO, respectively, enables cellular-resolution longitudinal imaging of changes in ophthalmic structure and function. We present a non-contact multimodal OCT+cSLO small animal imaging system with extended working distance to the pupil, which enables imaging during and after intraocular injection. While injections are routinely performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the location and volume delivered is not precisely controlled and difficult to reproduce. Animals were imaged using a custom-built OCT engine and scan-head combined with a modified commercial cSLO scan-head. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. When combined with imagesegmentation, we believe OCT can be used to precisely identify injection locations and quantify injection volumes. Fluorescence cSLO can provide complementary contrast for either fluorescently labeled compounds or transgenic cells for improved specificity. Our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections, which may be used for real-time image-guided injections.

  20. Structure/functional aspects of the human riboflavin transporter-3 (SLC52A3): role of the predicted glycosylation and substrate-interacting sites.

    Science.gov (United States)

    Subramanian, Veedamali S; Sabui, Subrata; Teafatiller, Trevor; Bohl, Jennifer A; Said, Hamid M

    2017-08-01

    The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted N -glycosylation sites at Asn 94 and Asn 168 led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser 16 , Ile 20 , Trp 24 , Phe 142 , Thr 314 , and Asn 315 Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe 142 mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.

  1. A multimodal microcharacterisation of trace-element zonation and crystallographic orientation in natural cassiterite by combining cathodoluminescence, EBSD, EPMA and contribution of confocal Raman-in-SEM imaging.

    Science.gov (United States)

    Wille, G; Lerouge, C; Schmidt, U

    2018-01-16

    In cassiterite, tin is associated with metals (titanium, niobium, tantalum, indium, tungsten, iron, manganese, mercury). Knowledge of mineral chemistry and trace-element distribution is essential for: the understanding of ore formation, the exploration phase, the feasibility of ore treatment, and disposal/treatment of tailings after the exploitation phase. However, the availability of analytical methods make these characterisations difficult. We present a multitechnical approach to chemical and structural data that includes scanning electron microscopy (SEM)-based imaging and microanalysis techniques such as: secondary and backscattered electrons, cathodoluminescence (CL), electron probe microanalyser (EPMA), electron backscattered diffraction (EBSD) and confocal Raman-imaging integrated in a SEM (RISE). The presented results show the complementarity of the used analytical techniques. SEM, CL, EBSD, EPMA provide information from the interaction of an electron beam with minerals, leading to atomistic information about their composition, whereas RISE, Raman spectroscopy and imaging completes the studies with information about molecular vibrations, which are sensitive to structural modifications of the minerals. The correlation of Raman bands with the presence/absence of Nb, Ta, Fe (heterovalent substitution) and Ti (homovalent substitution) is established at a submicrometric scale. Combination of the different techniques makes it possible to establish a direct link between chemical and crystallographic data of cassiterite. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

  2. Improving diagnostic sensitivity of combined dermoscopy and reflectance confocal microscopy imaging through double reader concordance evaluation in telemedicine settings: A retrospective study of 1000 equivocal cases.

    Directory of Open Access Journals (Sweden)

    A M Witkowski

    Full Text Available Reflectance confocal microscopy (RCM is an imaging device that permits non-invasive visualization of cellular morphology and has been shown to improve diagnostic accuracy of dermoscopically equivocal cutaneous lesions. The application of double reader concordance evaluation of dermoscopy-RCM image sets in retrospective settings and its potential application to telemedicine evaluation has not been tested in a large study population.To improve diagnostic sensitivity of RCM image diagnosis using a double reader concordance evaluation approach; to reduce mismanagement of equivocal cutaneous lesions in retrospective consultation and telemedicine settings.1000 combined dermoscopy-RCM image sets were evaluated in blind by 10 readers with advanced training and internship in dermoscopy and RCM evaluation. We compared sensitivity and specificity of single reader evaluation versus double reader concordance evaluation as well as the effect of diagnostic confidence on lesion management in a retrospective setting.Single reader evaluation resulted in an overall sensitivity of 95.2% and specificity of 76.3%, with misdiagnosis of 8 melanomas, 4 basal cell carcinomas and 2 squamous cell carcinomas. Combined double reader evaluation resulted in an overall sensitivity of 98.3% and specificity of 65.5%, with misdiagnosis of 1 in-situ melanoma and 2 basal cell carcinomas.Evaluation of dermoscopy-RCM image sets of cutaneous lesions by single reader evaluation in retrospective settings is limited by sensitivity levels that may result in potential mismanagement of malignant lesions. Double reader blind concordance evaluation may improve the sensitivity of diagnosis and management safety. The use of a second check can be implemented in telemedicine settings where expert consultation and second opinions may be required.

  3. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique

    Directory of Open Access Journals (Sweden)

    Zheng Ming H

    2008-07-01

    the collagen network in the superficial zone during early physiological alteration of articular cartilage. The fibre confocal imaging technology used in this study has allowed developing confocal arthroscopy for in vivo studying the chondrocytes in different depth of articular cartilage. Therefore, the current study has potential to develop an in vivo 3D histology for diagnosis of early osteoarthritis.

  4. Ultrasensitive and selective gold film-based detection of mercury (II) in tap water using a laser scanning confocal imaging-surface plasmon resonance system in real time.

    Science.gov (United States)

    Zhang, Hongyan; Yang, Liquan; Zhou, Bingjiang; Liu, Weimin; Ge, Jiechao; Wu, Jiasheng; Wang, Ying; Wang, Pengfei

    2013-09-15

    An ultrasensitive and selective detection of mercury (II) was investigated using a laser scanning confocal imaging-surface plasmon resonance system (LSCI-SPR). The detection limit was as low as 0.01ng/ml for Hg(2+) ions in ultrapure and tap water based on a T-rich, single-stranded DNA (ssDNA)-modified gold film, which can be individually manipulated using specific T-Hg(2+)-T complex formation. The quenching intensity of the fluorescence images for rhodamine-labeled ssDNA fitted well with the changes in SPR. The changes varied with the Hg(2+) ion concentration, which is unaffected by the presence of other metal ions. The coefficients obtained for ultrapure and tap water were 0.99902 and 0.99512, respectively, for the linear part over a range of 0.01-100ng/ml. The results show that the double-effect sensor has potential for practical applications with ultra sensitivity and selectivity, especially in online or real-time monitoring of Hg(2+) ions pollution in tap water with the further improvement of portable LSCI-SPR instrument. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. A Marked Poisson Process Driven Latent Shape Model for 3D Segmentation of Reflectance Confocal Microscopy Image Stacks of Human Skin

    Science.gov (United States)

    Ghanta, Sindhu; Jordan, Michael I.; Kose, Kivanc; Brooks, Dana H.; Rajadhyaksha, Milind; Dy, Jennifer G.

    2016-01-01

    Segmenting objects of interest from 3D datasets is a common problem encountered in biological data. Small field of view and intrinsic biological variability combined with optically subtle changes of intensity, resolution and low contrast in images make the task of segmentation difficult, especially for microscopy of unstained living or freshly excised thick tissues. Incorporating shape information in addition to the appearance of the object of interest can often help improve segmentation performance. However, shapes of objects in tissue can be highly variable and design of a flexible shape model that encompasses these variations is challenging. To address such complex segmentation problems, we propose a unified probabilistic framework that can incorporate the uncertainty associated with complex shapes, variable appearance and unknown locations. The driving application which inspired the development of this framework is a biologically important segmentation problem: the task of automatically detecting and segmenting the dermal-epidermal junction (DEJ) in 3D reflectance confocal microscopy (RCM) images of human skin. RCM imaging allows noninvasive observation of cellular, nuclear and morphological detail. The DEJ is an important morphological feature as it is where disorder, disease and cancer usually start. Detecting the DEJ is challenging because it is a 2D surface in a 3D volume which has strong but highly variable number of irregularly spaced and variably shaped “peaks and valleys”. In addition, RCM imaging resolution, contrast and intensity vary with depth. Thus a prior model needs to incorporate the intrinsic structure while allowing variability in essentially all its parameters. We propose a model which can incorporate objects of interest with complex shapes and variable appearance in an unsupervised setting by utilizing domain knowledge to build appropriate priors of the model. Our novel strategy to model this structure combines a spatial Poisson process

  6. Safety and performance analysis of acriflavine and methylene blue for in vivo imaging of precancerous lesions using fibered confocal fluorescence microscopy (FCFM): an experimental study.

    Science.gov (United States)

    Obstoy, Bérengère; Salaun, Mathieu; Veresezan, Liana; Sesboüé, Richard; Bohn, Pierre; Boland, François-Xavier; Thiberville, Luc

    2015-03-31

    Fibered confocal fluorescence microscopy (FCFM) allows in vivo investigation of pulmonary microstructures. However, the bronchial epithelium can only be imaged using exogenous fluorophores. The objective of this study is to compare methylene blue (MB) and acriflavine genotoxicity and to assess FCFM performance for in vivo imaging of precancerous lesions. Genotoxicity was assessed using the comet assay on both cultured human lymphocytes and NCI-H460 cells, which had been exposed to MB or acriflavine before being illuminated at 660 or 488 nm, respectively. FCFM was performed on precancerous lesions in the hamster cheek pouch model, following topical application of the fluorophores. FCFM data were analyzed according to histology. No genotoxicity was found using 0.01% (w/v) MB after illumination at 660 nm for 2 and 15 min (5 mW). Acriflavine exposure (0.025%) led to DNA damages, increasing from 2 to 15 min of light exposure at 448 nm in both lymphocytes (83.4 to 88%, p = 0.021) and NCI H460 cell populations (79.9 to 84.6%, p = 0.045). In total, 11 invasive carcinoma, 24 reserve cell hyperplasia, and 17 dysplasia lesions were imaged using FCFM in vivo. With both fluorophores, the cellular density increased from hyperplasia to high-grade dysplasia (p < 0.05). With MB, the cellular diameter significantly decreased (48.9 to 13.9 μm) from hyperplasia to carcinoma (p < 0.05). In this model, a cut-off diameter of 30 μm enabled the diagnosis of high-grade lesions with a sensitivity of 94.7% and a specificity of 97%. Methylene blue can be used safely to image precancerous lesions in vivo. This study does not support the use of acriflavine in humans.

  7. Confocal Adaptive Optics Imaging of Peripapillary Nerve Fiber Bundles: Implications for Glaucomatous Damage Seen on Circumpapillary OCT Scans.

    Science.gov (United States)

    Hood, Donald C; Chen, Monica F; Lee, Dongwon; Epstein, Benjamin; Alhadeff, Paula; Rosen, Richard B; Ritch, Robert; Dubra, Alfredo; Chui, Toco Y P

    2015-04-01

    To improve our understanding of glaucomatous damage as seen on circumpapillary disc scans obtained with frequency-domain optical coherence tomography (fdOCT), fdOCT scans were compared to images of the peripapillary retinal nerve fiber (RNF) bundles obtained with an adaptive optics-scanning light ophthalmoscope (AO-SLO). The AO-SLO images and fdOCT scans were obtained on 6 eyes of 6 patients with deep arcuate defects (5 points ≤-15 db) on 10-2 visual fields. The AO-SLO images were montaged and aligned with the fdOCT images to compare the RNF bundles seen with AO-SLO to the RNF layer thickness measured with fdOCT. All 6 eyes had an abnormally thin (1% confidence limit) RNF layer (RNFL) on fdOCT and abnormal (hyporeflective) regions of RNF bundles on AO-SLO in corresponding regions. However, regions of abnormal, but equal, RNFL thickness on fdOCT scans varied in appearance on AO-SLO images. These regions could be largely devoid of RNF bundles (5 eyes), have abnormal-appearing bundles of lower contrast (6 eyes), or have isolated areas with a few relatively normal-appearing bundles (2 eyes). There also were local variations in reflectivity of the fdOCT RNFL that corresponded to the variations in AO-SLO RNF bundle appearance. Relatively similar 10-2 defects with similar fdOCT RNFL thickness profiles can have very different degrees of RNF bundle damage as seen on fdOCT and AO-SLO. While the results point to limitations of fdOCT RNFL thickness as typically analyzed, they also illustrate the potential for improving fdOCT by attending to variations in local intensity.

  8. Characterization of X-linked Hypohidrotic Ectodermal Dysplasia (XL-HED) Hair and Sweat Gland Phenotypes Using Phototrichogram Analysis and Live Confocal Imaging

    Science.gov (United States)

    Jones, Kyle B.; Goodwin, Alice F.; Landan, Maya; Seidel, Kerstin; Tran, Dong-Kha; Hogue, Jacob; Chavez, Miquella; Fete, Mary; Yu, Wenli; Hussein, Tarek; Johnson, Ramsey; Huttner, Kenneth; Jheon, Andrew H.; Klein, Ophir D.

    2015-01-01

    Hypohidrotic ectodermal dysplasia (HED) is the most common type of ectodermal dysplasia (ED), which encompasses a large group of syndromes that share several phenotypic features such as missing or malformed ectodermal structures, including skin, hair, sweat glands, and teeth. X-linked hypohidrotic ectodermal dysplasia (XL-HED) is associated with mutations in ectodysplasin (EDA1). Hypohidrosis due to hypoplastic sweat glands and thin, sparse hair are phenotypic features that significantly affect the daily lives of XL-HED individuals and therefore require systematic analysis. We sought to determine the quality of life of individuals with XL-HED and to quantify sweat duct and hair phenotypes using confocal imaging, pilocarpine iontophoresis, and phototrichogram analysis. Using these highly sensitive and non-invasive techniques, we demonstrated that 11/12 XL-HED individuals presented with a complete absence of sweat ducts and that none produced sweat. We determined that the thin hair phenotype observed in XL-HED was due to multiple factors, such as fewer terminal hairs with decreased thickness and slower growth rate, as well as fewer follicular units and fewer hairs per unit. The precise characterization of XL-HED phenotypes using sensitive and non-invasive techniques presented in our study will improve upon larger genotype-phenotype studies and in the assessment of future therapies in XL-HED. PMID:23687000

  9. Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

    Science.gov (United States)

    König, Alexander; Glebe, Dieter

    2017-01-01

    To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

  10. A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.

    Science.gov (United States)

    Swearingen, Matthew C; Mehta, Ajeet; Mehta, Amar; Nistico, Laura; Hill, Preston J; Falzarano, Anthony R; Wozniak, Daniel J; Hall-Stoodley, Luanne; Stoodley, Paul

    2016-02-01

    Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Lipid droplets formation in human endothelial cells in response to polyunsaturated fatty acids and 1-methyl-nicotinamide (MNA); confocal Raman imaging and fluorescence microscopy studies.

    Science.gov (United States)

    Majzner, Katarzyna; Chlopicki, Stefan; Baranska, Malgorzata

    2016-04-01

    In this work the formation of lipid droplets (LDs) in human endothelial cells culture in response to the uptake of polyunsaturated fatty acids (PUFAs) was studied. Additionally, an effect of 1-methylnicotinamide (MNA) on the process of LDs formation was investigated. LDs have been previously described structurally and to some degree biochemically, however neither the precise function of LDs nor the factors responsible for LD induction have been clarified. Lipid droplets, sometimes referred in the literature as lipid bodies are organelles known to regulate neutrophil, eosinophil, or tumor cell functions but their presence and function in the endothelium is largely unexplored. 3D linear Raman spectroscopy was used to study LDs formation in vitro in a single endothelial cell. The method provides information about distribution and size of LDs as well as their composition. The incubation of endothelial cells with various PUFAs resulted in formation of LDs. As a complementary method for LDs identification a fluorescence microscopy was applied. Fluorescence measurements confirmed the Raman results suggesting endothelial cells uptake of PUFAs and subsequent LDs formation in the cytoplasm of the endothelium. Furthermore, MNA seem to potentiate intracellular uptake of PUFAs to the endothelium that may bear physiological and pharmacological significance. Confocal Raman imaging of HAoEC cell with LDs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Hallmarks of glycosylation in cancer.

    Science.gov (United States)

    Munkley, Jennifer; Elliott, David J

    2016-06-07

    Aberrant glycosylation plays a fundamental role in key pathological steps of tumour development and progression. Glycans have roles in cancer cell signalling, tumour cell dissociation and invasion, cell-matrix interactions, angiogenesis, metastasis and immune modulation. Aberrant glycosylation is often cited as a 'hallmark of cancer' but is notably absent from both the original hallmarks of cancer and from the next generation of emerging hallmarks. This review discusses how glycosylation is clearly an enabling characteristic that is causally associated with the acquisition of all the hallmark capabilities. Rather than aberrant glycosylation being itself a hallmark of cancer, another perspective is that glycans play a role in every recognised cancer hallmark.

  13. Sensitivity and specificity of a new scoring system for diabetic macular oedema detection using a confocal laser imaging system

    Science.gov (United States)

    Tong, L; Ang, A; Vernon, S; Zambarakji, H; Bhan, A; Sung, V; Page, S

    2001-01-01

    AIM—To assess the use of the Heidelberg retina tomograph (HRT) in screening for sight threatening diabetic macular oedema in a hospital diabetic clinic, using a new subjective analysis system (SCORE).
METHODS—200 eyes of 100 consecutive diabetic patients attending a diabetologist's clinic were studied, all eyes had an acuity of 6/9 or better. All patients underwent clinical examination by an ophthalmologist. Using the HRT, one good scan was obtained for each eye centred on the fovea. A System for Classification and Ordering of Retinal Edema (SCORE) was developed using subjective assessment of the colour map and the reflectivity image. The interobserver agreement of using this method to detect macular oedema was assessed by two observers (ophthalmic trainees) who were familiarised with SCORE by studying standard pictures of eyes not in the study. All scans were graded from 0-6 and test positive cases were defined as having a SCORE value of 0-2. The sensitivity of SCORE was assessed by pooling the data with an additional 88 scans of 88 eyes in order to reduce the confidence interval of the index.
RESULTS—12 eyes in eight out of the 100 patients had macular oedema clinically. Three scans in three patients could not be analysed because of poor scan quality. In the additional group of scans 76 out of 88 eyes had macular oedema clinically. The scoring system had a specificity of 99% (95% CI 96-100) and sensitivity of 67% (95% CI 57-76). The predictive value of a negative test was 87% (95% CI 82-99), and that of a positive test was 95% (95% CI 86-99). The mean difference of the SCORE value between two observers was -0.2 (95% CI -0.5 to +0.07).
CONCLUSIONS—These data suggest that SCORE is potentially useful for detecting diabetic macular oedema in hospital diabetic patients.

 PMID:11133709

  14. Re-scan confocal microscopy: scanning twice for better resolution.

    Science.gov (United States)

    De Luca, Giulia M R; Breedijk, Ronald M P; Brandt, Rick A J; Zeelenberg, Christiaan H C; de Jong, Babette E; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A; Stallinga, Sjoerd; Manders, Erik M M

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

  15. 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.

  16. 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...

  17. Confocal filtering in cathodoluminescence microscopy of nanostructures

    Science.gov (United States)

    Narváez, Angela C.; Weppelman, I. Gerward C.; Moerland, Robert J.; Hoogenboom, Jacob P.; Kruit, Pieter

    2014-06-01

    Cathodoluminescence (CL) microscopy allows optical characterization of nanostructures at high spatial resolution. At the nanoscale, a main challenge of the technique is related to the background CL generated within the sample substrate. Here, we implement confocal detection of the CL signal to minimize the background contribution to the measurement. Nano-phosphors were used as point sources to evaluate the filtering capabilities of our confocal CL system, obtaining an axial intensity profile with 2.7 μm full width at half maximum for the central peak, in good correspondence with theoretical expectations. Considering the electron interaction volume, we found that the confocal filter becomes effective for electron energies above 20 keV, when using a 25 μm pinhole (0.86 Airy units). To illustrate our approach, we present confocal CL imaging of gold nanowires and triangular shaped plates deposited on an indium-tin oxide covered glass substrate, comparing the images with those obtained in standard unfiltered CL detection. The results show that confocal CL microscopy is a valuable tool for the investigation of nanostructures on highly cathodoluminescent substrates, widely used in biological and optical applications.

  18. Confocal filtering in cathodoluminescence microscopy of nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Narváez, Angela C., E-mail: a.c.narvaez@tudelft.nl, E-mail: j.p.hoogenboom@tudelft.nl; Weppelman, I. Gerward C.; Moerland, Robert J.; Hoogenboom, Jacob P., E-mail: a.c.narvaez@tudelft.nl, E-mail: j.p.hoogenboom@tudelft.nl; Kruit, Pieter [Imaging Physics, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628CJ Delft (Netherlands)

    2014-06-23

    Cathodoluminescence (CL) microscopy allows optical characterization of nanostructures at high spatial resolution. At the nanoscale, a main challenge of the technique is related to the background CL generated within the sample substrate. Here, we implement confocal detection of the CL signal to minimize the background contribution to the measurement. Nano-phosphors were used as point sources to evaluate the filtering capabilities of our confocal CL system, obtaining an axial intensity profile with 2.7 μm full width at half maximum for the central peak, in good correspondence with theoretical expectations. Considering the electron interaction volume, we found that the confocal filter becomes effective for electron energies above 20 keV, when using a 25 μm pinhole (0.86 Airy units). To illustrate our approach, we present confocal CL imaging of gold nanowires and triangular shaped plates deposited on an indium-tin oxide covered glass substrate, comparing the images with those obtained in standard unfiltered CL detection. The results show that confocal CL microscopy is a valuable tool for the investigation of nanostructures on highly cathodoluminescent substrates, widely used in biological and optical applications.

  19. Integrated Photoacoustic and Fluorescence Confocal Microscopy

    OpenAIRE

    Wang, Yu; Maslov, Konstantin; Kim, Chulhong; Hu, Song; Wang, Lihong V.

    2010-01-01

    We have developed a dual-modality imaging system by integrating optical-resolution photoacoustic microscopy and fluorescence confocal microscopy to provide optical absorption and fluorescence contrasts simultaneously. By sharing the same laser source and objective lens, intrinsically registered photoacoustic and fluorescence images are acquired in a single scan. The micrometer resolution allows imaging of both blood and lymphatic vessels down to the capillary level. Simultaneous photoacoustic...

  20. The new confocal heavy ion microprobe beamline at ANSTO: The first microprobe resolution tests and applications for elemental imaging and analysis

    Science.gov (United States)

    Pastuovic, Z.; Siegele, R.; Cohen, D. D.; Mann, M.; Ionescu, M.; Button, D.; Long, S.

    2017-08-01

    The Centre for Accelerator Science facility at ANSTO has been expanded with the new NEC 6 MV ;SIRIUS; accelerator system in 2015. In this paper we present a detailed description of the new nuclear microprobe-Confocal Heavy Ion Micro-Probe (CHIMP) together with results of the microprobe resolution testing and the elemental analysis performed on typical samples of mineral ore deposits and hyper-accumulating plants regularly measured at ANSTO. The CHIMP focusing and scanning systems are based on the OM-150 Oxford quadrupole triplet and the OM-26 separated scan-coil doublet configurations. A maximum ion rigidity of 38.9 amu-MeV was determined for the following nuclear microprobe configuration: the distance from object aperture to collimating slits of 5890 mm, the working distance of 165 mm and the lens bore diameter of 11 mm. The overall distance from the object to the image plane is 7138 mm. The CHIMP beamline has been tested with the 3 MeV H+ and 6 MeV He2+ ion beams. The settings of the object and collimating apertures have been optimized using the WinTRAX simulation code for calculation of the optimum acceptance settings in order to obtain the highest possible ion current for beam spot sizes of 1 μm and 5 μm. For optimized aperture settings of the CHIMP the beam brightness was measured to be ∼0.9 pA μm-2 mrad-2 for 3 MeV H+ ions, while the brightness of ∼0.4 pA μm-2 mrad-2 was measured for 6 MeV He2+ ions. The smallest beam sizes were achieved using a microbeam with reduced particle rate of 1000 Hz passing through the object slit apertures several micrometers wide. Under these conditions a spatial resolution of ∼0.6 μm × 1.5 μm for 3 MeV H+ and ∼1.8 μm × 1.8 μm for 6 MeV He2+ microbeams in horizontal (and vertical) dimension has been achieved. The beam sizes were verified using STIM imaging on 2000 and 1000 mesh Cu electron microscope grids.

  1. Confocal laser endomicroscopy

    DEFF Research Database (Denmark)

    Karstensen, John Gásdal; Săftoiu, Adrian; Brynskov, Jørn

    2016-01-01

    BACKGROUND AND STUDY AIMS: Confocal laser endomicroscopy (CLE) has been shown to predict relapse in ulcerative colitis in remission, but little is currently known about its role in Crohn's disease. The aim of this study was to identify reproducible CLE features in patients with Crohn's disease...

  2. 4Pi-confocal microscopy of live cells

    Science.gov (United States)

    Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

    2002-06-01

    By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

  3. Model wavefront sensor for adaptive confocal microscopy

    Science.gov (United States)

    Booth, Martin J.; Neil, Mark A. A.; Wilson, Tony

    2000-05-01

    A confocal microscope permits 3D imaging of volume objects by the inclusion of a pinhole in the detector path which eliminates out of focus light. This configuration is however very sensitive to aberrations induced by the specimen or the optical system and would therefore benefit from an adaptive optics approach. We present a wavefront sensor capable of measuring directly the Zernike components of an aberrated wavefront and show that it is particularly applicable to the confocal microscope since only those wavefronts originating in the focal region contribute to the measured aberration.

  4. 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...

  5. Confocal Endomicroscopy of Colorectal Polyps

    Directory of Open Access Journals (Sweden)

    Vivian M. Ussui

    2012-01-01

    Full Text Available Confocal laser endomicroscopy (CLE is one of several novel methods that provide real-time, high-resolution imaging at a micron scale via endoscopes. CLE has the potential to be a disruptive technology in that it can change the current algorithms that depend on biopsy to perform surveillance of high-risk conditions. Furthermore, it allows on-table decision making that has the potential to guide therapy in real time and reduce the need for repeated procedures. CLE and related technologies are often termed “virtual biopsy” as they simulate the images seen in traditional histology. However, the imaging of living tissue allows more than just pragmatic convenience; it also allows imaging of living tissue such as active capillary circulation, cellular death, and vascular and endothelial translocation, thus extending beyond what is capable in traditional biopsy. Immediate potential applications of CLE are to guide biopsy sampling in Barrett's esophagus and inflammatory bowel disease surveillance, evaluation of colorectal polyps, and intraductal imaging of the pancreas and bile duct. Data on these applications is rapidly emerging, and more is needed to clearly demonstrate the optimal applications of CLE. In this paper, we will focus on the role of CLE as applied to colorectal polyps detected during colonoscopy.

  6. Glycosylation: a hallmark of cancer?

    Science.gov (United States)

    Vajaria, Bhairavi N; Patel, Prabhudas S

    2017-04-01

    The hallmarks of cancer are characterized by functional capabilities that allow cancer cells to survive, proliferate and disseminate during the multistep tumorigenesis. Cancer being a cellular disease, changes in cellular glycoproteins play an important role in malignant transformation and cancer progression. The present review summarizes various studies that depicted correlation of glycosylation with tumor initiation, progression and metastasis, which are helpful in early diagnosis, disease monitoring and prognosis. The results are further strengthened by our reports, which depicted alterations in sialylation and fucosylation in different cancers. Alterations in glycosyltransferases are also involved in formation of various tumor antigens (e.g. Sialyl Lewis x) which serves as ligand for the cell adhesion molecule, selectin which is involved in adhesion of cancer cells to vascular endothelium and thus contributes to hematogenous metastasis. Increased glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC)s are also involved in loss of E-cadherin, a key molecule implicated in metastatic dissemination of cells. The present review also summarizes the correlation of glycosylation with all the hallmarks of cancer. The enormous progress in the design of novel inhibitors of pathway intermediates of sialylation and fucosylation can prove wonders in combating the dreadful disease. The results provide the evidence that altered glycosylation is linked to tumor initiation, progression and metastasis. Hence, it can be considered as a new hallmark of cancer development and strategies to develop novel glycosylation targeted molecules should be strengthened.

  7. Functional importance of PAI-1 glycosylation

    DEFF Research Database (Denmark)

    Christensen, Anni; Naessens, Dominik; Skottrup, Peter

    2001-01-01

    Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-glycosylated PAI-1 could be conferred upon PAI-1 expressed in HEK293 cells by mutational inactivation of one or the other glycosylation site. These findings reveal a novel functional role for glycosylation of a serpin. The glycosylation sites are localised between a-helix H and b-strand 2C and b-strand 3C and a...

  8. Molecular confocal laser endomicroscopy

    DEFF Research Database (Denmark)

    Karstensen, John Gásdal; Klausen, Pia Helene; Saftoiu, Adrian

    2014-01-01

    While flexible endoscopy is essential for macroscopic evaluation, confocal laser endomicroscopy (CLE) has recently emerged as an endoscopic method enabling visualization at a cellular level. Two systems are currently available, one based on miniprobes that can be inserted via a conventional...... during on-going endoscopy), a novel world of molecular evaluation opens up. The method of molecular CLE could potentially be used for estimating the expression of important receptors in carcinomas, subsequently resulting in immediate individualization of treatment regimens, but also for improving...

  9. 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.

  10. Confocal Raman microspectroscopy

    International Nuclear Information System (INIS)

    Puppels, G.J.

    1991-01-01

    Raman spectroscopy is a technique that provides detailed structural information about molecules studied. In the field of molecular biophysics it has been extensively used for characterization of nucleic acids and proteins and for investigation of interactions between these molecules. It was felt that this technique would have great potential if it could be applied for in situ study of these molecules and their interactions, at the level of single living cell or a chromosome. To make this possible a highly sensitive confocal Raman microspectrometer (CRM) was developed. The instrument is described in detail in this thesis. It incorporates a number of recent technological developments. First, it employs a liquid nitrogen cooled CCD-camera. This type of detector, first used in astronomy, is the ultimate detector for Raman spectroscopy because it combines high quantum efficiency light detection with photon-noise limited operation. Second, an important factor in obtaining a high signal throughput of the spectrometer was the development of a new type of Raman notch filter. In the third place, the confocal detection principle was applied in the CRM. This limits the effective measuring volume to 3 . (author). 279 refs., 48 figs., 11 tabs

  11. Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking.

    Science.gov (United States)

    Pinaud, Fabien; Michalet, Xavier; Iyer, Gopal; Margeat, Emmanuel; Moore, Hsiao-Ping; Weiss, Shimon

    2009-06-01

    Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

  12. A near-infrared confocal scanner

    International Nuclear Information System (INIS)

    Lee, Seungwoo; Yoo, Hongki

    2014-01-01

    In the semiconductor industry, manufacturing of three-dimensional (3D) packages or 3D integrated circuits is a high-performance technique that requires combining several functions in a small volume. Through-silicon vias, which are vertical electrical connections extending through a wafer, can be used to direct signals between stacked chips, thus increasing areal density by stacking and connecting multiple patterned chips. While defect detection is essential in the semiconductor manufacturing process, it is difficult to identify defects within a wafer or to monitor the bonding results between bonded surfaces because silicon and many other semiconductor materials are opaque to visible wavelengths. In this context, near-infrared (NIR) imaging is a promising non-destructive method to detect defects within silicon chips, to inspect bonding between chips and to monitor the chip alignment since NIR transmits through silicon. In addition, a confocal scanner provides high-contrast, optically-sectioned images of the specimen due to its ability to reject out-of-focus noise. In this study, we report an NIR confocal scanner that rapidly acquires high-resolution images with a large field of view through silicon. Two orthogonal line-scanning images can be acquired without rotating the system or the specimen by utilizing two orthogonally configured resonant scanning mirrors. This NIR confocal scanner can be efficiently used as an in-line inspection system when manufacturing semiconductor devices by rapidly detecting defects on and beneath the surface. (paper)

  13. Enhanced SCAP glycosylation by inflammation induces macrophage foam cell formation.

    Directory of Open Access Journals (Sweden)

    Chao Zhou

    Full Text Available Inflammatory stress promotes foam cell formation by disrupting LDL receptor feedback regulation in macrophages. Sterol Regulatory Element Binding Proteins (SREBPs Cleavage-Activating Protein (SCAP glycosylation plays crucial roles in regulating LDL receptor and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR feedback regulation. The present study was to investigate if inflammatory stress disrupts LDL receptor and HMGCoAR feedback regulation by affecting SCAP glycosylation in THP-1 macrophages. Intracellular cholesterol content was assessed by Oil Red O staining and quantitative assay. The expression of molecules controlling cholesterol homeostasis was examined using real-time quantitative RT-PCR and Western blotting. The translocation of SCAP from the endoplasmic reticulum (ER to the Golgi was detected by confocal microscopy. We demonstrated that exposure to inflammatory cytokines increased lipid accumulation in THP-1 macrophages, accompanying with an increased SCAP expression even in the presence of a high concentration of LDL. These inflammatory cytokines also prolonged the half-life of SCAP by enhancing glycosylation of SCAP due to the elevated expression of the Golgi mannosidase II. This may enhance translocation and recycling of SCAP between the ER and the Golgi, escorting more SREBP2 from the ER to the Golgi for activation by proteolytic cleavages as evidenced by an increased N-terminal of SREBP2 (active form. As a consequence, the LDL receptor and HMGCoAR expression were up-regulated. Interestingly, these effects could be blocked by inhibitors of Golgi mannosidases. Our results indicated that inflammation increased native LDL uptake and endogenous cholesterol de novo synthesis, thereby causing foam cell formation via increasing transcription and protein glycosylation of SCAP in macrophages. These data imply that inhibitors of Golgi processing enzymes might have a potential vascular-protective role in prevention of atherosclerotic foam

  14. Enzymatic glycosylation of multivalent scaffolds

    Czech Academy of Sciences Publication Activity Database

    Bojarová, Pavla; Rosencrantz, R. R.; Elling, L.; Křen, Vladimír

    2013-01-01

    Roč. 42, č. 11 (2013), s. 4774-4797 ISSN 0306-0012 R&D Projects: GA MŠk(CZ) LD13042; GA ČR GAP207/10/0321 Institutional support: RVO:61388971 Keywords : N-ACETYLGLUCOSAMINYLTRANSFERASE-III * MUCIN TANDEM REPEAT * NEIGHBORING RESIDUE GLYCOSYLATION Subject RIV: CC - Organic Chemistry Impact factor: 30.425, year: 2013

  15. Dual filtered backprojection for micro-rotation confocal microscopy

    International Nuclear Information System (INIS)

    Laksameethanasan, Danai; Brandt, Sami S; Renaud, Olivier; Shorte, Spencer L

    2009-01-01

    Micro-rotation confocal microscopy is a novel optical imaging technique which employs dielectric fields to trap and rotate individual cells to facilitate 3D fluorescence imaging using a confocal microscope. In contrast to computed tomography (CT) where an image can be modelled as parallel projection of an object, the ideal confocal image is recorded as a central slice of the object corresponding to the focal plane. In CT, the projection images and the 3D object are related by the Fourier slice theorem which states that the Fourier transform of a CT image is equal to the central slice of the Fourier transform of the 3D object. In the micro-rotation application, we have a dual form of this setting, i.e. the Fourier transform of the confocal image equals the parallel projection of the Fourier transform of the 3D object. Based on the observed duality, we present here the dual of the classical filtered back projection (FBP) algorithm and apply it in micro-rotation confocal imaging. Our experiments on real data demonstrate that the proposed method is a fast and reliable algorithm for the micro-rotation application, as FBP is for CT application

  16. High-resolution confocal imaging of wall ingrowth deposition in plant transfer cells: Semi-quantitative analysis of phloem parenchyma transfer cell development in leaf minor veins of Arabidopsis.

    Science.gov (United States)

    Nguyen, Suong T T; McCurdy, David W

    2015-04-23

    Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy. Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level. The simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically

  17. Continuous and simultaneous measurement of the tank-treading motion of red blood cells and the surrounding flow using translational confocal micro-particle image velocimetry (micro-PIV) with sub-micron resolution

    International Nuclear Information System (INIS)

    Oishi, M; Utsubo, K; Kinoshita, H; Fujii, T; Oshima, M

    2012-01-01

    In this study, a translational confocal micro-particle image velocimetry (PIV) system is introduced to measure the microscopic interaction between red blood cells (RBCs) and the surrounding flow. Since the macroscopic behavior of RBCs, such as the tank-treading motion, is closely related to the axial migration and other flow characteristics in arterioles, the measurement method must answer the conflicting demands of sub-micron resolution, continuous measurement and applicability for high-speed flow. In order to avoid loss of the measurement target, i.e. RBCs, from the narrow field of view during high-magnification measurement, the translation stage with the flow device moves in the direction opposite the direction of flow. The proposed system achieves the measurement of higher absolute velocities compared with a conventional confocal micro-PIV system without the drawbacks derived from stage vibration. In addition, we have applied a multicolor separation unit, which can measure different phases simultaneously using different fluorescent particles, in order to clarify the interaction between RBCs and the surrounding flow. Based on our measurements, the tank-treading motion of RBCs depends on the shear stress gradient of the surrounding flow. Although, the relationship between the tank-treading frequency and the shear rate of the surrounding flow is of the same order as in the previous uniform shear rate experiments, our results reveal the remarkable behavior of the non-uniform membrane velocities and lateral velocity component of flow around the RBCs. (paper)

  18. Nanomorphology of polythiophene–fullerene bulk-heterojunction films investigated by structured illumination optical imaging and time-resolved confocal microscopy

    International Nuclear Information System (INIS)

    Hao, X-T; Hirvonen, L M; Smith, T A

    2013-01-01

    Structured illumination microscopy (SIM) and time-resolved confocal fluorescence microscopy are applied to investigate the nanomorphology of thin films comprising typical blends of the conjugated polymer, poly (3-hexylthiophene) (P3HT), and [6, 6]-phenyl C 61 -butyric acid methyl ester (PCBM), used for organic photovoltaic applications. SIM provides evidence for the presence of a thin emissive region around the crystalline regions of PCBM and at the tips of rod-like domains. The time-resolved measurements show that the emission surrounding the PCBM rods is longer lived than the bulk of the film. The two modes of microscopy provide complementary evidence indicating that electron–hole separation is inhibited between the polymer and the large PCBM-rich domains in these regions. We show here that structured illumination microscopy is a viable method of gaining additional information from these photovoltaic materials, despite their weak emission. (paper)

  19. 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

  20. 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.

  1. N-glycosylation in sugarcane

    Directory of Open Access Journals (Sweden)

    Maia Ivan G.

    2001-01-01

    Full Text Available The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

  2. A clearer view of the insect brain – combining bleaching with standard whole-mount immunocytochemistry allows confocal imaging of pigment-covered brain areas for 3D reconstruction.

    Directory of Open Access Journals (Sweden)

    Anna Lisa Stöckl

    2015-09-01

    Full Text Available In the study of insect neuroanatomy, three-dimensional reconstructions of neurons and neuropils have become a standard technique. As images have to be obtained from whole-mount brain preparations, pigmentation on the brain surface poses a serious challenge to imaging. In insects, this is a major problematic in the first visual neuropil of the optic lobe, the lamina, which is obstructed by the pigment of the retina as well as by the pigmented fenestration layer. This has prevented inclusion of this major processing center of the insect visual system into most neuroanatomical brain atlases and hinders imaging of neurons within the lamina by confocal microscopy. It has recently been shown that hydrogen peroxide bleaching is compatible with immunohistochemical labeling in insect brains, and we therefore developed a simple technique for removal of pigments on the surface of insect brains by chemical bleaching. We show that our technique enables imaging of the pigment-obstructed regions of insect brains when combined with standard protocols for both anti-synapsin-labeled as well as neurobiotin-injected samples. This method can be combined with different fixation procedures, as well as different fluorophore excitation wavelengths without negative effects on staining quality. It can therefore serve as an effective addition to most standard histology protocols used in insect neuroanatomy.

  3. Fused oblique incidence reflectometry and confocal fluorescence microscopy

    Science.gov (United States)

    Risi, Matthew D.; Rouse, Andrew R.; Gmitro, Arthur F.

    2011-03-01

    Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure, but relies on exogenous fluorophores, has a relatively limited penetration depth (100 μm) and field of view (700 μm), and produces a high rate of detailed information to the user. A new catheter based multi-modal system has been designed that combines confocal imaging and oblique incidence reflectometry (OIR), which is a non-invasive method capable of rapidly extracting tissue absorption, μa, and reduced scattering, μ's, spectra from tissue. The system builds on previous developments of a custom slit-scan multi-spectral confocal microendoscope and is designed to rapidly switch between diffuse spectroscopy and confocal fluorescence imaging modes of operation. An experimental proof-of-principle catheter has been developed that consists of a fiber bundle for traditional confocal fluorescence imaging and a single OIR source fiber which is manually redirected at +/- 26 degrees. Diffusely scattered light from each orientation of the source fiber is collected via the fiber bundle, with a frame of data representing spectra collected at a range of distances from the OIR source point. Initial results with intralipid phantoms show good agreement to published data over the 550-650 nm spectral range. We successfully imaged and measured the optical properties of rodent cardiac muscle.

  4. Sub-Airy Confocal Adaptive Optics Scanning Ophthalmoscopy.

    Science.gov (United States)

    Sredar, Nripun; Fagbemi, Oladipo E; Dubra, Alfredo

    2018-04-01

    To demonstrate the viability of improving transverse image resolution in reflectance scanning adaptive optics ophthalmoscopy using sub-Airy disk confocal detection. The foveal cone mosaic was imaged in five human subjects free of known eye disease using two custom adaptive optics scanning light ophthalmoscopes (AOSLOs) in reflectance with 7.75 and 4.30 mm pupil diameters. Confocal pinholes of 0.5, 0.6, 0.8, and 1.0 Airy disk diameters (ADDs) were used in a retinal conjugate plane before the light detector. Average cone photoreceptor intensity profile width and power spectrum were calculated for the resulting images. Detected energy using a model eye was recorded for each pinhole size. The cone photoreceptor mosaic is better resolved with decreasing confocal pinhole size, with the high spatial frequency content of the images enhanced in both the large- and small-pupil AOSLOs. The average cone intensity profile width was reduced by ∼15% with the use of a 0.5 ADD pinhole when compared to a 1.0 ADD, with an accompanying reduction in signal greater than a factor of four. The use of sub-Airy disk confocal pinhole detection without increasing retinal light exposure results in a substantial improvement in image resolution at the cost of larger than predicted signal reduction. Improvement in transverse resolution using sub-Airy disk confocal detection is a practical and low-cost approach that is applicable to all point- and line-scanning ophthalmoscopes, including optical coherence tomographers.

  5. Fluorescence confocal polarizing microscopy

    Indian Academy of Sciences (India)

    Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). 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 ...

  6. Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

    Science.gov (United States)

    Siegel, Nisan; Brooker, Gary

    2014-09-22

    FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

  7. 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

  8. Prediction of glycosylation sites using random forests

    Directory of Open Access Journals (Sweden)

    Hirst Jonathan D

    2008-11-01

    Full Text Available Abstract Background Post translational modifications (PTMs occur in the vast majority of proteins and are essential for function. Prediction of the sequence location of PTMs enhances the functional characterisation of proteins. Glycosylation is one type of PTM, and is implicated in protein folding, transport and function. Results We use the random forest algorithm and pairwise patterns to predict glycosylation sites. We identify pairwise patterns surrounding glycosylation sites and use an odds ratio to weight their propensity of association with modified residues. Our prediction program, GPP (glycosylation prediction program, predicts glycosylation sites with an accuracy of 90.8% for Ser sites, 92.0% for Thr sites and 92.8% for Asn sites. This is significantly better than current glycosylation predictors. We use the trepan algorithm to extract a set of comprehensible rules from GPP, which provide biological insight into all three major glycosylation types. Conclusion We have created an accurate predictor of glycosylation sites and used this to extract comprehensible rules about the glycosylation process. GPP is available online at http://comp.chem.nottingham.ac.uk/glyco/.

  9. Flagellar glycosylation in Clostridium botulinum.

    Science.gov (United States)

    Twine, Susan M; Paul, Catherine J; Vinogradov, Evgeny; McNally, David J; Brisson, Jean-Robert; Mullen, James A; McMullin, David R; Jarrell, Harold C; Austin, John W; Kelly, John F; Logan, Susan M

    2008-09-01

    Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

  10. Spectral confocal reflection microscopy using a white light source

    Science.gov (United States)

    Booth, M.; Juškaitis, R.; Wilson, T.

    2008-08-01

    We present a reflection confocal microscope incorporating a white light supercontinuum source and spectral detection. The microscope provides images resolved spatially in three-dimensions, in addition to spectral resolution covering the wavelength range 450-650nm. Images and reflection spectra of artificial and natural specimens are presented, showing features that are not normally revealed in conventional microscopes or confocal microscopes using discrete line lasers. The specimens include thin film structures on semiconductor chips, iridescent structures in Papilio blumei butterfly scales, nacre from abalone shells and opal gemstones. Quantitative size and refractive index measurements of transparent beads are derived from spectral interference bands.

  11. Functional importance of PAI-1 glycosylation

    DEFF Research Database (Denmark)

    Christensen, Anni; Naessens, Dominik; Skottrup, Peter

    susceptible PAI-1 variant was not necessarily the one used when raising the antibody. This and other observations indicated that the carbohydrate moieties or the glycosylation sites are unlikely to be part of the epitopes for these antibodies. The antibody susceptibility characteristic for non......Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...

  12. Nano-displacement measurement based on virtual pinhole confocal method

    International Nuclear Information System (INIS)

    Li, Long; Kuang, Cuifang; Xue, Yi; Liu, Xu

    2013-01-01

    A virtual pinhole confocal system based on charge-coupled device (CCD) detection and image processing techniques is built to measure axial displacement with 10 nm resolution, preeminent flexibility and excellent robustness when facing spot drifting. Axial displacement of the sample surface is determined by capturing the confocal laser spot using a CCD detector and quantifying the energy collected by programmable virtual pinholes. Experiments indicate an applicable measuring range of 1000 nm (Gaussian fitting r = 0.9902) with a highly linear range of 500 nm (linear fitting r = 0.9993). A concentric subtraction algorithm is introduced to further enhance resolution. Factors affecting measuring precision, sensitivity and signal-to-noise ratio are discussed using theoretical deductions and diffraction simulations. The virtual pinhole technique has promising applications in surface profiling and confocal imaging applications which require easily-customizable pinhole configurations. (paper)

  13. Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy.

    Science.gov (United States)

    Sivaguru, Mayandi; Fried, Glenn; Sivaguru, Barghav S; Sivaguru, Vignesh A; Lu, Xiaochen; Choi, Kyung Hwa; Saif, M Taher A; Lin, Brian; Sadayappan, Sakthivel

    2015-11-01

    The ability to image the entire adult mouse heart at high resolution in 3-D would provide enormous advantages in the study of heart disease. However, a technique for imaging nuclear/cellular detail as well as the overall structure of the entire heart in 3-D with minimal effort is lacking. To solve this problem, we modified the benzyl alcohol:benzyl benzoate (BABB) clearing technique by labeling mouse hearts with periodic acid Schiff (PAS) stain. We then imaged the hearts with a combination of two-photon fluorescence microscopy and automated tile-scan imaging/stitching. Utilizing the differential spectral properties of PAS, we could identify muscle and nuclear compartments in the heart. We were also able to visualize the differences between a 3-month-old normal mouse heart and a mouse heart that had undergone heart failure due to the expression of cardiac myosin binding protein-C (cMyBP-C) gene mutation (t/t). Using 2-D and 3-D morphometric analysis, we found that the t/t heart had anomalous ventricular shape, volume, and wall thickness, as well as a disrupted sarcomere pattern. We further validated our approach using decellularized hearts that had been cultured with 3T3 fibroblasts, which were tracked using a nuclear label. We were able to detect the 3T3 cells inside the decellularized intact heart tissue, achieving nuclear/cellular resolution in 3-D. The combination of labeling, clearing, and two-photon microscopy together with tiling eliminates laborious and time-consuming physical sectioning, alignment, and 3-D reconstruction.

  14. Pigment organization effects on energy transfer and Chl a emission imaged in the diatoms C. meneghiniana and P. tricornutum in vivo: a confocal laser scanning fluorescence (CLSF) microscopy and spectroscopy study.

    Science.gov (United States)

    Premvardhan, Lavanya; Réfrégiers, Matthieu; Büchel, Claudia

    2013-09-26

    The (auto)fluorescence from three diatom strains, Cyclotella meneghiniana (Cm), Phaeodactylum tricornutum 1a (Pt1a), and Phaeodactylum UTex (PtUTex), has been imaged in vivo to submicrometer resolution using confocal laser scanning fluorescence (CLSF) microscopy. The diatoms are excited at 473 and 532 nm, energy primarily absorbed by the carotenoid fucoxanthin (Fx) found within the fucoxanthin chlorophyll a/c proteins (FCPs). On the basis of the fluorescence spectra measured in each image voxel, we obtain information about the spatial and energetic distribution of the terminal Chl a emitters, localized in the FCPs and the reaction centers of the PSII protein complexes, and the nature and location of the primary absorbers that are linked to these emitters; 532 nm excites the highly efficient Fx(red) light harvesters, and lesser amounts of Fx(green)s, that are enriched in some FCPs and preferentially transfer energy to PSII, compared to 473 nm, which excites almost equal amounts of all three previously identified sets of Fx--Fx(red), Fx(green) and Fx(blue)--as well as Chl c. The heterogeneous Chl a emission observed from the (C)LSF images indicates that the different Fx's serve different final emitters in P. tricornutum and suggest, at least in C. meneghiniana , a localization of FCPs with relatively greater Fx(red) content at the chloroplast edges, but with overall higher FCP concentration in the interior of the plastid. To better understand our results, the concentration-dependent ensemble-averaged diatom solution spectra are compared to the (auto)fluorescence spectra of individual diatoms, which indicate that pigment packing effects at an intracellular level do affect the diatoms' spectral properties, in particular, concerning a 710 nm emission band apparent under stress conditions. A species-specific response of the spectral signature to the incident light is also discussed in terms of the presence of a silica shell in Cm but not in Pt1a nor PtUTex.

  15. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY

    Science.gov (United States)

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

  16. Effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1

    International Nuclear Information System (INIS)

    Watanabe, Ayahisa; Nishijima, Ken-ichi; Zhao, Songji; Tamaki, Nagara; Kuge, Yuji; Tanaka, Yoshikazu; Itoh, Takeshi; Takemoto, Hiroshi

    2012-01-01

    Glycosylation is generally applicable as a strategy for increasing the activity of bioactive proteins. In this study, we examined the effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1 (GLP-1) as a bioactive peptide for type 2 diabetes. Noninvasive imaging studies were performed using a gamma camera after the intravenous administration of 123 I-GLP-1 or 123 I-α2, 6-sialyl N-acetyllactosamine (glycosylated) GLP-1 in rats. In ex vivo biodistribution studies using 125 I-GLP-1 or 125 I-glycosylated GLP-1, organ samples were measured for radioactivity. Plasma samples were added to 15% trichloroacetic acid (TCA) to obtain TCA-insoluble and TCA-soluble fractions. The radioactivity in the TCA-insoluble and TCA-soluble fractions was measured. In the noninvasive imaging studies, a relatively high accumulation level of 123 I-GLP-1 was found in the liver, which is the major organ to eliminate exogenous GLP-1. The area under the time-activity curve (AUC) of 123 I-glycosylated GLP-1 in the liver was significantly lower (89%) than that of 123 I-GLP-1. These results were consistent with those of ex vivo biodistribution studies using 125 I-labeled peptides. The AUC of 125 I-glycosylated GLP-1 in the TCA-insoluble fraction was significantly higher (1.7-fold) than that of GLP-1. This study demonstrated that glycosylation significantly decreased the distribution of radiolabeled GLP-1 into the liver and increased the concentration of radiolabeled GLP-1 in plasma. These results suggested that glycosylation is a useful strategy for decreasing the distribution into the liver of bioactive peptides as desirable pharmaceuticals. (author)

  17. Evidence for highly localized damage in internal tin and powder-in-tube Nb{sub 3}Sn strands rolled before reaction obtained from coupled magneto-optical imaging and confocal laser scanning microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Polyanskii, A A; Lee, P J; Jewell, M C; Larbalestier, D C [Applied Superconductivity Center, National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310 (United States); Barzi, E; Turrioni, D; Zlobin, A V [Fermi National Accelerator Laboratory, Batavia, IL 60510 (United States)

    2009-09-15

    Nb{sub 3}Sn strands for high-current, high-field magnets must be cabled before reaction while the conductor is still composed of ductile components. Even though still in the ductile, deformable state, significant damage can occur in this step, which expresses itself by inhomogeneous A15 formation, Sn leakage or even worse effects during later reaction. In this study, we simulate cabling damage by rolling recent high performance powder-in-tube (PIT) and internal tin (IT) strands in controlled increments, applying standard Nb{sub 3}Sn reaction heat treatments, and then examining the local changes using magneto-optical imaging (MOI), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). These combined characterizations allow any local damage to the filament architecture to be made clear. MOI directly reveals the local variation of superconductivity while CLSM is extremely sensitive in revealing Sn leakage beyond the diffusion barrier into the stabilizing Cu. These techniques reveal a markedly different response to deformation by the PIT and IT strands. The study demonstrates that these tools can provide a local, thorough, and detailed view of how strands degrade and thus complement more complex extracted strand studies.

  18. Confocal stereology: an efficient tool for measurement of microscopic structures

    Czech Academy of Sciences Publication Activity Database

    Kubínová, Lucie; Janáček, Jiří

    2015-01-01

    Roč. 360, č. 1 (2015), s. 13-28 ISSN 0302-766X R&D Projects: GA MŠk(CZ) LH13028 Institutional support: RVO:67985823 Keywords : 3-D images * confocal microscopy * geometrical characteristics * spatial probes * stereology Subject RIV: EA - Cell Biology Impact factor: 2.948, year: 2015

  19. Analysis of endoplasmic reticulum of tobacco cells using confocal microscopy

    Czech Academy of Sciences Publication Activity Database

    Radochová, Barbora; Janáček, Jiří; Schwarzerová, K.; Demjénová, E.; Tomori, Z.; Karen, Petr; Kubínová, Lucie

    2005-01-01

    Roč. 24, č. 11 (2005), s. 181-185 ISSN 1580-3139 R&D Projects: GA AV ČR(CZ) KJB6011309 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal microscopy * endoplasmic reticulum * image analysis Subject RIV: EA - Cell Biology

  20. 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.

  1. Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination

    International Nuclear Information System (INIS)

    Kim, Taejoong; Gweon, DaeGab; Lee, Jun-Hee

    2009-01-01

    Confocal microscopy is an optical imaging technique used to reconstruct three-dimensional images without physical sectioning. As with other optical microscopes, the lateral resolution of the confocal microscope cannot surpass the diffraction limit. This paper presents a novel imaging system, structured illumination confocal scanning microscopy (SICSM), that uses structured illumination to improve the lateral resolution of the confocal microscope. The SICSM can easily be implemented by introducing a structured illumination generating optics to conventional line-scanning fluorescence confocal microscopy. In this paper, we report our analysis of the lateral and axial resolutions of the SICSM by use of mathematical imaging theory. Numerical simulation results show that the lateral resolution of the SICSM is 1.43-fold better than that of the confocal microscope. In the axial direction, however, the resolution of the SICSM is ∼15% poorer than that of the confocal microscope. This deterioration arises because of a decrease in the axial cut-off frequency caused by the process of generating structured illumination. We propose the use of imaging conditions under which a compromise between the axial and lateral resolutions is chosen. Finally, we show simulated images of diversely shaped test objects to demonstrate the lateral and axial resolution performance of the SICSM

  2. 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 confocal images require less than 2 min for acquisition and allow for evaluation of invasive tumor cellularity in breast core needle biopsy specimens with moderate agreement to histologic images. We show that confocal fluorescence microscopy can be performed immediately following specimen acquisition and could indicate the need for additional biopsies at the initial visit.

  3. Usnea barbata CO2-supercritical extract in alkyl polyglucoside-based emulsion system: contribution of Confocal Raman imaging to the formulation development of a natural product.

    Science.gov (United States)

    Zugic, Ana; Lunter, Dominique Jasmin; Daniels, Rolf; Pantelic, Ivana; Tasic Kostov, Marija; Tadic, Vanja; Misic, Dusan; Arsic, Ivana; Savic, Snezana

    2016-08-01

    Topical treatment of skin infections is often limited by drawbacks related to both antimicrobial agents and their vehicles. In addition, considering the growing promotion of natural therapeutic products, our objective was to develop and evaluate naturally-based emulsion system, as prospective topical formulation for skin infections-treatment. Therefore, alkyl polyglucoside surfactants were used for stabilization of a vehicle serving as potential carrier for supercritical CO2-extract of Usnea barbata, lichen with well-documented antimicrobial activity, incorporated using two protocols and three concentrations. Comprehensive physicochemical characterization suggested possible involvement of extract's particles in stabilization of the investigated system. Raman spectral imaging served as the key method in disclosing extract's particles potential to participate in the microstructure of the tested emulsion system via three mechanisms: (1) particle-particle aggregation, (2) adsorption at the oil-water interface and (3) hydrophobic particle-surfactant interactions. Stated extract-vehicle interaction proved to be correlated to the preparation procedure and extract concentration on one hand and to affect the physicochemical and biopharmaceutical features of investigated system, on the other hand. Thereafter, formulation with the best preliminary stability and liberation profile was selected for further efficiency and in vivo skin irritation potential evaluation, implying pertinent in vitro antimicrobial activity against G+ bacteria and overall satisfying preliminary safety profile.

  4. Spinning-disk confocal microscopy: present technology and future trends.

    Science.gov (United States)

    Oreopoulos, John; Berman, Richard; Browne, Mark

    2014-01-01

    Live-cell imaging requires not only high temporal resolution but also illumination powers low enough to minimize photodamage. Traditional single-point laser scanning confocal microscopy (LSCM) is generally limited by both the relatively slow speed at which it can acquire optical sections by serial raster scanning (a few Hz) and the higher potential for phototoxicity. These limitations have driven the development of rapid, parallel forms of confocal microscopy, the most popular of which is the spinning-disk confocal microscope (SDCM). Here, we briefly introduce the SDCM technique, discuss its strengths and weaknesses against LSCM, and update the reader on some recent developments in SDCM technology that improve its performance and expand its utility for life science research now and in the future. © 2014 Elsevier Inc. All rights reserved.

  5. A Simple Model for Nonlinear Confocal Ultrasonic Beams

    Science.gov (United States)

    Zhang, Dong; Zhou, Lin; Si, Li-Sheng; Gong, Xiu-Fen

    2007-01-01

    A confocally and coaxially arranged pair of focused transmitter and receiver represents one of the best geometries for medical ultrasonic imaging and non-invasive detection. We develop a simple theoretical model for describing the nonlinear propagation of a confocal ultrasonic beam in biological tissues. On the basis of the parabolic approximation and quasi-linear approximation, the nonlinear Khokhlov-Zabolotskaya-Kuznetsov (KZK) equation is solved by using the angular spectrum approach. Gaussian superposition technique is applied to simplify the solution, and an analytical solution for the second harmonics in the confocal ultrasonic beam is presented. Measurements are performed to examine the validity of the theoretical model. This model provides a preliminary model for acoustic nonlinear microscopy.

  6. 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.

  7. Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

    Science.gov (United States)

    Yamakoshi, Yasuo; Nagano, Takatoshi; Hu, Jan Cc; Yamakoshi, Fumiko; Simmer, James P

    2011-02-03

    Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr

  8. Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

    Directory of Open Access Journals (Sweden)

    Yamakoshi Fumiko

    2011-02-01

    Full Text Available Abstract Background Dentin sialophosphoprotein (Dspp is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp, the N-terminal domain of dentin sialophosphoprotein (Dspp, is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were

  9. Ultrasensitive and selective detection of mercury (II) in serum based on the gold film sensor using a laser scanning confocal imaging-surface plasmon resonance system in real time

    Science.gov (United States)

    Liu, Sha; Zhang, Hongyan; Liu, Weimin; Wang, Pengfei

    2015-10-01

    Hg2+ ions are one of the most toxic heavy metal ion pollutants, and are caustic and carcinogenic materials with high cellular toxicity. The Hg2+ ions can accumulate in the human body through the food chain and cause serious and permanent damage to the brain with both acute and chronic toxicity. According to the US Environment Protection Agency (EPA) guidelines, Hg2+ ions must be at concentrations below 1 ng/ml (10 nM) in drinking water. If the Hg2+ ions are higher than 2.5 ng/ml in serum, that will bring mercury poisoning. The traditional testing for Hg2+ ions includes atomic absorption, atomic fluorescence, and inductively coupled plasma mass spectrometry. These methods are usually coupled with gas chromatography, high-performance liquid chromatography, and capillary electrophoresis. However, these instrument-based techniques are rather complicated, time-consuming, costly, and unsuitable for online and portable use. An ultrasensitive and selective detection of mercury (II) in serum was investigated using a laser scanning confocal imaging-surface plasmon resonance system (LSCI-SPR). The detection limit was as low as 0.01 ng/ml for Hg2+ ions in fetal calf serum and that is lower than that was required Hg2+ ions must be at concentrations below 1 ng/ml by the US Environment Protection Agency (EPA) guidelines. This sensor was designed on a T-rich, single-stranded DNA (ssDNA)-modified gold film, which can be individually manipulated using specific T-Hg2+-T complex formation. The quenching intensity of the fluorescence images for rhodamine-labeled ssDNA fitted well with the changes in SPR. The changes varied with the Hg2+ ion concentration, which is unaffected by the presence of other metal ions. A good liner relation was got with the coefficients of 0.9116 in 30% fetal calf serums with the linear part over a range of 0.01 ng/ml to10 ng/ml.

  10. Identification of nodal tissue in the living heart using rapid scanning fiber-optics confocal microscopy and extracellular fluorophores.

    Science.gov (United States)

    Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

    2013-09-01

    Risks associated with pediatric reconstructive heart surgery include injury of the sinoatrial node (SAN) and atrioventricular node (AVN), requiring cardiac rhythm management using implantable pacemakers. These injuries are the result of difficulties in identifying nodal tissues intraoperatively. Here we describe an approach based on confocal microscopy and extracellular fluorophores to quantify tissue microstructure and identify nodal tissue. Using conventional 3-dimensional confocal microscopy we investigated the microstructural arrangement of SAN, AVN, and atrial working myocardium (AWM) in fixed rat heart. AWM exhibited a regular striated arrangement of the extracellular space. In contrast, SAN and AVN had an irregular, reticulated arrangement. AWM, SAN, and AVN tissues were beneath a thin surface layer of tissue that did not obstruct confocal microscopic imaging. Subsequently, we imaged tissues in living rat hearts with real-time fiber-optics confocal microscopy. Fiber-optics confocal microscopy images resembled images acquired with conventional confocal microscopy. We investigated spatial regularity of tissue microstructure from Fourier analysis and second-order image moments. Fourier analysis of fiber-optics confocal microscopy images showed that the spatial regularity of AWM was greater than that of nodal tissues (37.5 ± 5.0% versus 24.3 ± 3.9% for SAN and 23.8 ± 3.7% for AVN; Pfiber-optics confocal microscopy. Application of the approach in pediatric reconstructive heart surgery may reduce risks of injuring nodal tissues.

  11. Speckle-illuminated fluorescence confocal microscopy, using a digital micro-mirror device

    International Nuclear Information System (INIS)

    Jiang, Shi-Hong; Walker, John G

    2009-01-01

    An implementation of a speckle-illuminated fluorescence confocal microscope using a digital micro-mirror device (DMD) is described. The DMD not only projects a sequence of imaged binary speckle patterns onto the specimen at a very high frame rate but also operates as a spatial light modulator to perform real-time optical data processing. Frame averaging is accomplished by CCD charge accumulation during a single exposure. The recorded time-averaged image is a confocal image plus an unwanted non-confocal image which can be removed by recording a separate image. Experimental results with image acquisition within a fraction of a second are shown. Images of a thin biological sample are also shown to demonstrate practical application of the technique

  12. 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.

  13. EUS-Guided Needle-Based Confocal Laser Endomicroscopy

    DEFF Research Database (Denmark)

    Bhutani, Manoop S; Koduru, Pramoda; Joshi, Virendra

    2015-01-01

    Endoscopic ultrasound (EUS) has emerged as an excellent tool for imaging the gastrointestinal tract, as well as surrounding structures. EUS-guided fine-needle aspiration (EUS-FNA) has become the standard of care for the tissue sampling of a variety of masses and lymph nodes within and around...... the gut, providing further diagnostic and staging information. Confocal laser endomicroscopy (CLE) is a novel endoscopic method that enables imaging at a subcellular level of resolution during endoscopy, allowing up to 1000-fold magnification of tissue and providing an optical biopsy. A new procedure...... that has been developed in the past few years is needle-based confocal laser endomicroscopy (nCLE), which involves a mini-CLE probe that can be passed through a 1 9-gauge needle during EUS-FNA. This enables the real-time visualization of tissue at a microscopic level, with the potential to further improve...

  14. Design considerations of a real-time clinical confocal microscope

    Science.gov (United States)

    Masters, Barry R.

    1991-06-01

    A real-time clinical confocal light microscope provides the ophthalmologist with a new tool for the observation of the cornea and the ocular lens. In addition, the ciliary body, the iris, and the sclera can be observed. The real-time light microscopic images have high contrast and resolution. The transverse resolution is about one half micron and the range resolution is one micron. The following observations were made with visible light: corneal epithelial cells, wing cells, basal cells, Bowman's membrane, nerve fibers, basal lamina, fibroblast nuclei, Descemet's membrane, endothelial cells. Observation of the in situ ocular lens showed lens capsule, lens epithelium, lens fibrils, the interior of lens fibrils. The applications of the confocal microscope include: eye banking, laser refractive surgery, observation of wound healing, observation of the iris, the sciera, the ciliary body, the ocular lens, and the intraocular lens. Digital image processing can produce three-dimensional reconstructions of the cornea and the ocular lens.

  15. Immunolocalisation of members of the polypeptide N-acetylgalactosaminyl transferase (ppGalNAc-T) family is consistent with biologically relevant altered cell surface glycosylation in breast cancer

    DEFF Research Database (Denmark)

    Brooks, Susan A; Carter, Tracey M; Bennett, Eric P

    2007-01-01

    understood, may mediate the synthesis of varied glycoforms of cellular proteins with different biological activities. Disruptions in glycosylation are a common feature of cancer and may have functional significance. Immunocytochemistry with confocal scanning laser microscopy was employed to detect members...... of the ppGalNAc-T family, ppGalNAc-T1, -T2, -T3, -T4 and -T6 in a range of breast cell lines. The cells were chosen to represent a range of phenotypes from 'normal'/benign (HMT 3,522), primary, non-metastatic breast cancer (BT 474), to aggressive, metastatic breast cancer (ZR75-1, T47D, MCF-7, DU 4...... tightly restricted ppGalNAc-T's may result in initiation of O-linked glycosylation at normally unoccupied potential glycosylation sites leading to altered glycoforms of proteins with changed biological activity which may contribute to the pathogenesis of cancer....

  16. CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

    Science.gov (United States)

    Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

    2015-02-07

    FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

  17. 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.

  18. fasting blood glucose and glycosylated haemoglobin levels

    African Journals Online (AJOL)

    Prince Acheampong

    (HbA1c) levels of diabetes mellitus patients as an index of glycaemic control. It was a prospective case- finding study using laboratory and general practice records. ... range of glycosylated haemoglobins, and the cut-off values for some clinical .... quality of glycaemic control by glycated haemoglobin in out-patient diabetic ...

  19. Is glycosylated haemoglobin a marker of fertility?

    DEFF Research Database (Denmark)

    Hjollund, N H; Jensen, Tina Kold; Bonde, Jens Peter

    1999-01-01

    We performed a follow-up study of time to pregnancy in a population of first-time pregnancy planners without previous reproductive experience. The objective of this paper is to report and discuss a finding of a strong relationship between glycosylated haemoglobin (HbA1C) and fertility. A total...

  20. 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

  1. 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.

  2. Surface glycosylation profiles of urine extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Jared Q Gerlach

    Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

  3. 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.

  4. Detection of UV-induced pigmentary and epidermal changes over time using in vivo reflectance confocal microscopy

    NARCIS (Netherlands)

    Middelkamp-Hup, Maritza A.; Park, H.-Y.; Lee, Jin; Gilchrest, Barbara A.; Gonzalez, Salvador

    2006-01-01

    In vivo reflectance confocal microscopy (RCM) provides high-resolution optical sections of the skin in its native state, without needing to fix or section the tissue. Melanin provides an excellent contrast for RCM, giving a bright signal in the confocal images. The pigmented guinea-pig is a common

  5. Competition between folding and glycosylation in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Bruun, A W; Kielland-Brandt, Morten

    1996-01-01

    Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...

  6. Control of mucin-type O-glycosylation

    DEFF Research Database (Denmark)

    Bennett, Eric P; Mandel, Ulla; Clausen, Henrik

    2012-01-01

    residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue...... in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The Gal...

  7. The challenge of diagnosing seborrheic keratosis by reflectance confocal microscopy.

    Science.gov (United States)

    Guo, A; Chen, J; Yang, C; Ding, Y; Zeng, Q; Tan, L

    2018-05-24

    Seborrheic keratosis (SK) is one of the most common skin tumors seen by dermatologists. It should be differentiated with many diseases, especially skin tumors. Reflectance confocal microscopy (RCM) has been applied for evaluation of SK. There are a few studies that describe the RCM of SK. The aim of the study was to find the challenge of diagnosing seborrheic keratosis by reflectance confocal microscopy. A total of 390 patients with a clinical suspicious diagnosis of seborrheic keratosis were enrolled in this study, and lesions from each patient were imaged with RCM. Thirty-seven of these patients performed a biopsy in order to be given a histological diagnosis. We retrospectively analyzed the outcomes of RCM diagnosis and histological diagnosis, and then found the RCM characteristics of biopsy-proven lesions. According to RCM images, 258 of 390 (66.2%) patients were diagnosed with SK, 97 of 390 (24.9%) patients could not be diagnosed by the dermatologist according to RCM. Of all 37 biopsied lesions, 23 were SK, 6 were actinic keratosis, 2 were basal cell carcinoma, and 2 were squamous cell carcinoma. It is challenge to diagnose seborrheic keratosis by reflectance confocal microscopy. It may due to the variable clinical and RCM appearances of SK, and limited depth of RCM. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter

    2003-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla......The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous...... with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

  9. How the confocal laser scanning microscope entered biological research.

    Science.gov (United States)

    Amos, W B; White, J G

    2003-09-01

    A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.

  10. Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

    Science.gov (United States)

    Cushway, T R; Lanzetta, M; Cox, G; Trickett, R; Owen, E R

    1996-01-01

    Fibre-optic technology combined with confocality produces a microscope capable of optical thin sectioning. In this original study, tibial nerves have been stained in a rat model with a vital dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide, and analysed by fibre-optic confocal microscopy to produce detailed images of nerve ultrastructure. Schwann cells, nodes of Ranvier and longitudinal myelinated sheaths enclosing axons were clearly visible. Single axons appeared as brightly staining longitudinal structures. This allowed easy tracing of multiple signal axons within the nerve tissue. An accurate measurement of internodal lengths was easily accomplished. This technique is comparable to current histological techniques, but does not require biopsy, thin sectioning or tissue fixing. This study offers a standard for further in vivo microscopy, including the possibility of monitoring the progression of nerve regeneration following microsurgical neurorraphy.

  11. Efficient synthesis of glycosylated phenazine natural products and analogs with DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors

    DEFF Research Database (Denmark)

    Laursen, Jane B.; Petersen, Lars; Jensen, K.J.

    2003-01-01

    Inspired by the occurrence and function of phenazines in natural products, new glycosylated analogs were designed and synthesized. DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors were used in an efficient and easily-handled glycosylation protocol compatible with combinatorial chemistry....... Benzoylated D-glucose, D-galactose and L-quinovose DISAL glycosyl donors were synthesized in high yields and used under mild conditions to glycosylate methyl saphenate and 2-hydroxyphenazine. The glycosides were screened for biological activity and one compound showed inhibitory activity towards topoisomerase...

  12. Confocal laser feedback tomography for skin cancer detection.

    Science.gov (United States)

    Mowla, Alireza; Du, Benjamin Wensheng; Taimre, Thomas; Bertling, Karl; Wilson, Stephen; Soyer, H Peter; Rakić, Aleksandar D

    2017-09-01

    Tomographic imaging of soft tissue such as skin has a potential role in cancer detection. The penetration of infrared wavelengths makes a confocal approach based on laser feedback interferometry feasible. We present a compact system using a semiconductor laser as both transmitter and receiver. Numerical and physical models based on the known optical properties of keratinocyte cancers were developed. We validated the technique on three phantoms containing macro-structural changes in optical properties. Experimental results were in agreement with numerical simulations and structural changes were evident which would permit discrimination of healthy tissue and tumour. Furthermore, cancer type discrimination was also able to be visualized using this imaging technique.

  13. 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.

  14. Reflectance confocal microscopy features of thin versus thick melanomas.

    Science.gov (United States)

    Kardynal, Agnieszka; Olszewska, Małgorzata; de Carvalho, Nathalie; Walecka, Irena; Pellacani, Giovanni; Rudnicka, Lidia

    2018-01-24

    In vivo reflectance confocal microscopy (RCM) plays an increasingly important role in differential diagnosis of melanoma. The aim of the study was to assess typical confocal features of thin (≤1mm according to Breslow index) versus thick (>1mm) melanomas. 30 patients with histopathologically confirmed cutaneous melanoma were included in the study. Reflectance confocal microscopy was performed with Vivascope equipment prior to excision. Fifteen melanomas were thin (Breslow thickness ≤ 1mm) and 15 were thick melanomas (Breslow thickness >1mm). In the RCM examination, the following features were more frequently observed in thin compared to thick melanomas: edged papillae (26.7% vs 0%, p=0.032) and areas with honeycomb or cobblestone pattern (33.3% vs 6.7%, p=0.068). Both features are present in benign melanocytic lesions, so in melanoma are good prognostic factors. The group of thick melanomas compared to the group of thin melanomas in the RCM images presented with greater frequency of roundish cells (100% vs 40%, p=0.001), non-edged papillae (100% vs 60%, p=0.006), numerous pagetoid cells (73.3% vs 33.3%, p=0.028), numerous atypical cells at dermal-epidermal junction (53.3% vs 20%, p=0.058) and epidermal disarray (93.3% vs 66.7%, p=0.068). Non-invasive imaging methods helps in deepening of knowledge about the evolution and biology of melanoma. The most characteristic features for thin melanomas in confocal examination are: fragments of cobblestone or honeycomb pattern and edged papillae (as good prognostic factors). The features of thick melanomas in RCM examination are: roundish cells, non-edged papillae, numerous pagetoid cells at dermal-epidermal junction and epidermal disarray.

  15. Confocal scanning microscope for nuclear photoemulsion

    International Nuclear Information System (INIS)

    Batusov, Yu.A.; Kovalev, Yu.S.; Soroko, L.M.

    2005-01-01

    The application of the confocal scanning microscope to the objects in the nuclear photoemulsion is described. An array of 27 microtomograms of single silver grain is shown. The cross sections of the same particle track of diameter 1 μm, detected by means of the confocal scanning microscope with open and annular apertures, are presented. It was shown that the confocal scanning microscope opens indeed new opportunities for the nuclear photoemulsion technique to get previously inaccessible information for physics of the short-living particles

  16. Congenital disorders of glycosylation: The Saudi experience.

    Science.gov (United States)

    Alsubhi, Sarah; Alhashem, Amal; Faqeih, Eissa; Alfadhel, Majid; Alfaifi, Abdullah; Altuwaijri, Waleed; Alsahli, Saud; Aldhalaan, Hesham; Alkuraya, Fowzan S; Hundallah, Khalid; Mahmoud, Adel; Alasmari, Ali; Mutairi, Fuad Al; Abduraouf, Hanem; AlRasheed, Layan; Alshahwan, Saad; Tabarki, Brahim

    2017-10-01

    We retrospectively reviewed Saudi patients who had a congenital disorder of glycosylation (CDG). Twenty-seven Saudi patients (14 males, 13 females) from 13 unrelated families were identified. Based on molecular studies, the 27 CDG patients were classified into different subtypes: ALG9-CDG (8 patients, 29.5%), ALG3-CDG (7 patients, 26%), COG6-CDG (7 patients, 26%), MGAT2-CDG (3 patients, 11%), SLC35A2-CDG (1 patient), and PMM2-CDG (1 patient). All the patients had homozygous gene mutations. The combined carrier frequency of CDG for the encountered founder mutations in the Saudi population is 11.5 per 10,000, which translates to a minimum disease burden of 14 patients per 1,000,000. Our study provides comprehensive epidemiologic information and prevalence figures for each of these CDG in a large cohort of congenital disorder of glycosylation patients. © 2017 Wiley Periodicals, Inc.

  17. Optimal Synthetic Glycosylation of a Therapeutic Antibody.

    Science.gov (United States)

    Parsons, Thomas B; Struwe, Weston B; Gault, Joseph; Yamamoto, Keisuke; Taylor, Thomas A; Raj, Ritu; Wals, Kim; Mohammed, Shabaz; Robinson, Carol V; Benesch, Justin L P; Davis, Benjamin G

    2016-02-12

    Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non-natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or "glycorandomization") were readily generated.

  18. Involvement of Aberrant Glycosylation in Thyroid Cancer

    Directory of Open Access Journals (Sweden)

    Eiji Miyoshi

    2010-01-01

    Full Text Available Glycosylation is one of the most common posttranslational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharides structures are associated with many physiological and pathological events, including cell growth, migration and differentiation, and tumor invasion. Therefore, functional glycomics, which is a comprehensive study of the structures and functions of glycans, is attracting the increasing attention of scientists in various fields of life science. In cases of thyroid cancer, the biological characters and prognosis are completely different in each type of histopathology, and their oligosaccharide structures as well as the expression of glycosyltransferases are also different. In this review, we summarized our previous papers on oligosaccharides and thyroid cancers and discussed a possible function of oligosaccharides in the carcinogenesis in thyroid cancer.

  19. Dengue Virus Glycosylation: What Do We Know?

    Directory of Open Access Journals (Sweden)

    Sally S. L. Yap

    2017-07-01

    Full Text Available In many infectious diseases caused by either viruses or bacteria, pathogen glycoproteins play important roles during the infection cycle, ranging from entry to successful intracellular replication and host immune evasion. Dengue is no exception. Dengue virus glycoproteins, envelope protein (E and non-structural protein 1 (NS1 are two popular sub-unit vaccine candidates. E protein on the virion surface is the major target of neutralizing antibodies. NS1 which is secreted during DENV infection has been shown to induce a variety of host responses through its binding to several host factors. However, despite their critical role in disease and protection, the glycosylated variants of these two proteins and their biological importance have remained understudied. In this review, we seek to provide a comprehensive summary of the current knowledge on protein glycosylation in DENV, and its role in virus biogenesis, host cell receptor interaction and disease pathogenesis.

  20. Nonenzymatic glycosylation of bovine myelin basic protein

    International Nuclear Information System (INIS)

    Hitz, J.B.

    1987-01-01

    In the CNS myelin sheath the nonenzymatic glycosylation reaction (at the early stage of the Amadori product) occurs only with the myelin basic protein and not with the other myelin proteins. This was observed in isolated bovine myelin by in vitro incubation with [ 14 C]-galactose and [ 14 C]-glucose. The respective in-vitro incorporation rates for purified bovine myelin basic protein with D-galactose, D-glucose and D-mannose were 7.2, 2.4 and 2.4 mmoles/mole myelin basic protein per day at 37 0 C. A more rapid, HPLC method was devised and characterized to specifically analyze for the Amadori product. The HPLC method was correlated to the [ 14 C]-sugar incorporation method for myelin basic protein under a set of standard reaction conditions using [ 14 C]-glucose and [ 14 C]-mannose with HPLC values at 1/6 and 1/5 of the [ 14 C]-sugar incorporation method. A novel myelin basic protein purification step has been developed that yields a relativity proteolytic free preparation that is easy to work with, being totally soluble at a neutral pH. Nine new spots appear for a trypsinized glycosylated MBP in the paper peptide map of which eight correspond to positions of the [ 3 H]-labeled Amadori product in affinity isolated peptides. These studies provide a general characterization of and a structural basis for investigations on nonenzymatically glycosylated MBP as well as identifying MBP as the only nonenzymatically glycosylated protein in the CNS myelin sheath which may accumulate during aging, diabetes, and demyelinating diseases in general

  1. Glycosyl-Nucleolipids as new bioinspired amphiphiles.

    Science.gov (United States)

    Latxague, Laurent; Patwa, Amit; Amigues, Eric; Barthélémy, Philippe

    2013-09-30

    Four new Glycosyl-NucleoLipid (GNL) analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry) indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower glim) compared to the first generation of GNFs.

  2. 3D Volumetric Analysis of Fluid Inclusions Using Confocal Microscopy

    Science.gov (United States)

    Proussevitch, A.; Mulukutla, G.; Sahagian, D.; Bodnar, B.

    2009-05-01

    Fluid inclusions preserve valuable information regarding hydrothermal, metamorphic, and magmatic processes. The molar quantities of liquid and gaseous components in the inclusions can be estimated from their volumetric measurements at room temperatures combined with knowledge of the PVTX properties of the fluid and homogenization temperatures. Thus, accurate measurements of inclusion volumes and their two phase components are critical. One of the greatest advantages of the Laser Scanning Confocal Microscopy (LSCM) in application to fluid inclsion analsyis is that it is affordable for large numbers of samples, given the appropriate software analysis tools and methodology. Our present work is directed toward developing those tools and methods. For the last decade LSCM has been considered as a potential method for inclusion volume measurements. Nevertheless, the adequate and accurate measurement by LSCM has not yet been successful for fluid inclusions containing non-fluorescing fluids due to many technical challenges in image analysis despite the fact that the cost of collecting raw LSCM imagery has dramatically decreased in recent years. These problems mostly relate to image analysis methodology and software tools that are needed for pre-processing and image segmentation, which enable solid, liquid and gaseous components to be delineated. Other challenges involve image quality and contrast, which is controlled by fluorescence of the material (most aqueous fluid inclusions do not fluoresce at the appropriate laser wavelengths), material optical properties, and application of transmitted and/or reflected confocal illumination. In this work we have identified the key problems of image analysis and propose some potential solutions. For instance, we found that better contrast of pseudo-confocal transmitted light images could be overlayed with poor-contrast true-confocal reflected light images within the same stack of z-ordered slices. This approach allows one to narrow

  3. Diversity in protein glycosylation among insect species.

    Directory of Open Access Journals (Sweden)

    Gianni Vandenborre

    Full Text Available BACKGROUND: A very common protein modification in multicellular organisms is protein glycosylation or the addition of carbohydrate structures to the peptide backbone. Although the Class of the Insecta is the largest animal taxon on Earth, almost all information concerning glycosylation in insects is derived from studies with only one species, namely the fruit fly Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the differences in glycoproteomes between insects belonging to several economically important insect orders were studied. Using GNA (Galanthus nivalis agglutinin affinity chromatography, different sets of glycoproteins with mannosyl-containing glycan structures were purified from the flour beetle (Tribolium castaneum, the silkworm (Bombyx mori, the honeybee (Apis mellifera, the fruit fly (D. melanogaster and the pea aphid (Acyrthosiphon pisum. To identify and characterize the purified glycoproteins, LC-MS/MS analysis was performed. For all insect species, it was demonstrated that glycoproteins were related to a broad range of biological processes and molecular functions. Moreover, the majority of glycoproteins retained on the GNA column were unique to one particular insect species and only a few glycoproteins were present in the five different glycoprotein sets. Furthermore, these data support the hypothesis that insect glycoproteins can be decorated with mannosylated O-glycans. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that oligomannose N-glycosylation events are highly specific depending on the insect species. In addition, we also demonstrated that protein O-mannosylation in insect species may occur more frequently than currently believed.

  4. Diffractive elements performance in chromatic confocal microscopy

    International Nuclear Information System (INIS)

    Garzon, J; Duque, D; Alean, A; Toledo, M; Meneses, J; Gharbi, T

    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.

  5. Confocal Laser Endomicroscopy in Inflammatory Bowel Disease

    DEFF Research Database (Denmark)

    Rasmussen, Ditlev Nytoft; Karstensen, John Gásdal; Riis, Lene Buhl

    2015-01-01

    included. Next, eligible studies were analysed with respect to several parameters, such as technique and clinical aim and definitions of outcomes. RESULTS: Confocal laser endomicroscopy has been used for a wide range of purposes in inflammatory bowel disease, covering assessment of inflammatory severity...... of confocal laser endomicroscopy for inflammatory bowel disease. METHODS: Available literature was searched systematically for studies applying confocal laser endomicroscopy in Crohn's disease or ulcerative colitis. Relevant literature was reviewed and only studies reporting original clinical data were...... of histological features such as colonic crypts, epithelial gaps and epithelial leakiness to fluorescein. CONCLUSIONS: Confocal laser endomicroscopy remains an experimental but emerging tool for assessment of inflammatory bowel disease. It is the only method that enables in vivo functional assessment...

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

    International Nuclear Information System (INIS)

    Weiswald, Louis-Bastien; Guinebretière, Jean-Marc; Richon, Sophie; Bellet, Dominique; Saubaméa, Bruno; Dangles-Marie, Virginie

    2010-01-01

    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. 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. 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 the luminal cell surface of colorectal cancer acini

  7. Quantification of confocal fluorescence microscopy for the detection of cervical intraepithelial neoplasia.

    Science.gov (United States)

    Sheikhzadeh, Fahime; Ward, Rabab K; Carraro, Anita; Chen, Zhao Yang; van Niekerk, Dirk; Miller, Dianne; Ehlen, Tom; MacAulay, Calum E; Follen, Michele; Lane, Pierre M; Guillaud, Martial

    2015-10-24

    Cervical cancer remains a major health problem, especially in developing countries. Colposcopic examination is used to detect high-grade lesions in patients with a history of abnormal pap smears. New technologies are needed to improve the sensitivity and specificity of this technique. We propose to test the potential of fluorescence confocal microscopy to identify high-grade lesions. We examined the quantification of ex vivo confocal fluorescence microscopy to differentiate among normal cervical tissue, low-grade Cervical Intraepithelial Neoplasia (CIN), and high-grade CIN. We sought to (1) quantify nuclear morphology and tissue architecture features by analyzing images of cervical biopsies; and (2) determine the accuracy of high-grade CIN detection via confocal microscopy relative to the accuracy of detection by colposcopic impression. Forty-six biopsies obtained from colposcopically normal and abnormal cervical sites were evaluated. Confocal images were acquired at different depths from the epithelial surface and histological images were analyzed using in-house software. The features calculated from the confocal images compared well with those features obtained from the histological images and histopathological reviews of the specimens (obtained by a gynecologic pathologist). The correlations between two of these features (the nuclear-cytoplasmic ratio and the average of three nearest Delaunay-neighbors distance) and the grade of dysplasia were higher than that of colposcopic impression. The sensitivity of detecting high-grade dysplasia by analysing images collected at the surface of the epithelium, and at 15 and 30 μm below the epithelial surface were respectively 100, 100, and 92 %. Quantitative analysis of confocal fluorescence images showed its capacity for discriminating high-grade CIN lesions vs. low-grade CIN lesions and normal tissues, at different depth of imaging. This approach could be used to help clinicians identify high-grade CIN in clinical

  8. 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

  9. Three-dimensional measurement and visualization of internal flow of a moving droplet using confocal micro-PIV.

    Science.gov (United States)

    Kinoshita, Haruyuki; Kaneda, Shohei; Fujii, Teruo; Oshima, Marie

    2007-03-01

    This paper presents a micro-flow diagnostic technique, 'high-speed confocal micro-particle image velocimetry (PIV)', and its application to the internal flow measurement of a droplet passing through a microchannel. A confocal micro-PIV system has been successfully constructed wherein a high-speed confocal scanner is combined with the conventional micro-PIV technique. The confocal micro-PIV system enables us to obtain a sequence of sharp and high-contrast cross-sectional particle images at 2000 frames s(-1). This study investigates the confocal depth, which is a significant parameter to determine the out-of-plane measurement resolution in confocal micro-PIV. Using the present confocal micro-PIV system, we can measure velocity distributions of micro-flows in a 228 microm x 171 microm region with a confocal depth of 1.88 microm. We also propose a three-dimensional velocity measurement method based on the confocal micro-PIV and the equation of continuity. This method enables us to measure three velocity components in a three-dimensional domain of micro flows. The confocal micro-PIV system is applied to the internal flow measurement of a droplet. We have measured three-dimensional distributions of three-component velocities of a droplet traveling in a 100 microm (width) x 58 microm (depth) channel. A volumetric velocity distribution inside a droplet is obtained by the confocal micro-PIV and the three-dimensional flow structure inside the droplet is investigated. The measurement results suggest that a three-dimensional and complex circulating flow is formed inside the droplet.

  10. Confocal endomicroscopy: Is it time to move on?

    Science.gov (United States)

    Robles-Medranda, Carlos

    2016-01-10

    Confocal laser endomicroscopy permits in-vivo microscopy evaluation during endoscopy procedures. It can be used in all the parts of the gastrointestinal tract and includes: Esophagus, stomach, small bowel, colon, biliary tract through and endoscopic retrograde cholangiopancreatography and pancreas through needles during endoscopic ultrasound procedures. Many researches demonstrated a high correlation of results between confocal laser endomicroscopy and histopathology in the diagnosis of gastrointestinal lesions; with accuracy in about 86% to 96%. Moreover, in spite that histopathology remains the gold-standard technique for final diagnosis of any diseases; a considerable number of misdiagnosis rate could be present due to many factors such as interpretation mistakes, biopsy site inaccuracy, or number of biopsies. Theoretically; with the diagnostic accuracy rates of confocal laser endomicroscopy could help in a daily practice to improve diagnosis and treatment management of the patients. However, it is still not routinely used in the clinical practice due to many factors such as cost of the procedure, lack of codification and reimbursement in some countries, absence of standard of care indications, availability, physician image-interpretation training, medico-legal problems, and the role of the pathologist. These limitations are relative, and solutions could be found based on new researches focused to solve these barriers.

  11. Reflectance confocal microscopy: an effective tool for monitoring ultraviolet B phototherapy in psoriasis.

    NARCIS (Netherlands)

    Wolberink, E.A.W.; Erp, P.E.J. van; Boer-van Huizen, R.T. de; Kerkhof, P.C.M. van de; Gerritsen, M.J.P.

    2012-01-01

    Background In vivo reflectance confocal microscopy (RCM) is a novel, noninvasive imaging technique which enables imaging of skin at a cellular resolution comparable to conventional microscopy. Objectives We performed a pilot study to evaluate RCM as a noninvasive tool for monitoring ultraviolet (UV)

  12. Halide-mediated regioselective 6-O-glycosylation of unprotected hexopyranosides with perbenzylated glycosyl bromide donors

    DEFF Research Database (Denmark)

    Niedbal, Dominika Alina; Madsen, Robert

    2016-01-01

    The regio- and stereoselective glycosylation at the 6-position in 2,3,4,6-unprotected hexopyranosides has been investigated with dibutyltin oxide as the directing agent. Perbenzylated hexopyranosyl bromides were employed as the donors and the glycosylations were promoted by tetrabutylammonium...... bromide. The couplings were completely selective for both glucose and galactose donors and acceptors as long as the stannylene acetal of the acceptor was soluble in dichloromethane. This gave rise to a number of 1,2-cis-linked disaccharides in reasonable yields. Mannose donors and acceptors, on the other...

  13. Glycosylation in HIV-1 envelope glycoprotein and its biological implications

    KAUST Repository

    Ho, Yung Shwen

    2013-08-01

    Glycosylation of HIV-1 envelope proteins (Env gp120/gp41) plays a vital role in viral evasion from the host immune response, which occurs through the masking of key neutralization epitopes and the presentation of the Env glycosylation as \\'self\\' to the host immune system. Env glycosylation is generally conserved, yet its continual evolution plays an important role in modulating viral infectivity and Env immunogenicity. Thus, it is believed that Env glycosylation, which is a vital part of the HIV-1 architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may assist in effectively designing Env-based immunogens and in clearly understanding HIV vaccines. This review provides an in-depth perspective of various aspects of Env glycosylation in the context of HIV-1 pathogenesis. © 2013 Future Medicine Ltd.

  14. Signal and noise modeling in confocal laser scanning fluorescence microscopy.

    Science.gov (United States)

    Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf E; Aach, Til

    2012-01-01

    Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

  15. Links between CD147 Function, Glycosylation, and Caveolin-1

    OpenAIRE

    Tang, Wei; Chang, Sharon B.; Hemler, Martin E.

    2004-01-01

    Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-cataly...

  16. Application of Confocal Laser Scanning Microscopy in Biology and Medicine

    OpenAIRE

    I. A. Volkov; N. V. Frigo; L. F. Znamenskaya; O. R. Katunina

    2014-01-01

    Fluorescence confocal laser scanning microscopy and reflectance confocal laser scanning microscopy are up-to-date highend study methods. Confocal microscopy is used in cell biology and medicine. By using confocal microscopy, it is possible to study bioplasts and localization of protein molecules and other compounds relative to cell or tissue structures, and to monitor dynamic cell processes. Confocal microscopes enable layer-by-layer scanning of test items to create demonstrable 3D models. As...

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

    NARCIS (Netherlands)

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

    2017-01-01

    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

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

    NARCIS (Netherlands)

    De Luca, G.; Breedijk, R.; Hoebe, R.; Stallinga, S.; Manders, E.

    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

  19. Is glycosylated haemoglobin a marker of fertility?

    DEFF Research Database (Denmark)

    Hjollund, N H; Jensen, T K; Bonde, J P

    1999-01-01

    We performed a follow-up study of time to pregnancy in a population of first-time pregnancy planners without previous reproductive experience. The objective of this paper is to report and discuss a finding of a strong relationship between glycosylated haemoglobin (HbA1C) and fertility. A total...... concentration of inhibin A. No association was found between HbA1C and psychosocial distress. The reduced fertility among women with high HbA1C may be due to an association with subclinical polycystic ovaries as indicated by the hormonal profile....

  20. Glycosyl-Nucleolipids as New Bioinspired Amphiphiles

    Directory of Open Access Journals (Sweden)

    Philippe Barthélémy

    2013-09-01

    Full Text Available Four new Glycosyl-NucleoLipid (GNL analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower glim compared to the first generation of GNFs.

  1. A confocal optical microscope for detection of single impurities in a bulk crystal at cryogenic temperatures.

    Science.gov (United States)

    Karlsson, Jenny; Rippe, Lars; Kröll, Stefan

    2016-03-01

    A compact sample-scanning confocal optical microscope for detection of single impurities below the surface of a bulk crystal at cryogenic temperatures is described. The sample, lens, and scanners are mounted inside a helium bath cryostat and have a footprint of only 19 × 19 mm. Wide field imaging and confocal imaging using a Blu-ray lens immersed in liquid helium are demonstrated with excitation at 370 nm. A spatial resolution of 300 nm and a detection efficiency of 1.6% were achieved.

  2. 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.

  3. Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas.

    Science.gov (United States)

    Sanai, Nader; Snyder, Laura A; Honea, Norissa J; Coons, Stephen W; Eschbacher, Jennifer M; Smith, Kris A; Spetzler, Robert F

    2011-10-01

    Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5-ALA)-induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000× magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Intraoperative confocal microscopy can visualize cellular 5-ALA-induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors' institution.

  4. Glycosylation profiles of therapeutic antibody pharmaceuticals.

    Science.gov (United States)

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

    2011-11-01

    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Confocal laser endomicroscopy in ulcerative colitis

    DEFF Research Database (Denmark)

    Karstensen, John Gásdal; Săftoiu, Adrian; Brynskov, Jørn

    2016-01-01

    BACKGROUND AND AIMS: Confocal laser endomicroscopy enables real-time in vivo microscopy during endoscopy and can predict relapse in patients with inflammatory bowel disease in remission. However, little is known about how endomicroscopic features change with time. The aim of this longitudinal study...... was to correlate colonic confocal laser endomicroscopy (CLE) in ulcerative colitis with histopathology and macroscopic appearance before and after intensification of medical treatment. METHODS: Twenty-two patients with ulcerative colitis in clinical relapse and 7 control subjects referred for colonoscopy were...

  6. 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.

  7. Method Development in the Regioselective Glycosylation of Unprotected Carbohydrates

    DEFF Research Database (Denmark)

    Niedbal, Dominika Alina

    and the glycosylations were promoted by tetrabutylammonium bromide. The couplings were completely selective and gave rise to a number of 1,6-linked disaccharides with 1,2- cis-linked orientation. Project 2: Boron-mediated glycosylation of unprotected carbohydrates Boron-mediated regioselective Koenigs...

  8. N-linked glycosylation of the immunoglobulin variable region

    NARCIS (Netherlands)

    van de Bovenkamp, Fleur S.; Derksen, Ninotska I. L.; Ooijevaar-de Heer, Pleuni; van Schie, Karin A.; Kruithof, Simone; Berkowska, Magdalena A.; van der Schoot, C. Ellen; Ijspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E. M.; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S. Marieke; Vidarsson, Gestur; Rispens, Theo

    2018-01-01

    N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we

  9. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...

  10. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower......Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...

  11. Nutritional Therapies in Congenital Disorders of Glycosylation (CDG

    Directory of Open Access Journals (Sweden)

    Peter Witters

    2017-11-01

    Full Text Available Congenital disorders of glycosylation (CDG are a group of more than 130 inborn errors of metabolism affecting N-linked, O-linked protein and lipid-linked glycosylation. The phenotype in CDG patients includes frequent liver involvement, especially the disorders belonging to the N-linked protein glycosylation group. There are only a few treatable CDG. Mannose-Phosphate Isomerase (MPI-CDG was the first treatable CDG by high dose mannose supplements. Recently, with the successful use of d-galactose in Phosphoglucomutase 1 (PGM1-CDG, other CDG types have been trialed on galactose and with an increasing number of potential nutritional therapies. Current mini review focuses on therapies in glycosylation disorders affecting liver function and dietary intervention in general in N-linked glycosylation disorders. We also emphasize now the importance of early screening for CDG in patients with mild hepatopathy but also in cholestasis.

  12. Fluorescence imaging of reactive oxygen species by confocal laser scanning microscopy for track analysis of synchrotron X-ray photoelectric nanoradiator dose: X-ray pump-optical probe.

    Science.gov (United States)

    Jeon, Jae Kun; Han, Sung Mi; Kim, Jong Ki

    2016-09-01

    Bursts of emissions of low-energy electrons, including interatomic Coulomb decay electrons and Auger electrons (0-1000 eV), as well as X-ray fluorescence produced by irradiation of large-Z element nanoparticles by either X-ray photons or high-energy ion beams, is referred to as the nanoradiator effect. In therapeutic applications, this effect can damage pathological tissues that selectively take up the nanoparticles. Herein, a new nanoradiator dosimetry method is presented that uses probes for reactive oxygen species (ROS) incorporated into three-dimensional gels, on which macrophages containing iron oxide nanoparticles (IONs) are attached. This method, together with site-specific irradiation of the intracellular nanoparticles from a microbeam of polychromatic synchrotron X-rays (5-14 keV), measures the range and distribution of OH radicals produced by X-ray emission or superoxide anions ({\\rm{O}}_2^-) produced by low-energy electrons. The measurements are based on confocal laser scanning of the fluorescence of the hydroxyl radical probe 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF) or the superoxide probe hydroethidine-dihydroethidium (DHE) that was oxidized by each ROS, enabling tracking of the radiation dose emitted by the nanoradiator. In the range 70 µm below the irradiated cell, ^\\bullet{\\rm{OH}} radicals derived mostly from either incident X-ray or X-ray fluorescence of ION nanoradiators are distributed along the line of depth direction in ROS gel. In contrast, {\\rm{O}}_2^- derived from secondary electron or low-energy electron emission by ION nanoradiators are scattered over the ROS gel. ROS fluorescence due to the ION nanoradiators was observed continuously to a depth of 1.5 mm for both oxidized APF and oxidized DHE with relatively large intensity compared with the fluorescence caused by the ROS produced solely by incident primary X-rays, which was limited to a depth of 600 µm, suggesting dose enhancement as well as more

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Zavislan James M

    2009-08-01

    Full Text Available Abstract 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

  15. 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

  16. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: LASER POWER MEASUREMENTS

    Science.gov (United States)

    Laser power abstract The reliability of the confocal laser-scanning microscope (CLSM) to obtain intensity measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. The laser power test appears to be one ...

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

    Indian Academy of Sciences (India)

    journal of. August 2003 physics pp. 373–384. Fluorescence confocal polarizing ... and focal conic domains in flat samples of lamellar LCs are practically indistinguishable. ... or less) LC layer confined between two transparent plates. ... in studies of electro-optic effects such as the Frederiks effect, defects, surface anchoring,.

  18. ECM Proteins Glycosylation and Relation to Diabetes

    Science.gov (United States)

    Pernodet, Nadine; Bloomberg, Ayla; Sood, Vandana; Slutsky, Lenny; Ge, Shouren; Clark, Richard; Rafailovich, Miriam

    2004-03-01

    The chemical modification and crosslinking of proteins by sugar glycosylation contribute to the aging of tissue proteins, and acceleration of this reaction during hyperglycemia is implicated in the pathogenesis of diabetic complications, such as disorder of the wound healing. Advanced glycation endproducts (AGEs) formation and protein crosslinking are irreversible processes that alter the structural and functional properties of proteins, lipid components and nucleic acids. And the mechanism, by which it happens, is not clear. Fibrinogen and fibronectin are plasma proteins, which play a major role in human wound healing. Fibrinogen converts to an insoluble fibrin "gel" following a cut, which eventually forms a clot to prevent blood loss, to direct cell adhesion and migration for forming scars. Fibronectin is a critical protein for cell adhesion and migration in wound healing. The effects of glucose on the binding of these plasma proteins from the extra cellular matrix (ECM) were followed at different concentrations by atomic force microscopy and lateral force modulation to measure the mechanical response of the samples. Glucose solutions (1, 2, and 3mg/mL) were incubated with the protein (100 mg/ml) and silicon (Si) substrates spun with sulfonated polystyrene (SPS) 28% for five days. Data showed that not only the organization of the protein on the surface was affected but also its mechanical properties. At 3 mg/mL glucose, Fn fibers were observed to be harder than those of the control, in good agreement with our hypothesis that glycosylation hardens tissues by crosslinking of proteins in the ECM and might cause fibers to break more easily.

  19. N-glycosylation of Colorectal Cancer Tissues

    Science.gov (United States)

    Balog, Crina I. A.; Stavenhagen, Kathrin; Fung, Wesley L. J.; Koeleman, Carolien A.; McDonnell, Liam A.; Verhoeven, Aswin; Mesker, Wilma E.; Tollenaar, Rob A. E. M.; Deelder, André M.; Wuhrer, Manfred

    2012-01-01

    Colorectal cancer is the third most common cancer worldwide with an annual incidence of ∼1 million cases and an annual mortality rate of ∼655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers. PMID:22573871

  20. Pigmented Nodular Basal Cell Carcinomas in Differential Diagnosis with Nodular Melanomas: Confocal Microscopy as a Reliable Tool for In Vivo Histologic Diagnosis

    International Nuclear Information System (INIS)

    Casari, A.; Pellacani, G.; Seidenari, S.; Pepe, P.; Longo, C.; Cesinaro, A. M.; Beretti, F.

    2011-01-01

    Nodular basal cell carcinoma, especially when pigmented, can be in differential diagnosis with nodular melanomas, clinically and dermoscopically. Reflectance confocal microscopy is a relatively new imaging technique that permits to evaluate in vivo skin tumors with a nearly histological resolution. Here, we present four cases of challenging nodular lesions where confocal microscopy was able to clarify the diagnosis.

  1. Confocal microscopy studies of morphology and apoptosis: ovaries, limbs, embryos and insects

    Science.gov (United States)

    Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ap...

  2. Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

    Science.gov (United States)

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  3. Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

    Science.gov (United States)

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  4. Insight into the Microbial Multicellular Lifestyle via Flow-Cell Technology and Confocal Microscopy

    DEFF Research Database (Denmark)

    Pamp, Sünje Johanna; Sternberg, Claus; Tolker-Nielsen, Tim

    2009-01-01

    , industry, and human health. Accordingly a number of biofilm model systems, molecular tools, microscopic techniques, and image analysis programs have been employed for the study of biofilms under controlled and reproducible conditions. Studies using confocal laser scanning microscopy (CLSM) of biofilms...

  5. In vivo Diagnosis of Basal Cell Carcinoma Subtype by Reflectance Confocal Microscopy

    NARCIS (Netherlands)

    Peppelman, M.; Wolberink, E.A.W.; Blokx, W.A.M.; Kerkhof, P.C.M. van de; Erp, P.E.J. van; Gerritsen, M.J.P.

    2013-01-01

    Background: Reflectance confocal microscopy (RCM) is a noninvasive imaging technique. Currently, RCM is mainly used for the diagnosis of melanoma and nonmelanoma skin cancer including basal cell carcinoma (BCC). Until now, it has not been possible to distinguish between subtypes of BCC using RCM.

  6. Reflectance confocal microscopy: non-invasive distinction between actinic keratosis and squamous cell carcinoma

    NARCIS (Netherlands)

    Peppelman, M.; Nguyen, K.P.; Hoogedoorn, L.; Erp, P.E.J. van; Gerritsen, M.J.P.

    2015-01-01

    BACKGROUND: Early recognition of squamous cell carcinoma (SCC) is difficult. Non-invasive reflectance confocal microscopic (RCM) imaging of the skin is a promising diagnostic technique. Although several RCM features for SCC and AK have been described, it is not determined whether RCM has the ability

  7. Characterization of tissue autofluorescence in Barrett's esophagus by confocal fluorescence microscopy

    NARCIS (Netherlands)

    Kara, M. A.; DaCosta, R. S.; Streutker, C. J.; Marcon, N. E.; Bergman, J. J. G. H. M.; Wilson, B. C.

    2007-01-01

    High grade dysplasia and early cancer in Barrett's esophagus can be distinguished in vivo by endoscopic autofluorescence point spectroscopy and imaging from non-dysplastic Barrett's mucosa. We used confocal fluorescence microscopy for ex vivo comparison of autofluorescence in non-dysplastic and

  8. 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

  9. Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

    Science.gov (United States)

    Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple perform...

  10. A multi-axis confocal rheoscope for studying shear flow of structured fluids

    KAUST Repository

    Lin, Neil Y. C.; McCoy, Jonathan H.; Cheng, Xiang; Leahy, Brian; Israelachvili, Jacob N.; Cohen, Itai

    2014-01-01

    of confinement effects. By using our shear cell in conjunction with a biaxial force measurement device and a high-speed confocal microscope, we are able to measure the real-time biaxial stress while simultaneously imaging the material three-dimensional structure

  11. 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.

  12. Engineering Mammalian Mucin-type O-Glycosylation in Plants

    DEFF Research Database (Denmark)

    Yang, Zhang; Drew, Damian P; Jørgensen, Bodil

    2012-01-01

    -glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity...... was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon a2b. In plants, prolines in certain...... classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host...

  13. Functional Analysis of Glycosylation of Zika Virus Envelope Protein

    Directory of Open Access Journals (Sweden)

    Camila R. Fontes-Garfias

    2017-10-01

    Full Text Available Summary: Zika virus (ZIKV infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. : Zika virus (ZIKV causes devastating congenital abnormities and Guillain-Barré syndrome. Fontes-Garfias et al. showed that the glycosylation of ZIKV envelope protein plays an important role in infecting mosquito vectors and pathogenesis in mouse. Keywords: Zika virus, glycosylation, flavivirus entry, mosquito transmission, vaccine

  14. Quantifying risk of penile prosthesis infection with elevated glycosylated hemoglobin.

    Science.gov (United States)

    Wilson, S K; Carson, C C; Cleves, M A; Delk, J R

    1998-05-01

    Elevation of glycosylated hemoglobin above levels of 11.5 mg.% has been considered a contraindication to penile prosthesis implantation in diabetic patients. We determine the predictive value of glycosylated hemoglobin A1C in penile prosthesis infections in diabetic and nondiabetic patients to confirm or deny this prevalent opinion. We conducted a 2-year prospective study of 389 patients, including 114 diabetics, who underwent 3-piece penile prosthesis implantation. All patients had similar preoperative preparation without regard to diabetic status, control or glycosylated hemoglobin A1C level. Risk of infection was statistically analyzed for diabetics versus nondiabetics, glycosylated hemoglobin A1C values above and below 11.5 mg.%, insulin dependent versus oral medication diabetics, and fasting blood sugars above and below 180 mg.%. Prosthesis infections developed in 10 diabetics (8.7%) and 11 nondiabetics (4.0%). No increased infection rate was observed in diabetics with high fasting sugars or diabetics on insulin. There was no statistically significant increased infection risk with increased levels of glycosylated hemoglobin A1C among all patients or among only the diabetics. In fact, there was no meaningful difference in the median or mean level of glycosylated hemoglobin A1C in the infected and noninfected patients regardless of diabetes. Use of glycosylated hemoglobin A1C values to identify and exclude surgical candidates with increased risk of infections is not proved by this study. Elevation of fasting sugar or insulin dependence also does not increase risk of infection in diabetics undergoing prosthesis implantation.

  15. Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

    Science.gov (United States)

    de Jongh, Harmen H. J.; Robles, Carlos López; Nordlee, Julie A.; Lee, Poi-Wah; Baumert, Joseph L.; Hamilton, Robert G.; Taylor, Steve L.; Koppelman, Stef J.

    2013-01-01

    Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing. PMID:23878817

  16. Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

    Directory of Open Access Journals (Sweden)

    Harmen H. J. de Jongh

    2013-01-01

    Full Text Available Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa, the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein’s digestibility is not affected by such processing.

  17. Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts.

    Science.gov (United States)

    Eklund, Erik A; Merbouh, Nabyl; Ichikawa, Mie; Nishikawa, Atsushi; Clima, Jessica M; Dorman, James A; Norberg, Thomas; Freeze, Hudson H

    2005-11-01

    Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

  18. Confocal detection of Rayleigh scattering for residual stress measurement in chemically tempered glass

    Energy Technology Data Exchange (ETDEWEB)

    Hödemann, S., E-mail: siim.hodemann@ut.ee; Möls, P.; Kiisk, V.; Saar, R.; Kikas, J. [Institute of Physics, University of Tartu, Wilhelm Ostwald st., Tartu 50411 (Estonia); Murata, T. [Nippon Electric Glass Co., 7-1 Seiran 2-chome, Otsu-shi, Shiga 520-8639 (Japan)

    2015-12-28

    A new optical method is presented for evaluation of the stress profile in chemically tempered (chemically strengthened) glass based on confocal detection of scattered laser beam. Theoretically, a lateral resolution of 0.2 μm and a depth resolution of 0.6 μm could be achieved by using a confocal microscope with high-NA immersion objective. The stress profile in the 250 μm thick surface layer of chemically tempered lithium aluminosilicate glass was measured with a high spatial resolution to illustrate the capability of the method. The confocal method is validated using transmission photoelastic and Na{sup +} ion concentration profile measurement. Compositional influence on the stress-optic coefficient is calculated and discussed. Our method opens up new possibilities for three-dimensional scattered light tomography of mechanical imaging in birefringent materials.

  19. Association between dermoscopic and reflectance confocal microscopy features of cutaneous melanoma with BRAF mutational status.

    Science.gov (United States)

    Bombonato, C; Ribero, S; Pozzobon, F C; Puig-Butille, J A; Badenas, C; Carrera, C; Malvehy, J; Moscarella, E; Lallas, A; Piana, S; Puig, S; Argenziano, G; Longo, C

    2017-04-01

    Melanomas harbouring common genetic mutations might share certain morphological features detectable with dermoscopy and reflectance confocal microscopy. BRAF mutational status is crucial for the management of metastatic melanoma. To correlate the dermoscopic characteristics of primary cutaneous melanomas with BRAF mutational status. Furthermore, a subset of tumours has also been analysed for the presence of possible confocal features that might be linked with BRAF status. Retrospectively acquired dermoscopic and confocal images of patients with melanoma in tertiary referral academic centres: Skin Cancer Unit in Reggio Emilia and at the Melanoma Unit in Barcelona. Kruskal-Wallis test, logistic regressions, univariate and multivariate analyses have been performed to find dermoscopic and confocal features significantly correlated with BRAF mutational status. Dermoscopically, the presence of irregular peripheral streaks and ulceration were positive predictors of BRAF-mutated melanomas with a statistically significance value, while dotted vessels were more represented in wild-type melanomas. None of the evaluated reflectance confocal microscopy features were correlated with genetic profiling. Ulceration and irregular peripheral streaks represent dermoscopic feature indicative for BRAF-mutated melanoma, while dotted vessels are suggestive for wild-type melanoma. © 2016 European Academy of Dermatology and Venereology.

  20. Confocal soft X-ray scanning transmission microscopy: setup, alignment procedure and limitations

    International Nuclear Information System (INIS)

    Späth, Andreas; Raabe, Jörg; Fink, Rainer H.

    2015-01-01

    A conventional STXM setup has been upgraded with a second micro zone plate and aligned to confocal geometry. Two confocal geometries (in-line and off-axis) have been evaluated and a discussion on prospects and limitations is presented. Zone-plate-based scanning transmission soft X-ray microspectroscopy (STXM) is a well established technique for high-contrast imaging of sufficiently transparent specimens (e.g. ultrathin biological tissues, polymer materials, archaeometric specimens or magnetic thin films) with spatial resolutions in the regime of 20 nm and high spectroscopic or chemical sensitivity. However, due to the relatively large depth of focus of zone plates, the resolution of STXM along the optical axis so far stays unambiguously behind for thicker X-ray transparent specimens. This challenge can be addressed by the implementation of a second zone plate in the detection pathway of the beam, resulting in a confocal arrangement. Within this paper a first proof-of-principle study for a confocal STXM (cSTXM) and an elaborate alignment procedure in transmission and fluorescence geometry are presented. Based on first confocal soft X-ray micrographs of well known specimens, the advantage and limitation of cSTXM as well as further development potentials for future applications are discussed

  1. Confocal soft X-ray scanning transmission microscopy: setup, alignment procedure and limitations

    Energy Technology Data Exchange (ETDEWEB)

    Späth, Andreas [Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Egerlandstraße 3, 91058 Erlangen (Germany); Raabe, Jörg [Paul Scherrer Institut, 5232 Villigen (Switzerland); Fink, Rainer H., E-mail: rainer.fink@fau.de [Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Egerlandstraße 3, 91058 Erlangen (Germany); Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Egerlandstraße 3, 91058 Erlangen (Germany)

    2015-01-01

    A conventional STXM setup has been upgraded with a second micro zone plate and aligned to confocal geometry. Two confocal geometries (in-line and off-axis) have been evaluated and a discussion on prospects and limitations is presented. Zone-plate-based scanning transmission soft X-ray microspectroscopy (STXM) is a well established technique for high-contrast imaging of sufficiently transparent specimens (e.g. ultrathin biological tissues, polymer materials, archaeometric specimens or magnetic thin films) with spatial resolutions in the regime of 20 nm and high spectroscopic or chemical sensitivity. However, due to the relatively large depth of focus of zone plates, the resolution of STXM along the optical axis so far stays unambiguously behind for thicker X-ray transparent specimens. This challenge can be addressed by the implementation of a second zone plate in the detection pathway of the beam, resulting in a confocal arrangement. Within this paper a first proof-of-principle study for a confocal STXM (cSTXM) and an elaborate alignment procedure in transmission and fluorescence geometry are presented. Based on first confocal soft X-ray micrographs of well known specimens, the advantage and limitation of cSTXM as well as further development potentials for future applications are discussed.

  2. N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation.

    Science.gov (United States)

    Stavenhagen, Kathrin; Kayili, H Mehmet; Holst, Stephanie; Koeleman, Carolien A M; Engel, Ruchira; Wouters, Diana; Zeerleder, Sacha; Salih, Bekir; Wuhrer, Manfred

    2018-06-01

    Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N - and O -glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O -glycosylated N-terminal region. Five novel and five known O -glycosylation sites were identified, carrying mainly core1-type O -glycans. In addition, we detected a heavily O -glycosylated portion spanning from Thr 82 -Ser 121 with up to 16 O -glycans attached. Likewise, all known six N -glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N -glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Whole-body ring-shaped confocal photoacoustic computed tomography of small animals in vivo

    Science.gov (United States)

    Xia, Jun; Chatni, Muhammad R.; Maslov, Konstantin; Guo, Zijian; Wang, Kun; Anastasio, Mark; Wang, Lihong V.

    2012-05-01

    We report a novel small-animal whole-body imaging system called ring-shaped confocal photoacoustic computed tomography (RC-PACT). RC-PACT is based on a confocal design of free-space ring-shaped light illumination and 512-element full-ring ultrasonic array signal detection. The free-space light illumination maximizes the light delivery efficiency, and the full-ring signal detection ensures a full two-dimensional view aperture for accurate image reconstruction. Using cylindrically focused array elements, RC-PACT can image a thin cross section with 0.10 to 0.25 mm in-plane resolutions and 1.6 s/frame acquisition time. By translating the mouse along the elevational direction, RC-PACT provides a series of cross-sectional images of the brain, liver, kidneys, and bladder.

  4. High harmonic terahertz confocal gyrotron with nonuniform electron beam

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Wenjie; Guan, Xiaotong; Yan, Yang [THz Research Center, School of Physical Electronics, University of Electronic Science and Technology of China, Chengdu 610054 (China)

    2016-01-15

    The harmonic confocal gyrotron with nonuniform electron beam is proposed in this paper in order to develop compact and high power terahertz radiation source. A 0.56 THz third harmonic confocal gyrotron with a dual arc section nonuniform electron beam has been designed and investigated. The studies show that confocal cavity has extremely low mode density, and has great advantage to operate at high harmonic. Nonuniform electron beam is an approach to improve output power and interaction efficiency of confocal gyrotron. A dual arc beam magnetron injection gun for designed confocal gyrotron has been developed and presented in this paper.

  5. Glycosylation in HIV-1 envelope glycoprotein and its biological implications

    KAUST Repository

    Ho, Yung Shwen; Saksena, Nitin K.

    2013-01-01

    architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may

  6. [The role of protein glycosylation in immune system].

    Science.gov (United States)

    Ząbczyńska, Marta; Pocheć, Ewa

    2015-01-01

    Glycosylation is one of the most frequent post-translational modifications of proteins. The majority of cell surface and secreted proteins involved in immune response is glycosylated. The structural diversity of glycans depends on monosaccharide composition, type of glycosidic linkage and branching. These structural modifications determine a great variability of glycoproteins. The oligosaccharide components of proteins are regulated mostly by expression of glycosyltransferases and glycosidases and many environmental factors. Glycosylation influences the function of all immune cells. Glycans play a crucial role in intercellular contacts and leukocytes migration. These interactions are important in activation and proliferation of leukocytes and during immune response. The key immune proteins, such as TCR, MHC, TLR and antibodies are glycosylated. Sugars on the surface of pathogens and self-surface glycoproteins are recognized by special carbohydrate binding proteins called lectins. Changes of glycan structure are common in many pathological processes occurring in immune system, therefore they are used as molecular markers of different diseases.

  7. Enzymatic Glycosylation of Small Molecules: Challenging Substrates Require Tailored Catalysts

    Czech Academy of Sciences Publication Activity Database

    Desmet, T.; Soetaert, W.; Bojarová, Pavla; Křen, Vladimír; Dijkhuizen, L.; Eastwick-Field, V.; Schiller, A.

    2012-01-01

    Roč. 18, č. 35 (2012), s. 10786-10801 ISSN 0947-6539 Institutional support: RVO:61388971 Keywords : acceptor specificity * enzyme engineering * glycosylation Subject RIV: CE - Biochemistry Impact factor: 5.831, year: 2012

  8. GLYCOSYLATED YGHJ POLYPEPTIDES FROM ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC)

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to glycosylated YghJ polypeptides from or derived from enterotoxigenic Escherichia coli (ETEC) that are immunogenic. In particular, the present invention relates to compositions or vaccines comprising the polypeptides and their application in immunization, vaccination...

  9. IN VITRO STUDY ON INHIBITION OF GLYCOSYLATION OF ...

    African Journals Online (AJOL)

    Administrator

    complications of diabetes mellitus (Makita et al., 1991). Apart from protein ... enzymes; inhibition of regulatory molecule binding; crosslinking of glycosylated .... further investigation specific bio active compound responsible for such activities.

  10. Design and analysis of a cross-type structured-illumination confocal microscope for high speed and high resolution

    International Nuclear Information System (INIS)

    Kim, Young-Duk; Ahn, MyoungKi; Kim, Taejoong; Gweon, DaeGab; Yoo, Hongki

    2012-01-01

    There have been many studies about a super resolution microscope for many years. A super resolution microscope can detect the physical phenomena or morphology of a biological sample more precisely than conventional microscopes. The structured-illumination microscope (SIM) is one of the technologies that demonstrate super resolution. However, the conventional SIM requires more time to obtain one resolution-enhanced image than other super resolution microscopes. More specifically, the conventional SIM uses three images with a 120° phase difference for each direction and three different directions are image-processed to make one resolution enhancement by increasing the optical transfer function in three directions. In this paper, we present a novel cross structured-illumination confocal microscope (CSICM) that takes the advantage of the technology of both SIM and the confocal microscope. The CSICM uses only two directions with three phase difference images, for a total of six images. By reducing the number of images that must be obtained, the total image acquisition time and image reconstruction time in obtaining the final output images can be decreased, and the confocal microscope provides axial information of the sample automatically. We demonstrate our method of cross illumination and evaluate the performance of the CSICM and compare it to the conventional SIM and the confocal microscope. (paper)

  11. A new method for depth profiling reconstruction in confocal microscopy

    Science.gov (United States)

    Esposito, Rosario; Scherillo, Giuseppe; Mensitieri, Giuseppe

    2018-05-01

    Confocal microscopy is commonly used to reconstruct depth profiles of chemical species in multicomponent systems and to image nuclear and cellular details in human tissues via image intensity measurements of optical sections. However, the performance of this technique is reduced by inherent effects related to wave diffraction phenomena, refractive index mismatch and finite beam spot size. All these effects distort the optical wave and cause an image to be captured of a small volume around the desired illuminated focal point within the specimen rather than an image of the focal point itself. The size of this small volume increases with depth, thus causing a further loss of resolution and distortion of the profile. Recently, we proposed a theoretical model that accounts for the above wave distortion and allows for a correct reconstruction of the depth profiles for homogeneous samples. In this paper, this theoretical approach has been adapted for describing the profiles measured from non-homogeneous distributions of emitters inside the investigated samples. The intensity image is built by summing the intensities collected from each of the emitters planes belonging to the illuminated volume, weighed by the emitters concentration. The true distribution of the emitters concentration is recovered by a new approach that implements this theoretical model in a numerical algorithm based on the Maximum Entropy Method. Comparisons with experimental data and numerical simulations show that this new approach is able to recover the real unknown concentration distribution from experimental profiles with an accuracy better than 3%.

  12. Diversity and functions of protein glycosylation in insects.

    Science.gov (United States)

    Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

    2017-04-01

    The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Materials and corrosion characterization using the confocal resonator

    Energy Technology Data Exchange (ETDEWEB)

    Tigges, C.P.; Sorensen, N.R.; Hietala, V.M.; Plut, T.A. [and others

    1997-05-01

    Improved characterization and process control is important to many Sandia and DOE programs related to manufacturing. Many processes/structures are currently under-characterized including thin film growth, corrosion and semiconductor structures, such as implant profiles. A sensitive tool is required that is able to provide lateral and vertical imaging of the electromagnetic properties of a sample. The confocal resonator is able to characterize the surface and near-surface impedance of materials. This device may be applied to a broad range of applications including in situ evaluation of thin film processes, physical defect detection/characterization, the characterization of semiconductor devices and corrosion studies. In all of these cases, the technology should work as a real-time process diagnostic or as a feedback mechanism regarding the quality of a manufacturing process. This report summarizes the development and exploration of several diagnostic applications.

  14. Ex vivo confocal microscopy: an emerging technique in dermatology

    Science.gov (United States)

    Perrot, Jean Luc; Labeille, Bruno; Cambazard, Frédéric; Rubegni, Pietro

    2018-01-01

    This review aims to give an overview of the current available applications of ex vivo confocal microscopy (EVCM) in dermatology. EVCM is a relatively new imaging technique that allows microscopic examination of freshly excised unfixed tissue. It enables a rapid examination of the skin sample directly in the surgery room and thus represents an alternative to the intraoperative micrographic control of the surgical margins of cutaneous tumors by standard microscopic examination on cryopreserved sections during Mohs surgery. Although this technique has mainly been developed for the margin’s control of basal cell carcinoma, many other skin tumors have been studied, including melanoma. Use of EVCM is continuing to evolve, and many possible applications are under investigation, such as the study of nails and hair diseases and the diagnosis of skin infections. PMID:29785327

  15. [Contribution of confocal microscopy and anterior chamber OCT to the study of corneal endothelial pathologies].

    Science.gov (United States)

    Fayol, N; Labbé, A; Dupont-Monod, S; Dupas, B; Baudouin, C

    2007-04-01

    To describe the appearance of various endothelial diseases with in vivo confocal microscopy and anterior chamber optical coherence tomography (AC OCT). In this study, ten patients with five different corneal endothelial pathologies were evaluated. Three patients had cornea guttata, three had corneal endothelial precipitates, two had irido-corneo-endothelial (ICE) syndrome, one had endothelial folds, and one had breaks in the Descemet membrane. All patients had bilateral ophthalmologic examinations, in vivo confocal microscopy, and AC OCT analysis. In cases of cornea guttata, AC OCT showed a finely embossed line corresponding to the empty intercellular cavities found with in vivo confocal microscopy. Corneal endothelium precipitates had the aspect of round formations suspended with the endothelium. Iris atrophy and irido-corneal synechiae resulting from ICE syndrome were precisely visualized with the AC OCT. High-resolution images of the anterior segment could be obtained using the AC OCT. Associated with in vivo confocal microscopy, these two new imaging techniques provide a precise evaluation of endothelial pathologies.

  16. 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.

  17. [Non-enzymatic glycosylation of dietary protein in vitro].

    Science.gov (United States)

    Bednykh, B S; Evdokimov, I A; Sokolov, A I

    2015-01-01

    Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation

  18. Prion propagation in cells expressing PrP glycosylation mutants.

    Science.gov (United States)

    Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-04-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

  19. Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

    DEFF Research Database (Denmark)

    Norén, K; Hansen, Gert Helge; Clausen, H

    1997-01-01

    In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...... the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms...

  20. 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

  1. Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

    Science.gov (United States)

    Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

    2017-10-31

    Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein

    DEFF Research Database (Denmark)

    Sherlock, O.; Dobrindt, U.; Jensen, J.B.

    2006-01-01

    a novel member to this exclusive group, namely, antigen 43 (Ag43), a self-recognizing autotransporter protein. By mass spectrometry Ag43 was demonstrated to be glycosylated by addition of heptose residues at several positions in the passenger domain. Glycosylation of Ag43 by the action of the Aah and Tib......C glycosyltransferases was observed in laboratory strains. Importantly, Ag43 was also found to be glycosylated in a wild-type strain, suggesting that Ag43-glycosylation may be a widespread phenomenon. Glycosylation of Ag43 does not seem to interfere with its self-associating properties. However, the glycosylated form...

  3. Fluorescence lifetime measurement with confocal endomicroscopy for direct analysis of tissue biochemistry in vivo

    Directory of Open Access Journals (Sweden)

    Youngjae Won

    2016-08-01

    Full Text Available Confocal endomicroscopy is a powerful tool for in vivo real-time imaging at cellular resolution inside a living body without tissue resection. Microscopic fluorescence lifetime measurement can provide information about localized biochemical conditions such as pH and the concentrations of oxygen and calcium. We hypothesized that combining these techniques could assist accurate cancer discrimination by providing both biochemical and morphological information. We designed a dual-mode experimental setup for confocal endomicroscopic imaging and fluorescence lifetime measurement and applied it to a mouse xenograft model of activated human pancreatic cancer generated by subcutaneous injection of AsPC-1 tumor cells. Using this method with pH-sensitive sodium fluorescein injection, we demonstrated discrimination between normal and cancerous tissues in a living mouse. With further development, this method may be useful for clinical cancer detection.

  4. Confocal Laser Endomicroscopy in the Study of Colonic Mucosa in IBD Patients: A Review

    Directory of Open Access Journals (Sweden)

    Francesca Salvatori

    2012-01-01

    Full Text Available Confocal laser endomicroscopy (CLE is one of several novel methods that provide real-time, high-resolution imaging at a micronscale via endoscopes. CLE and related technologies are often termed “virtual biopsy” as they simulate the images seen in traditional histology. Recently, the use of CLE was reported in the study of colonic mucosa in patients with inflammatory bowel diseases and in particular in patients affected by ulcerative colitis. CLE has the potential to have an important role in management of IBD patients as it can be used to assess the grading of colitis and in detection of microscopic colitis in endoscopically silent segments. Moreover, CLE can be used in surveillance programs especially in high-risk patients. This report aims to evaluate the current data on the application of confocal endomicroscopy in clinical gastroenterology and particularly in the study of colonic mucosa in UC patients.

  5. In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial pressure.

    Science.gov (United States)

    Wang, Yu; Hu, Song; Maslov, Konstantin; Zhang, Yu; Xia, Younan; Wang, Lihong V

    2011-04-01

    We developed dual-modality microscope integrating photoacoustic microscopy (PAM) and fluorescence confocal microscopy (FCM) to noninvasively image hemoglobin oxygen saturation (sO₂) and oxygen partial pressure (pO₂) in vivo in single blood vessels with high spatial resolution. While PAM measures sO₂ by imaging hemoglobin optical absorption at two wavelengths, FCM quantifies pO₂ using phosphorescence quenching. The variations of sO₂ and pO₂ values in multiple orders of vessel branches under hyperoxic (100% oxygen) and normoxic (21% oxygen) conditions correlate well with the oxygen-hemoglobin dissociation curve. In addition, the total concentration of hemoglobin is imaged by PAM at an isosbestic wavelength.

  6. Methods for studying biofilm formation: flow cells and confocal laser scanning microscopy

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim; Sternberg, Claus

    2014-01-01

    In this chapter methods for growing and analyzing biofilms under hydrodynamic conditions in flow cells are described. Use of flow cells allows for direct microscopic investigation of biofilm formation. The flow in these chambers is essentially laminar, which means that the biofilms can be grown u......, inoculation of the flow cells, running of the system, confocal laser scanning microscopy and image analysis, and disassembly and cleaning of the system....

  7. Toward real-time virtual biopsy of oral lesions using confocal laser endomicroscopy interfaced with embedded computing.

    Science.gov (United States)

    Thong, Patricia S P; Tandjung, Stephanus S; Movania, Muhammad Mobeen; Chiew, Wei-Ming; Olivo, Malini; Bhuvaneswari, Ramaswamy; Seah, Hock-Soon; Lin, Feng; Qian, Kemao; Soo, Khee-Chee

    2012-05-01

    Oral lesions are conventionally diagnosed using white light endoscopy and histopathology. This can pose a challenge because the lesions may be difficult to visualise under white light illumination. Confocal laser endomicroscopy can be used for confocal fluorescence imaging of surface and subsurface cellular and tissue structures. To move toward real-time "virtual" biopsy of oral lesions, we interfaced an embedded computing system to a confocal laser endomicroscope to achieve a prototype three-dimensional (3-D) fluorescence imaging system. A field-programmable gated array computing platform was programmed to enable synchronization of cross-sectional image grabbing and Z-depth scanning, automate the acquisition of confocal image stacks and perform volume rendering. Fluorescence imaging of the human and murine oral cavities was carried out using the fluorescent dyes fluorescein sodium and hypericin. Volume rendering of cellular and tissue structures from the oral cavity demonstrate the potential of the system for 3-D fluorescence visualization of the oral cavity in real-time. We aim toward achieving a real-time virtual biopsy technique that can complement current diagnostic techniques and aid in targeted biopsy for better clinical outcomes.

  8. Confocal fluorescence techniques in industrial application

    Science.gov (United States)

    Eggeling, Christian; Gall, Karsten; Palo, Kaupo; Kask, Peet; Brand, Leif

    2003-06-01

    The FCS+plus family of evaluation tools for confocal fluorescence spectroscopy, which was developed during recent years, offers a comprehensive view to a series of fluorescence properties. Originating in fluorescence correlation spectroscopy (FCS) and using similar experimental equipment, a system of signal processing methods such as fluorescence intensity distribution analysis (FIDA) was created to analyze in detail the fluctuation behavior of fluorescent particles within a small area of detection. Giving simultaneous access to molecular parameters like concentration, translational and rotational diffusion, molecular brightness, and multicolor coincidence, this portfolio was enhanced by more traditional techniques of fluorescence lifetime as well as time-resolved anisotropy determination. The cornerstones of the FCS+plus methodology will be shortly described. The inhibition of a phosphatase enzyme activity gives a comprehensive industrial application that demonstrates FCS+plus' versatility and its potential for pharmaceutical drug discovery.

  9. Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

    NARCIS (Netherlands)

    Wang, Guanbo; de Jong, Rob N; van den Bremer, Ewald T J; Parren, Paul W H I; Heck, Albert J R

    2017-01-01

    The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

  10. Evaluation and purchase of confocal microscopes: Numerous factors to consider

    Science.gov (United States)

    The purchase of a confocal microscope can be a complex and difficult decision for an individual scientist, group or evaluation committee. This is true even for scientists that have used confocal technology for many years. The task of reaching the optimal decision becomes almost i...

  11. DISAL glycosyl donors for the synthesis of a linear hexasaccharide under mild conditions

    DEFF Research Database (Denmark)

    Petersen, Lars; Laursen, Jane B.; Larsen, K.

    2003-01-01

    The new class of glycosyl donors with a methyl 3,5-dinitrosalicylate (DISAL) anomeric leaving group has proved efficient for glycosylation under strictly neutral, mildly basic, or mildly acidic conditions. Here, we report the synthesis of novel DISAL disaccharide glycosyl donors prepared by easy...... nucleophilic aromatic substitution. These DISAL donors proved efficient in the synthesis of a starch-related hexasaccharide under very mild conditions. Glycosylations proceeded with alpha-selectivity and were compatible with Trt protecting groups....

  12. Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy.

    OpenAIRE

    Williams, S K; Devenny, J J; Bitensky, M W

    1981-01-01

    Microvessels isolated from rat epididymal fat exhibit differential vesicular ingestion rates for unmodified and non-enzymatically glycosylated rat albumin. While unmodified rat albumin is excluded from ingestion by endothelial micropinocytic vesicles, glycosylated albumin is avidly taken up by endocytosis. Interaction of albumin and glycosylated albumin with endothelium was studied with a double-label fluorescence assay of micropinocytosis. When glycosylated albumin was present at a concentra...

  13. Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

    Science.gov (United States)

    Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

    2012-05-01

    Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  14. 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

  15. N-Glycosylation of Carnosinase Influences Protein Secretion and Enzyme Activity Implications for Hyperglycemia

    NARCIS (Netherlands)

    Riedl, Eva; Koeppel, Hannes; Pfister, Frederick; Peters, Verena; Sauerhoefer, Sibylle; Sternik, Paula; Brinkkoetter, Paul; Zentgraf, Hanswalter; Navis, Gerjan; Henning, Robert H.; Van Den Born, Jacob; Bakker, Stephan J. L.; Janssen, Bart; van der Woude, Fokko J.; Yard, Benito A.

    OBJECTIVE-The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1

  16. Effect of Cola acuminate on Blood Glucose and Glycosylated ...

    African Journals Online (AJOL)

    The levels of blood glucose and glycosylated haemoglobin (GHB) were studied in 42 Wistar rats divided into three groups; controls, group A and group B. Control rats consumed only feeds, group A consumed 0.04g of Cola acuminate, while group B consumed 0.08g of Cola acuminate mixed with their feeds daily for six ...

  17. Perinatal and early infantile symptoms in congenital disorders of glycosylation

    NARCIS (Netherlands)

    Funke, S.; Gardeitchik, T.; Kouwenberg, D.; Mohamed, M.; Wortmann, S.B.; Korsch, E.; Adamowicz, M.; Al-Gazali, L.; Wevers, R.A.; Horvath, A.; Lefeber, D.J.; Morava, E.

    2013-01-01

    Congenital disorders of glycosylation (CDG) are a rapidly growing family of inborn errors. Screening for CDG in suspected cases is usually performed in the first year of life by serum transferrin isoelectric focusing or mass spectrometry. Based on the transferrin analysis patients can be

  18. SnapShot: O-Glycosylation Pathways across Kingdoms

    DEFF Research Database (Denmark)

    Joshi, Hiren J.; Narimatsu, Yoshiki; Schjoldager, Katrine T.

    2018-01-01

    O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans modulate the structure, stability, and function of proteins and serve generalized as well as highly specific roles in most biological processes. This ShapShot presents types of......-glycans found in different organisms and their principle biosynthetic pathways...

  19. Chapter Three -- Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels

    Energy Technology Data Exchange (ETDEWEB)

    Greene, Eric R. [Univ. of Colorado, Boulder, CO (United States). Dept. of Chemistry and Biochemistry and BioFrontiers Inst.; Himmel, Michael E. [National Renewable Energy Lab. (NREL), Golden, CO (United States). Biosciences Center; Beckham, Gregg T. [National Renewable Energy Lab. (NREL), Golden, CO (United States). National Bioenergy Center; Tan, Zhongping [Univ. of Colorado, Boulder, CO (United States). Dept. of Chemistry and Biochemistry and BioFrontiers Inst.

    2015-10-24

    Methods for the manipulation of glycan structures have been recently reported that employ genetic tuning of glycan-active enzymes expressed from homogeneous and heterologous fungal hosts. Taken together, these studies have enabled new strategies for the exploitation of protein glycosylation for the production of enhanced cellulases for biofuel production.

  20. Predictive glycoengineering of biosimilars using a Markov chain glycosylation model

    DEFF Research Database (Denmark)

    Spahn, Philipp N.; Hansen, Anders Holmgaard; Kol, Stefan

    2017-01-01

    Biosimilar drugs must closely resemble the pharmacological attributes of innovator products to ensure safetyand efficacy to obtain regulatory approval. Glycosylation is one critical quality attribute that must be matched, but it is inherently difficult to control due to the complexity of its...

  1. Glycosylation patterns of kidney proteins differ in rat diabetic nephropathy.

    Science.gov (United States)

    Ravidà, Alessandra; Musante, Luca; Kreivi, Marjut; Miinalainen, Ilkka; Byrne, Barry; Saraswat, Mayank; Henry, Michael; Meleady, Paula; Clynes, Martin; Holthofer, Harry

    2015-05-01

    Diabetic nephropathy often progresses to end-stage kidney disease and, ultimately, to renal replacement therapy. Hyperglycemia per se is expected to have a direct impact on the biosynthesis of N- and O-linked glycoproteins. This study aims to establish the link between protein glycosylation and progression of experimental diabetic kidney disease using orthogonal methods. Kidneys of streptozotocin-diabetic and control rats were harvested at three different time points post streptozotocin injection. A panel of 12 plant lectins was used in the screening of lectin blots. The lectins UEAI, PHA-E, GSI, PNA, and RCA identified remarkable disease-associated differences in glycoprotein expression. Lectin affinity chromatography followed by mass spectrometric analyses led to the identification of several glycoproteins involved in salt-handling, angiogenesis, and extracellular matrix degradation. Our data confirm a substantial link between glycosylation signature and diabetes progression. Furthermore, as suggested by our findings on dipeptidyl peptidase-IV, altered protein glycosylation may reflect changes in biochemical properties such as enzymatic activity. Thus, our study demonstrates the unexplored potential of protein glycosylation analysis in the discovery of molecules linked to diabetic kidney disease.

  2. Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

    DEFF Research Database (Denmark)

    Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

    2003-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla...

  3. 21 CFR 864.7470 - Glycosylated hemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glycosylated hemoglobin assay. 864.7470 Section 864.7470 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7470...

  4. Deciphering a pathway of Halobacterium salinarum N-glycosylation

    Science.gov (United States)

    Kandiba, Lina; Eichler, Jerry

    2015-01-01

    Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered. PMID:25461760

  5. Confocal histopathology of irritant contact dermatitis in vivo and the impact of skin color (black vs white)

    NARCIS (Netherlands)

    Hicks, Shari P.; Swindells, Kirsty J.; Middelkamp-Hup, Maritza A.; Sifakis, Martine A.; González, Ernesto; González, Salvador

    2003-01-01

    BACKGROUND: The pathogenesis of irritant contact dermatitis and its modulation according to skin color is not well understood. Reflectance confocal microscopy (RCM) enables high-resolution, real-time, in-vivo imaging of human skin. OBJECTIVE: The goal of our study was to use RCM to determine whether

  6. Confocal Raman Microspectroscopy of Oral Streptococci

    Science.gov (United States)

    Beier, Brooke D.

    Raman spectroscopy has been used in a variety of applications throughout the field of biomedical optics. It has the ability to acquire chemically-specific information in a non-invasive manner, without the need for exogenous markers. This makes it useful in the identification of bacterial species, as well as in the study of tissues and other cells. In this work, a species identification model has been created in order to discriminate between the oral bacterial species Streptococcus sanguinis and Streptococcus mutans. These are two of the most prevalent species within the human mouth and their relative concentrations can be an indicator of a patient's oral health and risk of tooth decay. They are predominantly found within plaque on the tooth's surface. To study a simplified model for dental plaque, we have examined S. sanguinis and S. mutans grown in biofilm forms. Raman spectroscopy has been implemented here through a confocal microscope. The optical system has been equipped with computationally controlled stages to allow for automated scanning, including autofocusing to probe a consistent depth within a sample. A spectrum has been acquired from each position within a scan and sent for spectral preprocessing before being submitted for species identification. This preprocessing includes an algorithm that has been developed to remove fluorescence features from known contaminants within the confocal volume, to include signal from a fluorescent substrate. Species classification has been accomplished using a principal component score-fed logistic regression model constructed from a variety of biofilm samples that have been transferred and allowed to dry, as might occur with the study of plaque samples. This binary classification model has been validated on other samples with identical preparations. The model has also been transferred to determine the species of hydrated biofilms studied in situ. Artificially mixed biofilms have been examined to test the spatial

  7. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots

    Science.gov (United States)

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J.; Rohrbach, Alexander

    2016-01-01

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

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

    Science.gov (United States)

    Wang, Peng; Behan, Gavin; Kirkland, Angus I; Nellist, Peter D; Cosgriff, Eireann C; D'Alfonso, Adrian J; Morgan, Andrew J; Allen, Leslie J; Hashimoto, Ayako; Takeguchi, Masaki; Mitsuishi, Kazutaka; Shimojo, Masayuki

    2011-06-01

    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. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots.

    Science.gov (United States)

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J; Rohrbach, Alexander

    2016-08-24

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection.

  10. N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase

    Czech Academy of Sciences Publication Activity Database

    Plíhal, Ondřej; Sklenář, Jan; Kmoníčková, J.; Man, Petr; Pompach, Petr; Havlíček, Vladimír; Křen, Vladimír; Bezouška, Karel

    2004-01-01

    Roč. 32, č. 5 (2004), s. 764-765 ISSN 0300-5127 R&D Projects: GA ČR GA203/04/1045 Institutional research plan: CEZ:AV0Z5020903 Keywords : asperillus oryzoe * glycosyl hydrolase * preproprotein Subject RIV: EE - Microbiology, Virology Impact factor: 2.267, year: 2004

  11. Clinical applications of in vivo fluorescence confocal laser scanning microscopy

    Science.gov (United States)

    Oh, Chilhwan; Park, Sangyong; Kim, Junhyung; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Chun, Byungseon; Gweon, Daegab

    2008-02-01

    Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In

  12. Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery.

    Science.gov (United States)

    Bennàssar, A; Vilata, A; Puig, S; Malvehy, J

    2014-02-01

    Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours. © 2013 British Association of Dermatologists.

  13. In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract

    Science.gov (United States)

    Piyawattanametha, Wibool; Ra, Hyejun; Qiu, Zhen; Friedland, Shai; Liu, Jonathan T. C.; Loewke, Kevin; Kino, Gordon S.; Solgaard, Olav; Wang, Thomas D.; Mandella, Michael J.; Contag, Christopher H.

    2012-02-01

    Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 μm2 and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.

  14. Investigation of phosphatidylcholine enhancing FITC-insulin across buccal mucosa by confocal laser scanning microscopy

    Science.gov (United States)

    Tian, Weiqun; Su, Li; Zeng, Shaoqun; Luo, Qingming; Gao, Qiuhua; Xu, Huibi

    2002-04-01

    The aim was to characterize the transport of fluorescein isothiocyanate (FITC)-labeled dextran and insulin with different resoluble compounds for peptides and proteins through buccal mucosa. The penetration rate of insulin molecules through porcine buccal mucosa (a nonkeratinized epithelium, comparable to human buccal mucosa) was investigated by measuring transbuccal fluxes and by analyzing the distribution of the fluorescent probe in the rabbit buccal mucosa epithelium, using confocal laser scanning microscopy for visualizing permeation pathways. The confocal images of the distribution pattern of FITC-dextran and FITC-insulin showed that the paracellular route is the major pathway of FITC-dextran through buccal mucosa epithelium, the intra-cellular route is the major pathway of FITC-insulin through buccal mucosa epithelium. The permeation rate can be increased by co-administration of soybean phosphatidylcholine (SPC).

  15. Analysis of cell-tissue grafts under weightless conditions using confocal fluorescence microscopy

    Science.gov (United States)

    Volova, L. T.; Milyakova, M. N.; Rossinskaya, V. V.; Boltovskaya, V. V.; Kulagina, L. N.; Kurganskaya, L. V.; Timchenko, P. E.; Timchenko, E. V.; Zherdeva Taskina, Larisa A.

    2015-03-01

    The research results of monitoring of viable cells in a cellular-tissue graft using confocal laser fluorescence microscopy at 488 nm and 561 nm with the use of fluorophore propidium iodide (propidium iodide, PI Sigma Aldrich USA) are presented. The processing of the received images was carried out using the software ANDOR. It is experimentally shown that the method of confocal fluorescence microscopy is one of the informational methods for detecting cells populated in a 3-D bio-carrier with a resolution of at least 400 nm. Analysis of the received micrographs suggests that the cells that were in a bio-carrier for 30 days in a synchronous ground-based experiment retained their viability compared to a similar space-based experiment in which the cells were hardly detected in a bio-carrier.

  16. A multi-axis confocal rheoscope for studying shear flow of structured fluids

    KAUST Repository

    Lin, Neil Y. C.

    2014-03-01

    We present a new design for a confocal rheoscope that enables uniform uniaxial or biaxial shear. The design consists of two precisely positioned parallel plates with a gap that can be adjusted down to 2 ±0.1 μm, allowing for the exploration of confinement effects. By using our shear cell in conjunction with a biaxial force measurement device and a high-speed confocal microscope, we are able to measure the real-time biaxial stress while simultaneously imaging the material three-dimensional structure. We illustrate the importance of the instrument capabilities by discussing the applications of this instrument in current and future research topics in colloidal suspensions. © 2014 AIP Publishing LLC.

  17. 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.

  18. Selective Bioparticle Retention and Characterization in a Chip-Integrated Confocal Ultrasonic Cavity

    DEFF Research Database (Denmark)

    Svennebring, J.; Manneberg, O.; Skafte-Pedersen, Peder

    2009-01-01

    We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field at the c......We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field...... sample feeding, a set of several manipulation functions performed in series is demonstrated: sample bypass-injection-aggregation and retention-positioning. Finally, we demonstrate transillumination microscopy imaging Of Ultrasonically trapped COS-7 cell aggregates. Biotechnol. Bioeng. 2009;103: 323-328....

  19. 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.

  20. Application of Reflectance Confocal Microscopy in Dermatology Practice

    Directory of Open Access Journals (Sweden)

    Ayşe Esra Koku Aksu

    2015-03-01

    Full Text Available In vivo reflectance confocal microscopy (RCM is a non-invasive method, imaging cellular structures in living skin at a level close to the histological resolution. It is easier to diagnose melanocytic and non-melanocytic skin tumors especially in difficult cases when RCM features have been identified. Determination of the cellular features, presence of cellular and structural atypia with RCM allows the discrimination of benign and malignant lesions. Preoperative differential diagnosis of malignant lesions, determining preoperative lesion borders in complicated cases, identification of local recurrence after excision of malignant lesions, monitoring the treatment efficacy in patients using topical treatment and who can not be operated, are the main areas of RCM in tumoral lesions. Besides, RCM is helpful in the establishing the diagnosis of inflammatory disease like psoriasis, contact dermatitis, lichen planus and in evaluation of therapeutic efficacy, detecting of infestation like tinea, skabiyes, demodicosis and determining the level of bullae in bullous disease. Due to being noninvasive, RCM is preferred in cosmetology, in clinical research and practice for the evaluation of the effectiveness of cosmetic products and cosmetic procedures.

  1. Confocal fluorescence microscopy for minimal-invasive tumor diagnosis

    International Nuclear Information System (INIS)

    Zenzinger, M.; Bille, J.

    2000-01-01

    The goal of the project ''stereotactic laser-neurosurgery'' is the development of a system for careful and minimal-invasive resection of brain tumors with ultrashort laser pulses through a thin probe. A confocal laser-scanning-microscope is integrated in the probe. In this paper, the simulation of its optical properties by a laboratory setup and the expansion by the ability for fluorescence microscopy are reported. For a valuation of the imaging properties, the point-spread-function in three dimensions and the axial depth-transfer-function were measured and thus, among other things, the resolving power and the capacity for depth discrimination were analysed. The microscope will enable intra-operative detection of tumor cells by the method of immunofluorescence. As a first model of the application in the brain, cell cultures, that fluorescein-labelled antibodies were bound to specifically, were used in this work. Due to the fluorescence signal, it was possible to detect and identify clearly the areas that had been marked in this manner, proving the suitability of the setup for minimal-invasive tumor diagnosis. (orig.)

  2. Simultaneous Confocal Scanning Laser Ophthalmoscopy Combined with High-Resolution Spectral-Domain Optical Coherence Tomography: A Review

    Directory of Open Access Journals (Sweden)

    Verônica Castro Lima

    2011-01-01

    Full Text Available We aimed to evaluate technical aspects and the clinical relevance of a simultaneous confocal scanning laser ophthalmoscope and a high-speed, high-resolution, spectral-domain optical coherence tomography (SDOCT device for retinal imaging. The principle of confocal scanning laser imaging provides a high resolution of retinal and choroidal vasculature with low light exposure. Enhanced contrast, details, and image sharpness are generated using confocality. The real-time SDOCT provides a new level of accuracy for assessment of the angiographic and morphological correlation. The combined system allows for simultaneous recordings of topographic and tomographic images with accurate correlation between them. Also it can provide simultaneous multimodal imaging of retinal pathologies, such as fluorescein and indocyanine green angiographies, infrared and blue reflectance (red-free images, fundus autofluorescence images, and OCT scans (Spectralis HRA + OCT; Heidelberg Engineering, Heidelberg, Germany. The combination of various macular diagnostic tools can lead to a better understanding and improved knowledge of macular diseases.

  3. Patterns of glycemic control using glycosylated hemoglobin in diabetics

    Directory of Open Access Journals (Sweden)

    Arunpreet Singh Kahlon

    2011-01-01

    Full Text Available Aim : Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. Objective : To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. Materials and Methods : A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Results : Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3% had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came

  4. Patterns of glycemic control using glycosylated hemoglobin in diabetics.

    Science.gov (United States)

    Kahlon, Arunpreet Singh; Pathak, Rambha

    2011-07-01

    Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS) is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3%) had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came out to be .311. This correlation was found to be statistically

  5. 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.

  6. 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.

  7. 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.

  8. Investigation of the petrophysical properties of a porous sandstone sample using confocal scanning laser microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Petford, N. [Kingston Univ., Centre for Earth and Environmental Science Research, Kingston (United Kingdom); Davidson, G. [University Coll., Dept. of Electronic and Electrical Engineering, London (United Kingdom); Miller, J.A. [Cambridge Univ., Dept. of Earth Sciences, Cambridge (United Kingdom)

    2001-05-01

    Confocal scanning laser microscopy (CSLM) is used to produce images of the two- and three-dimensional distribution and geometry of pore space in a reservoir sandstone and measure the 2D distribution of pore throat radii. Non-destructive serial sectioning of the rock using laser light at 100% illumination, combined with image thresholding and histogram equalization techniques allow the pore volume structure of the uppermost 100 {mu}m of the sample to be reconstructed. Negative imaging of the pore volume gave superior depth and feature resolution compared to positive (reflection) imaging. Artefacts encountered in applying classical Medial Axial Transforms to CSLM images include branch networks dominated by coordination numbers of 3. Skeletonization using Euclidean distance maps gives increased accuracy in the description of the pore network. Measured pore throat size distribution in the rock is strongly exponential and described by the expression y 219e{sup -0.25x} where y is the number of pore throats. (Author)

  9. Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Jeong-Hwan Lee

    Full Text Available Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(Ps, provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P SO57 with or without an endoplasmic reticulum (ER-retention peptide signal (Lys-Asp-Glu-Leu; KDEL in transgenic tobacco plants (Nicotiana tabacum were analyzed. The expression levels of mAb(P SO57 with KDEL (mAb(PK were significantly higher than those of mAb(P SO57 without KDEL (mAb(P regardless of the transcription level. The Fc domains of both purified mAb(P and mAb(PK and hybridoma-derived mAb (mAb(H had similar levels of binding activity to the FcγRI receptor (CD64. The mAb(PK had glycan profiles of both oligomannose (OM type (91.7% and Golgi type (8.3%, whereas the mAb(P had mainly Golgi type glycans (96.8% similar to those seen with mAb(H. Confocal analysis showed that the mAb(PK was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P with KDEL in the ER. Both mAb(P and mAb(PK disappeared with similar trends to mAb(H in BALB/c mice. In addition, mAb(PK was as effective as mAb(H at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

  10. Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation.

    Science.gov (United States)

    Romero-Fernandez, Wilber; Borroto-Escuela, Dasiel O; Alea, Mileidys Perez; Garcia-Mesa, Yoelvis; Garriga, Pere

    2011-12-01

    The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity. © The Author 2011. Published by Oxford University Press. All rights reserved.

  11. Macrocyclic bis-thioureas catalyze stereospecific glycosylation reactions.

    Science.gov (United States)

    Park, Yongho; Harper, Kaid C; Kuhl, Nadine; Kwan, Eugene E; Liu, Richard Y; Jacobsen, Eric N

    2017-01-13

    Carbohydrates are involved in nearly all aspects of biochemistry, but their complex chemical structures present long-standing practical challenges to their synthesis. In particular, stereochemical outcomes in glycosylation reactions are highly dependent on the steric and electronic properties of coupling partners; thus, carbohydrate synthesis is not easily predictable. Here we report the discovery of a macrocyclic bis-thiourea derivative that catalyzes stereospecific invertive substitution pathways of glycosyl chlorides. The utility of the catalyst is demonstrated in the synthesis of trans-1,2-, cis-1,2-, and 2-deoxy-β-glycosides. Mechanistic studies are consistent with a cooperative mechanism in which an electrophile and a nucleophile are simultaneously activated to effect a stereospecific substitution reaction. Copyright © 2017, American Association for the Advancement of Science.

  12. Glycosylation status of vitamin D binding protein in cancer patients.

    Science.gov (United States)

    Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

    2009-10-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

  13. 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.

  14. Confocal laser scanning microscopy in study of bone calcification

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► High-magnification images with depth selection, and thin sections were observed using CLSM. ► The direction and velocity of calcification of the bone was observed by administration of 2 fluorescent dyes. ► In dog femora grafted with coral blocks, newly-formed bone was observed in the coral block space with a rough surface. ► 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 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.

  15. Electrospray Ionization Mass Spectrometric Analysis of Highly Reactive Glycosyl Halides

    Directory of Open Access Journals (Sweden)

    Lajos Kovács

    2012-07-01

    Full Text Available Highly reactive glycosyl chlorides and bromides have been analysed by a routine mass spectrometric method using electrospray ionization and lithium salt adduct-forming agents in anhydrous acetonitrile solution, providing salient lithiated molecular ions [M+Li]+, [2M+Li]+ etc. The role of other adduct-forming salts has also been evaluated. The lithium salt method is useful for accurate mass determination of these highly sensitive compounds.

  16. Glycosyl azide-a novel substrate for enzymatic transgycosylations

    Czech Academy of Sciences Publication Activity Database

    Fialová, Pavla; Carmona, A. T.; Robina, I.; Ettrich, R.; Sedmera, Petr; Přikrylová, Věra; Hušáková, Lucie; Křen, Vladimír

    2005-01-01

    Roč. 46, - (2005), s. 8715-8718 ISSN 0040-4039 R&D Projects: GA ČR GA203/05/0172; GA MŠk OC D25.002 Grant - others:GA KONTAKT 1862/04 Institutional research plan: CEZ:AV0Z50200510 Keywords : enzyme catalysis * glycosyl azide * molecular modelling Subject RIV: EE - Microbiology, Virology Impact factor: 2.477, year: 2005

  17. Reduced apolipoprotein glycosylation in patients with the metabolic syndrome.

    Directory of Open Access Journals (Sweden)

    Olga V Savinova

    Full Text Available The purpose of this study was to compare the apolipoprotein composition of the three major lipoprotein classes in patients with metabolic syndrome to healthy controls.Very low density (VLDL, intermediate/low density (IDL/LDL, hereafter LDL, and high density lipoproteins (HDL fractions were isolated from plasma of 56 metabolic syndrome subjects and from 14 age-sex matched healthy volunteers. The apolipoprotein content of fractions was analyzed by one-dimensional (1D gel electrophoresis with confirmation by a combination of mass spectrometry and biochemical assays.Metabolic syndrome patients differed from healthy controls in the following ways: (1 total plasma--apoA1 was lower, whereas apoB, apoC2, apoC3, and apoE were higher; (2 VLDL--apoB, apoC3, and apoE were increased; (3 LDL--apoC3 was increased, (4 HDL--associated constitutive serum amyloid A protein (SAA4 was reduced (p<0.05 vs. controls for all. In patients with metabolic syndrome, the most extensively glycosylated (di-sialylated isoform of apoC3 was reduced in VLDL, LDL, and HDL fractions by 17%, 30%, and 25%, respectively (p<0.01 vs. controls for all. Similarly, the glycosylated isoform of apoE was reduced in VLDL, LDL, and HDL fractions by 15%, 26%, and 37% (p<0.01 vs. controls for all. Finally, glycosylated isoform of SAA4 in HDL fraction was 42% lower in patients with metabolic syndrome compared with controls (p<0.001.Patients with metabolic syndrome displayed several changes in plasma apolipoprotein composition consistent with hypertriglyceridemia and low HDL cholesterol levels. Reduced glycosylation of apoC3, apoE and SAA4 are novel findings, the pathophysiological consequences of which remain to be determined.

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

    Indian Academy of Sciences (India)

    Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). 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 ...

  19. Detection of site specific glycosylation in proteins using flow cytometry†

    Science.gov (United States)

    Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

    2009-01-01

    We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

  20. Glycosylation-Based Serum Biomarkers for Cancer Diagnostics and Prognostics.

    Science.gov (United States)

    Kirwan, Alan; Utratna, Marta; O'Dwyer, Michael E; Joshi, Lokesh; Kilcoyne, Michelle

    2015-01-01

    Cancer is the second most common cause of death in developed countries with approximately 14 million newly diagnosed individuals and over 6 million cancer-related deaths in 2012. Many cancers are discovered at a more advanced stage but better survival rates are correlated with earlier detection. Current clinically approved cancer biomarkers are most effective when applied to patients with widespread cancer. Single biomarkers with satisfactory sensitivity and specificity have not been identified for the most common cancers and some biomarkers are ineffective for the detection of early stage cancers. Thus, novel biomarkers with better diagnostic and prognostic performance are required. Aberrant protein glycosylation is well known hallmark of cancer and represents a promising source of potential biomarkers. Glycoproteins enter circulation from tissues or blood cells through active secretion or leakage and patient serum is an attractive option as a source for biomarkers from a clinical and diagnostic perspective. A plethora of technical approaches have been developed to address the challenges of glycosylation structure detection and determination. This review summarises currently utilised glycoprotein biomarkers and novel glycosylation-based biomarkers from the serum glycoproteome under investigation as cancer diagnostics and for monitoring and prognostics and includes details of recent high throughput and other emerging glycoanalytical techniques.

  1. Optimization of a colorimetric assay for glycosylated human serum albumin

    International Nuclear Information System (INIS)

    Bohney, J.P.; Feldhoff, R.C.

    1986-01-01

    The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100 0 C. A NaBH 4 reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with [ 3 H]glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation

  2. Glucosamine derived DISAL donors for stereoselective glycosylations under neutral conditions

    DEFF Research Database (Denmark)

    Grathe, S.; Thygesen, M.B.; Larsen, K.

    2005-01-01

    DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report the synthe......DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report...... the synthesis of new glucosamine DISAL donors, carrying N-TCP, -Troc, or -TFAc protecting groups, and their use in beta-(1,2-trans) selective glycosylations, primarily in NMP in the absence of any added Lewis acids, or in CH3NO2 with LiClO4. Finally, precise microwave heating proved effective in promoting...

  3. Semi-automated scoring of triple-probe FISH in human sperm using confocal microscopy.

    Science.gov (United States)

    Branch, Francesca; Nguyen, GiaLinh; Porter, Nicholas; Young, Heather A; Martenies, Sheena E; McCray, Nathan; Deloid, Glen; Popratiloff, Anastas; Perry, Melissa J

    2017-09-01

    Structural and numerical sperm chromosomal aberrations result from abnormal meiosis and are directly linked to infertility. Any live births that arise from aneuploid conceptuses can result in syndromes such as Kleinfelter, Turners, XYY and Edwards. Multi-probe fluorescence in situ hybridization (FISH) is commonly used to study sperm aneuploidy, however manual FISH scoring in sperm samples is labor-intensive and introduces errors. Automated scoring methods are continuously evolving. One challenging aspect for optimizing automated sperm FISH scoring has been the overlap in excitation and emission of the fluorescent probes used to enumerate the chromosomes of interest. Our objective was to demonstrate the feasibility of combining confocal microscopy and spectral imaging with high-throughput methods for accurately measuring sperm aneuploidy. Our approach used confocal microscopy to analyze numerical chromosomal abnormalities in human sperm using enhanced slide preparation and rigorous semi-automated scoring methods. FISH for chromosomes X, Y, and 18 was conducted to determine sex chromosome disomy in sperm nuclei. Application of online spectral linear unmixing was used for effective separation of four fluorochromes while decreasing data acquisition time. Semi-automated image processing, segmentation, classification, and scoring were performed on 10 slides using custom image processing and analysis software and results were compared with manual methods. No significant differences in disomy frequencies were seen between the semi automated and manual methods. Samples treated with pepsin were observed to have reduced background autofluorescence and more uniform distribution of cells. These results demonstrate that semi-automated methods using spectral imaging on a confocal platform are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods. © 2017 International Society for Advancement of Cytometry. © 2017

  4. Confocal Laser Endomicroscopy in Neurosurgery: A New Technique with Much Potential

    Directory of Open Access Journals (Sweden)

    David Breuskin

    2013-01-01

    Full Text Available Technical innovations in brain tumour diagnostic and therapy have led to significant improvements of patient outcome and recurrence free interval. The use of technical devices such as surgical microscopes as well as neuronavigational systems have helped localising tumours as much as fluorescent agents, such as 5-aminolaevulinic acid, have helped visualizing pathologically altered tissue. Nonetheless, intraoperative instantaneous frozen sections and histological diagnosis remain the only method of gaining certainty of the nature of the resected tissue. This technique is time consuming and does not provide close-to-real-time information. In gastroenterology, confocal endoscopy closed the gap between tissue resection and histological examination, providing an almost real-time histological diagnosis. The potential of this technique using a confocal laser endoscope EndoMAG1 by Karl Storz Company was evaluated by our group on pig brains, tumour tissue cell cultures, and fresh human tumour specimen. Here, the authors report for the first time on the results of applying this new technique and provide first confocal endoscopic images of various brain and tumour structures. In all, the technique harbours a very promising potential to provide almost real-time intraoperative diagnosis, but further studies are needed to provide evidence for the technique’s potential.

  5. Cement paste surface roughness analysis using coherence scanning interferometry and confocal microscopy

    International Nuclear Information System (INIS)

    Apedo, K.L.; Munzer, C.; He, H.; Montgomery, P.; Serres, N.; Fond, C.; Feugeas, F.

    2015-01-01

    Scanning electron microscopy and scanning probe microscopy have been used for several decades to better understand the microstructure of cementitious materials. Very limited work has been performed to date to study the roughness of cementitious materials by optical microscopy such as coherence scanning interferometry (CSI) and chromatic confocal sensing (CCS). The objective of this paper is to better understand how CSI can be used as a tool to analyze surface roughness and topography of cement pastes. Observations from a series of images acquired using this technique on both polished and unpolished samples are described. The results from CSI are compared with those from a STIL confocal microscopy technique (SCM). Comparison between both optical techniques demonstrates the ability of CSI to measure both polished and unpolished cement pastes. - Highlights: • Coherence scanning interferometry (CSI) was used to analyze cement paste surfaces. • The results from the CSI were compared with those from a confocal microscopy. • 3D roughness parameters were obtained using the window resizing method. • Polished and unpolished cement pastes were studied

  6. Development of confocal 3D micro-XRF spectrometer with dual Cr-Mo excitation

    International Nuclear Information System (INIS)

    Kouichi Tsuji; Kazuhiko Nakano

    2007-01-01

    A new 3D micro-XRF instrument based on a confocal setup using two independent poly-capillary x-ray lenses and two x-ray sources (Cr and Mo targets) was developed. A full poly-capillary x-ray lens was attached to each x-ray tube. Another half poly-capillary lens was attached to a silicon drift x-ray detector (SDD). The focal spots of the three lenses were adjusted to a common position. The depth resolutions that were evaluated by use of a 10-μm thick Au foil were approximately 90 μm for the x-ray energy of Au Lα. The effects of the dual Cr-Mo x-ray beam excitation were investigated. It was confirmed that the XRF intensity of light elements was increased by applying the Cr-target x-ray tube in a confocal configuration. In the proposed confocal configuration, 3D elemental mapping of the major elements of an amaranth seed was performed nondestructively at ambient air pressure. Each element of the seed showed different mapping images in the different depth layers. (authors)

  7. Cement paste surface roughness analysis using coherence scanning interferometry and confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Apedo, K.L., E-mail: apedo@unistra.fr [ICube, Université de Strasbourg, CNRS, 2 rue Boussingault, 67000 Strasbourg (France); Munzer, C.; He, H. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France); Montgomery, P. [ICube, Université de Strasbourg, CNRS, 23 rue du Loess, 67037 Strasbourg (France); Serres, N. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France); Fond, C. [ICube, Université de Strasbourg, CNRS, 2 rue Boussingault, 67000 Strasbourg (France); Feugeas, F. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France)

    2015-02-15

    Scanning electron microscopy and scanning probe microscopy have been used for several decades to better understand the microstructure of cementitious materials. Very limited work has been performed to date to study the roughness of cementitious materials by optical microscopy such as coherence scanning interferometry (CSI) and chromatic confocal sensing (CCS). The objective of this paper is to better understand how CSI can be used as a tool to analyze surface roughness and topography of cement pastes. Observations from a series of images acquired using this technique on both polished and unpolished samples are described. The results from CSI are compared with those from a STIL confocal microscopy technique (SCM). Comparison between both optical techniques demonstrates the ability of CSI to measure both polished and unpolished cement pastes. - Highlights: • Coherence scanning interferometry (CSI) was used to analyze cement paste surfaces. • The results from the CSI were compared with those from a confocal microscopy. • 3D roughness parameters were obtained using the window resizing method. • Polished and unpolished cement pastes were studied.

  8. Laser scanning confocal microscope with programmable amplitude, phase, and polarization of the illumination beam.

    Science.gov (United States)

    Boruah, B R; Neil, M A A

    2009-01-01

    We describe the design and construction of a laser scanning confocal microscope with programmable beam forming optics. The amplitude, phase, and polarization of the laser beam used in the microscope can be controlled in real time with the help of a liquid crystal spatial light modulator, acting as a computer generated hologram, in conjunction with a polarizing beam splitter and two right angled prisms assembly. Two scan mirrors, comprising an on-axis fast moving scan mirror for line scanning and an off-axis slow moving scan mirror for frame scanning, configured in a way to minimize the movement of the scanned beam over the pupil plane of the microscope objective, form the XY scan unit. The confocal system, that incorporates the programmable beam forming unit and the scan unit, has been implemented to image in both reflected and fluorescence light from the specimen. Efficiency of the system to programmably generate custom defined vector beams has been demonstrated by generating a bottle structured focal volume, which in fact is the overlap of two cross polarized beams, that can simultaneously improve both the lateral and axial resolutions if used as the de-excitation beam in a stimulated emission depletion confocal microscope.

  9. [Revealing the chemical changes of tea cell wall induced by anthracnose with confocal Raman microscopy].

    Science.gov (United States)

    Li, Xiao-li; Luo, Liu-bin; Hu, Xiao-qian; Lou, Bing-gan; He, Yong

    2014-06-01

    Healthy tea and tea infected by anthracnose were first studied by confocal Raman microscopy to illustrate chemical changes of cell wall in the present paper. Firstly, Raman spectra of both healthy and infected sample tissues were collected with spatial resolution at micron-level, and ultrastructure of healthy and infected tea cells was got from scanning electron microscope. These results showed that there were significant changes in Raman shift and Raman intensity between healthy and infected cell walls, indicating that great differences occurred in chemical compositions of cell walls between healthy and infected samples. In details, intensities at many Raman bands which were closely associated with cellulose, pectin, esters were reduced after infection, revealing that the content of chemical compounds such as cellulose, pectin, esters was decreased after infection. Subsequently, chemical imaging of both healthy and infected tea cell walls were realized based on Raman fingerprint spectra of cellulose and microscopic spatial structure. It was found that not only the content of cellulose was reduced greatly after infection, but also the ordered structure of cellulose was destroyed by anthracnose infection. Thus, confocal Raman microscopy was shown to be a powerful tool to detect the chemical changes in cell wall of tea caused by anthracnose without any chemical treatment or staining. This research firstly applied confocal Raman microscopy in phytopathology for the study of interactive relationship between host and pathogen, and it will also open a new way for intensive study of host-pathogen at cellular level.

  10. Development of confocal 3D micro-XRF spectrometer with dual Cr-Mo excitation

    Energy Technology Data Exchange (ETDEWEB)

    Kouichi Tsuji [Department of Applied Chemistry, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku Osaka 558-8585 (Japan); PRESTO-JST - Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan); Kazuhiko Nakano [Department of Applied Chemistry, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku Osaka 558-8585 (Japan)

    2007-05-15

    A new 3D micro-XRF instrument based on a confocal setup using two independent poly-capillary x-ray lenses and two x-ray sources (Cr and Mo targets) was developed. A full poly-capillary x-ray lens was attached to each x-ray tube. Another half poly-capillary lens was attached to a silicon drift x-ray detector (SDD). The focal spots of the three lenses were adjusted to a common position. The depth resolutions that were evaluated by use of a 10-{mu}m thick Au foil were approximately 90 {mu}m for the x-ray energy of Au L{alpha}. The effects of the dual Cr-Mo x-ray beam excitation were investigated. It was confirmed that the XRF intensity of light elements was increased by applying the Cr-target x-ray tube in a confocal configuration. In the proposed confocal configuration, 3D elemental mapping of the major elements of an amaranth seed was performed nondestructively at ambient air pressure. Each element of the seed showed different mapping images in the different depth layers. (authors)

  11. Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes

    Directory of Open Access Journals (Sweden)

    Anne Dell

    2010-01-01

    Full Text Available Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i oligosaccharyltransferase (OST-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring “en bloc” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

  12. A low error reconstruction method for confocal holography to determine 3-dimensional properties

    Energy Technology Data Exchange (ETDEWEB)

    Jacquemin, P.B., E-mail: pbjacque@nps.edu [Mechanical Engineering, University of Victoria, EOW 548,800 Finnerty Road, Victoria, BC (Canada); Herring, R.A. [Mechanical Engineering, University of Victoria, EOW 548,800 Finnerty Road, Victoria, BC (Canada)

    2012-06-15

    A confocal holography microscope developed at the University of Victoria uniquely combines holography with a scanning confocal microscope to non-intrusively measure fluid temperatures in three-dimensions (Herring, 1997), (Abe and Iwasaki, 1999), (Jacquemin et al., 2005). The Confocal Scanning Laser Holography (CSLH) microscope was built and tested to verify the concept of 3D temperature reconstruction from scanned holograms. The CSLH microscope used a focused laser to non-intrusively probe a heated fluid specimen. The focused beam probed the specimen instead of a collimated beam in order to obtain different phase-shift data for each scan position. A collimated beam produced the same information for scanning along the optical propagation z-axis. No rotational scanning mechanisms were used in the CSLH microscope which restricted the scan angle to the cone angle of the probe beam. Limited viewing angle scanning from a single view point window produced a challenge for tomographic 3D reconstruction. The reconstruction matrices were either singular or ill-conditioned making reconstruction with significant error or impossible. Establishing boundary conditions with a particular scanning geometry resulted in a method of reconstruction with low error referred to as 'wily'. The wily reconstruction method can be applied to microscopy situations requiring 3D imaging where there is a single viewpoint window, a probe beam with high numerical aperture, and specified boundary conditions for the specimen. The issues and progress of the wily algorithm for the CSLH microscope are reported herein. -- Highlights: Black-Right-Pointing-Pointer Evaluation of an optical confocal holography device to measure 3D temperature of a heated fluid. Black-Right-Pointing-Pointer Processing of multiple holograms containing the cumulative refractive index through the fluid. Black-Right-Pointing-Pointer Reconstruction issues due to restricting angular scanning to the numerical aperture of the

  13. A low error reconstruction method for confocal holography to determine 3-dimensional properties

    International Nuclear Information System (INIS)

    Jacquemin, P.B.; Herring, R.A.

    2012-01-01

    A confocal holography microscope developed at the University of Victoria uniquely combines holography with a scanning confocal microscope to non-intrusively measure fluid temperatures in three-dimensions (Herring, 1997), (Abe and Iwasaki, 1999), (Jacquemin et al., 2005). The Confocal Scanning Laser Holography (CSLH) microscope was built and tested to verify the concept of 3D temperature reconstruction from scanned holograms. The CSLH microscope used a focused laser to non-intrusively probe a heated fluid specimen. The focused beam probed the specimen instead of a collimated beam in order to obtain different phase-shift data for each scan position. A collimated beam produced the same information for scanning along the optical propagation z-axis. No rotational scanning mechanisms were used in the CSLH microscope which restricted the scan angle to the cone angle of the probe beam. Limited viewing angle scanning from a single view point window produced a challenge for tomographic 3D reconstruction. The reconstruction matrices were either singular or ill-conditioned making reconstruction with significant error or impossible. Establishing boundary conditions with a particular scanning geometry resulted in a method of reconstruction with low error referred to as “wily”. The wily reconstruction method can be applied to microscopy situations requiring 3D imaging where there is a single viewpoint window, a probe beam with high numerical aperture, and specified boundary conditions for the specimen. The issues and progress of the wily algorithm for the CSLH microscope are reported herein. -- Highlights: ► Evaluation of an optical confocal holography device to measure 3D temperature of a heated fluid. ► Processing of multiple holograms containing the cumulative refractive index through the fluid. ► Reconstruction issues due to restricting angular scanning to the numerical aperture of the beam. ► Minimizing tomographic reconstruction error by defining boundary

  14. Fasting serum glucose and glycosylated hemoglobin level in obesity.

    Science.gov (United States)

    Das, R K; Nessa, A; Hossain, M A; Siddiqui, N I; Hussain, M A

    2014-04-01

    Obesity is a condition in which the body fat stores are increased to an extent which impairs health and leads to serious health consequences. The amount of body fat is difficult to measure directly, and is usually determined from an indirect measure - the body mass index (BMI). Increased BMI in obese persons is directly associated with an increase in metabolic disease, such as type 2 diabetes mellitus. This Analytical cross sectional study was undertaken to assess the relation between obesity and glycemic control of body by measuring fasting serum glucose and glycosylated hemoglobin. This study was carried out in the Department of Physiology, Mymensingh Medical College, Mymensingh from 1st July 2011 to 30th June 2012 on 120 equally divided male and female persons within the age range of 25 to 55 years. Age more than 55 years and less than 25 years and diagnosed case of Hypothyroidism, Cushing's syndrome, polycystic ovary, Antipsychotic drug user and regular steroid users were excluded. Non probability purposive type of sampling technique was used for selecting the study subjects. Measurement of body mass index was done as per procedure. Fasting serum glucose was estimated by glucose oxidase method and Glycosylated hemoglobin by Boronate Affinity method. Statistical analysis was done by SPSS (version 17.0). Data were expressed as Mean±SE and statistical significance of difference among the groups were calculated by unpaired student's 't' test and Pearson's correlation coefficient tests were done as applicable. The Mean±SE of fasting serum glucose was significant at 1% level (P value obese group of BMI. There was no significant difference of glycosylated hemoglobin level between control and study groups. But there was positive correlation within each group. Fasting serum glucose also showed a bit stronger positive correlation with BMI. Both obese male and female persons showed higher levels of fasting serum glucose and glycosylated hemoglobin. The observed positive

  15. The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality

    DEFF Research Database (Denmark)

    Boskovski, Marko T; Yuan, Shiaulou; Pedersen, Nis Borbye

    2013-01-01

    to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with heterotaxy, and now demonstrate, in Xenopus tropicalis, that galnt11 activates Notch signalling. GALNT11 O-glycosylates human NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either...... by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus left-right organizer cilia and show that Galnt11-mediated Notch1 signalling modulates the spatial distribution and ratio...... of motile and immotile cilia at the left-right organizer. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. By contrast, Notch overexpression decreases this ratio, mimicking...

  16. Importance of N-Glycosylation on CD147 for Its Biological Functions

    Science.gov (United States)

    Bai, Yang; Huang, Wan; Ma, Li-Tian; Jiang, Jian-Li; Chen, Zhi-Nan

    2014-01-01

    Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation. PMID:24739808

  17. Importance of N-Glycosylation on CD147 for Its Biological Functions

    Directory of Open Access Journals (Sweden)

    Yang Bai

    2014-04-01

    Full Text Available Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation.

  18. Toward stable genetic engineering of human o-glycosylation in plants

    DEFF Research Database (Denmark)

    Yang, Zhang; Bennett, Eric Paul; Jørgensen, Bodil

    2012-01-01

    Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types...... an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating Gal......NAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O...

  19. The role of glycosylation in breast cancer metastasis and cancer control

    Directory of Open Access Journals (Sweden)

    Alexandra eKölbl

    2015-10-01

    Full Text Available AbstractGlycosylation and its correlation to the formation of remote metastasis in breast cancer had been an important scientific topic in the last 25 years. With the development of new analytical techniques new insights were gained on the mechanisms underlying metastasis formation and the role of aberrant glycosylation within. Mucin-1 and Galectin were recognized as key players in glycosylation. Interestingly, aberrant carbohydrate structures seem to support the development of brain metastasis in breast cancer patients, as changes in glycosylation structures facilitate an overcoming of blood-brain barrier. Changes in the gene expression of glycosyltransferases are the leading cause for a modification of carbohydrate chains, so that also altered gene expression plays a role for glycosylation. In consequence, glycosylation and changes within can be useful for cancer diagnosis, determination of tumour stage and prognosis, but can as well be targets for therapeutic strategies. Thus, further research on this topic would worth wile for cancer combating.

  20. Mitochondrial behavior during oogenesis in zebrafish: a confocal microscopy analysis.

    Science.gov (United States)

    Zhang, Yong-Zhong; Ouyang, Ying-Chun; Hou, Yi; Schatten, Heide; Chen, Da-Yuan; Sun, Qing-Yuan

    2008-03-01

    The behavior of mitochondria during early oogenesis remains largely unknown in zebrafish. We used three mitochondrial probes (Mito Tracker Red CMXRos, Mito Tracker Green FM, and JC-1) to stain early zebrafish oocyte mitochondria, and confocal microscopy to analyze mitochondrial aggregation and distribution. By using fluorescence recovery after photobleaching (FRAP), we traced mitochondrial movement. The microtubule assembly inhibitor nocodazole and microfilament inhibitor cytochalasin B (CB) were used to analyze the role of microtubules and microfilaments on mitochondrial movement. By using the dual emission probe, JC-1, and oxidative phosphorylation uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), we determined the distribution of active and inactive (low-active) mitochondria. Green/red fluorescence ratios of different sublocations in different oocyte groups stained by JC-1 were detected in merged (green and red) images. Our results showed that mitochondria exhibited a unique distribution pattern in early zebrafish oocytes. They tended to aggregate into large clusters in early stage I oocytes, but in a threadlike state in latter stage I oocytes. We detected a lower density mitochondrial area and a higher density mitochondrial area on opposite sides of the germinal vesicle. The green/red fluorescence ratios in different sublocations in normal oocytes were about 1:1. This implies that active mitochondria were distributed in all sublocations. FCCP treatment caused significant increases in the ratios. CB and nocodazole treatment caused an increase of the ratios in clusters and mitochondrial cloud, but not in dispersed areas. Mitochondria in different sublocations underwent fast dynamic movement. Inhibition or disruption of microtubules or microfilaments resulted in even faster mitochondrial free movement.

  1. Aqueous Colloid + Polymer Depletion System for Confocal Microscopy and Rheology

    Science.gov (United States)

    Park, Nayoung; Umanzor, Esmeralda J.; Conrad, Jacinta C.

    2018-05-01

    We developed a model depletion system with colloidal particles that were refractive index- and density-matched to 80 (w/w)% glycerol in water, and characterized the effect of interparticle interactions on the structure and dynamics of non-equilibrium phases. 2,2,2-trifluoroethyl methacrylate-co-tert-butyl methacrylate copolymer particles were synthesized following Kodger et al. (Sci. Rep. 5, 14635 (2015)). Particles were dispersed in glycerol/water solutions to generate colloidal suspensions with good control over electrostatic interactions and a moderately high background viscosity of 55 mPa-s. To probe the effects of charge screening and depletion attractions on the suspension phase behavior, we added NaCl and polyacrylamide (M_w = 186 kDa) at various concentrations to particle suspensions formulated at volume fractions of phi = 0.05 and 0.3 and imaged the suspensions using confocal microscopy. The particles were nearly hard spheres at a NaCl concentration of 20 mM, but aggregated when the concentration of NaCl was further increased. Changes in the particle structure and dynamics with increasing concentration of the depletant polyacrylamide followed the trends expected from earlier experiments on depletion-driven gelation. Additionally, we measured the viscosity and corrected first normal stress difference of suspensions formulated at phi = 0.4 with and without added polymer. The solvent viscosity was suitable for rheology measurements without the onset of instabilities such as secondary flows or edge fracture. These results validate this system as an alternative to one common model system, suspensions of poly(methyl methacrylate) particles and polystyrene depletants in organic solvents, for investigating phase behavior and flow properties in attractive colloidal suspensions.

  2. A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

    DEFF Research Database (Denmark)

    Larsen, Martin; Skottrup, Peter; Enghild, Jan Johannes

    Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... graphite powder micro-columns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and would be ideal for proteomics studies and verification of correct glycosylation of recombinant glycoproteins....

  3. Confocal and dermoscopic features of basal cell carcinoma in Gorlin-Goltz syndrome: A case report.

    Science.gov (United States)

    Casari, Alice; Argenziano, Giuseppe; Moscarella, Elvira; Lallas, Aimilios; Longo, Caterina

    2017-05-01

    Gorlin-Goltz (GS) syndrome is an autosomal dominant disease linked to a mutation in the PTCH gene. Major criteria include the onset of multiple basal cell carcinoma (BCC), keratocystic odontogenic tumours in the jaws and bifid ribs. Dermoscopy and reflectance confocal microscopy represent imaging tools that are able to increase the diagnostic accuracy of skin cancer in a totally noninvasive manner, without performing punch biopsies. Here we present a case of a young woman in whom the combined approach of dermoscopy and RCM led to the identification of multiple small inconspicuous lesions as BCC and thus to the diagnosis of GS syndrome. © 2016 The Australasian College of Dermatologists.

  4. Scanner component and head development for confocal microscopy using moving mirror technology

    Science.gov (United States)

    Loney, Gregory C.

    1993-12-01

    One of the challenges in designing a confocal microscope is choosing the scan system configuration. The selection is based largely on the microscope application and involves a few distinct schemes. One scheme, moving mirror using galvanometer and resonant scanners, has been shown to offer an excellent solution exhibited by the large number of commercial systems which utilize them. Perceived shortcomings, such as slow image acquisition, are being dispelled due to the advent of large angle, high frequency resonant scanners. These newer devices offer near video rate performance at good scan efficiency.

  5. Role of Confocal Laser Endomicroscopy in Detection of Residual Barrett's Esophagus after Radiofrequency Ablation

    Directory of Open Access Journals (Sweden)

    Giorgio Diamantis

    2011-01-01

    Full Text Available Endoscopic endoluminal radiofrequency ablation (RFA is a novel and promising modality for Barrett's esophagus (BE treatment. Actually the only surveillance method after the ablation treatment is random biopsies throughout the whole treated area. Confocal laser endomicroscopy (CLE is a new endoscopic imaging tool that permits high-resolution microscopic examination of the gastrointestinal tract. The technology has garnered increasing attention because of its ability to provide real-time “optical” biopsy specimens, with a very high sensitivity and specificity. This paper summarize the potential application of CLE in the surveillance of the reepithelialization of BE, after endoscopic RFA.

  6. Dark-field scanning confocal microscope for vertical particle tracks in nuclear emulsion

    International Nuclear Information System (INIS)

    Astakhov, A.Ya.; Batusov, Yu.A.; Soroko, L.M.; Tereshchenko, S.V.; Tereshchenko, V.V.

    1999-01-01

    The principle of the DArk-FIeld Scanning CONfocal (DAFISCON) microscope for selective observation of the vertical particle tracks in nuclear emulsion is described. The construction of the DAFISCON microscope, built on the basis of the 2D measurement microscope, is described. The results of the experimental testing of the DAFISCON microscope, accomplished at high density of the vertical particle tracks, are presented. The 2D plot and the 1D plot of the CCD dark-field image are given. The spatial resolution of our microscope can be increased by using the objective with higher aperture

  7. 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.

  8. Insights into esophagus tissue architecture using two-photon confocal microscopy

    Science.gov (United States)

    Liu, Nenrong; Wang, Yue; Feng, Shangyuan; Chen, Rong

    2013-08-01

    In this paper, microstructures of human esophageal mucosa were evaluated using the two-photon laser scanning confocal microscopy (TPLSCM), based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). The distribution of epithelial cells, muscle fibers of muscularis mucosae has been distinctly obtained. Furthermore, esophageal submucosa characteristics with cancer cells invading into were detected. The variation of collagen, elastin and cancer cells is very relevant to the pathology in esophagus, especially early esophageal cancer. Our experimental results indicate that the MPM technique has the much more advantages for label-free imaging, and has the potential application in vivo in the clinical diagnosis and monitoring of early esophageal cancer.

  9. Depth-variant blind restoration with pupil-phase constraints for 3D confocal microscopy

    International Nuclear Information System (INIS)

    Hadj, Saima Ben; Blanc-Féraud, Laure; Engler, Gilbert

    2013-01-01

    Three-dimensional images of confocal laser scanning microscopy suffer from a depth-variant blur, due to refractive index mismatch between the different mediums composing the system as well as the specimen, leading to optical aberrations. Our goal is to develop an image restoration method for 3D confocal microscopy taking into account the blur variation with depth. The difficulty is that optical aberrations depend on the refractive index of the biological specimen. The depth-variant blur function or the Point Spread Function (PSF) is thus different for each observation. A blind or semi-blind restoration method needs to be developed for this system. For that purpose, we use a previously developed algorithm for the joint estimation of the specimen function (original image) and the 3D PSF, the continuously depth-variant PSF is approximated by a convex combination of a set of space-invariant PSFs taken at different depths. We propose to add to that algorithm a pupil-phase constraint for the PSF estimation, given by the the optical instrument geometry. We thus define a blind estimation algorithm by minimizing a regularized criterion in which we integrate the Gerchberg-Saxton algorithm allowing to include these physical constraints. We show the efficiency of this method relying on some numerical tests

  10. Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

    DEFF Research Database (Denmark)

    Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

    2012-01-01

    Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing......, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo...

  11. Spectrally encoded confocal microscopy (SECM) for rapid assessment of breast excision specimens (Conference Presentation)

    Science.gov (United States)

    Brachtel, Elena F.; Johnson, Nicole B.; Huck, Amelia E.; Rice-Stitt, Travis L.; Vangel, Mark G.; Smith, Barbara L.; Tearney, Guillermo J.; Kang, DongKyun

    2016-03-01

    Unacceptably large percentage (20-40%) of breast cancer lumpectomy patients are required to undergo multiple surgeries when positive margins are found upon post-operative histologic assessment. If the margin status can be determined during surgery, surgeon can resect additional tissues to achieve tumor-free margin, which will reduce the need for additional surgeries. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to image the entire surgical margin within a short procedural time. Previously, SECM was shown to rapidly image a large area (10 mm by 10 mm) of human esophageal tissue within a short procedural time (15 seconds). When used in lumpectomy, SECM will be able to image the entire margin surface of ~30 cm2 in around 7.5 minutes. SECM images will then be used to determine margin status intra-operatively. In this paper, we present results from a study of testing accuracy of SECM for diagnosing malignant breast tissues. We have imaged freshly-excised breast specimens (N=46) with SECM. SECM images clearly visualized histomorphologic features associated with normal/benign and malignant breast tissues in a similar manner to histologic images. Diagnostic accuracy was tested by comparing SECM diagnoses made by three junior pathologists with corresponding histologic diagnoses made by a senior pathologist. SECM sensitivity and specificity were high, 0.91 and 0.93, respectively. Intra-observer agreement and inter-observer agreement were also high, 0.87 and 0.84, respectively. Results from this study showed that SECM has a potential to accurately determine margin status during breast cancer lumpectomy.

  12. Optimization of a colorimetric assay for glycosylated human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Bohney, J.P.; Feldhoff, R.C.

    1986-05-01

    The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.

  13. Characterization of kallikrein-related peptidase 4 glycosylations.

    Science.gov (United States)

    Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C; Simmer, James P

    2011-12-01

    Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. © 2011 Eur J Oral Sci.

  14. Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

    Directory of Open Access Journals (Sweden)

    Babst Benjamin A

    2009-12-01

    Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

  15. Noncontact ballistic motion measurement using a fiber-optic confocal sensor

    International Nuclear Information System (INIS)

    Shafir, E.; Berkovic, G.; Horovitz, Y.; Appelbaum, G.; Moshe, E.; Horovitz, E.; Skutelski, A.; Werdiger, M.; Perelmutter, L.; Sudai, M.

    2007-01-01

    A fiber-optic confocal sensor for noncontact ballistic measurements is described. Determination of motion at velocities of 1.7 km/s with an uncertainty as small as ±0.3% is demonstrated for both a projectile and a free-surface target. The fibers detect the passage of the object at their conjugate image points created by low F/ optics. This results in an output signal comprising a train of sharp pulses each precisely identifying when the ballistic object traverses an image point. Since the ballistic object does not contact the sensor at the time of imaging, the measurements do not perturb the motion, enabling multi-fragment measurement, as well as repetitive measurements of the same object point

  16. Optimising performance of a confocal fluorescence microscope with a differential pinhole

    International Nuclear Information System (INIS)

    Kakade, Rohan; Walker, John G; Phillips, Andrew J

    2016-01-01

    The signal-to-noise ratio (SNR)-resolution trade-off is of great importance to bio-imaging applications where the aim is to image the sample using as little light as possible without significantly sacrificing image quality. In this paper the inherent SNR-resolution tradeoff in Confocal Fluorescence Microscopy (CFM) systems is presented by means of an effective tradeoff curve. A CFM system that employs a differential pinhole detection scheme has recently been shown to offer increased resolution, but at the expense of SNR. An optimum profile for the differential pinhole is identified in this paper that offers improved performance over a conventional (circular pinhole) system. The performance enhancement is illustrated through computer simulation. (paper)

  17. In vivo assessment of the structure of skin microcirculation by reflectance confocal-laser-scanning microscopy

    Science.gov (United States)

    Sugata, Keiichi; Osanai, Osamu; Kawada, Hiromitsu

    2012-02-01

    One of the major roles of the skin microcirculation is to supply oxygen and nutrition to the surrounding tissue. Regardless of the close relationship between the microcirculation and the surrounding tissue, there are few non-invasive methods that can evaluate both the microcirculation and its surrounding tissue at the same site. We visualized microcapillary plexus structures in human skin using in vivo reflectance confocal-laser-scanning microscopy (CLSM), Vivascope 3000® (Lucid Inc., USA) and Image J software (National Institutes of Health, USA) for video image processing. CLSM is a non-invasive technique that can visualize the internal structure of the skin at the cellular level. In addition to internal morphological information such as the extracellular matrix, our method reveals capillary structures up to the depth of the subpapillary plexus at the same site without the need for additional optical systems. Video images at specific depths of the inner forearm skin were recorded. By creating frame-to-frame difference images from the video images using off-line video image processing, we obtained images that emphasize the brightness depending on changes of intensity coming from the movement of blood cells. Merging images from different depths of the skin elucidates the 3-dimensional fine line-structure of the microcirculation. Overall our results show the feasibility of a non-invasive, high-resolution imaging technique to characterize the skin microcirculation and the surrounding tissue.

  18. 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.

  19. Penetration and binding of monoclonal antibody in human osteosarcoma multicell spheroids. Comparison of confocal laser scanning microscopy and autoadiography

    International Nuclear Information System (INIS)

    Hjelstuen, M.H.; Rasch-Halvorsen, K.; Brekken, C.; Bruland, Oe.; Davies, C. de L.

    1996-01-01

    Penetration and binding of monoclonal antibody (MAb) in multicell osteosarcoma spheroids have been studied by autoradiography and confocal laser scanning microscopy (CLSM). Optical sectioning of the 3-dimensional spheroids was performed by CLSM. Owing to attenuation of fluorescence intensity, FITC-labelled MAb could not be detected at depths greater than 60 μm within the spheroids. The antibody uptake seen in autoradiographs and CLSM images 60 μm within the spheroids were essentially identical. MAb had reached all parts of the spheroids within 6 h. Quantitative measurements of the fluorescence intensity of FITC-labelled MAb seen in confocal images and measurements of MAb bound per cell using flow cytometry, showed that maximum uptake was reached after 6 h. The possibility to perform both quantatitive and qualitative measurements makes CLSM a promising method for studying antibody uptake in thick tissue samples. (orig.)

  20. 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 1confocal images, we developed a method to generate three-dimensional (3D) in silico gels 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

  1. Fabry-Perot confocal resonator optical associative memory

    Science.gov (United States)

    Burns, Thomas J.; Rogers, Steven K.; Vogel, George A.

    1993-03-01

    A unique optical associative memory architecture is presented that combines the optical processing environment of a Fabry-Perot confocal resonator with the dynamic storage and recall properties of volume holograms. The confocal resonator reduces the size and complexity of previous associative memory architectures by folding a large number of discrete optical components into an integrated, compact optical processing environment. Experimental results demonstrate the system is capable of recalling a complete object from memory when presented with partial information about the object. A Fourier optics model of the system's operation shows it implements a spatially continuous version of a discrete, binary Hopfield neural network associative memory.

  2. 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.

  3. Confocal microscopy as an early relapse marker for acanthamoeba keratitis.

    Science.gov (United States)

    Daas, Loay; Viestenz, Arne; Schnabel, Philipp Albert; Fries, Fabian N; Hager, Tobias; SzentmÁry, Nora; Seitz, Berthold

    2018-01-01

    Acanthameoba keratitis is a serious ophthalmological condition with a potentially vision-threatening prognosis. Early diagnosis and recognition of relapse, and the detection of persistent Acanthamoeba cysts, are essential for informing the prognosis and managing the condition. We suggest the use of in vivo confocal microscopy not only to identify the early signs of relapse after keratoplasty in patients with Acanthamoeba keratitis, but also as an additional follow-up tool after antimicrobial crosslinking. This study shows that in vivo confocal microscopy is, in experienced hands, a quick and reliable diagnostic tool. Clin. Anat. 31:60-63, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. Microscopia confocal in vivo na cistinose: relato de caso

    Directory of Open Access Journals (Sweden)

    Victor Gustavo

    2004-01-01

    Full Text Available A cistinose é doença autossômica recessiva rara caracterizada pelo acúmulo do aminoácido cistina livre dentro dos lisossomos e geralmente é fatal na primeira década de vida na ausência de transplante renal. O presente estudo tem por objetivo relatar os achados da microscopia confocal in vivo em paciente adulto com cistinose infantil. O exame de microscopia confocal in vivo revelou que há diferenças quanto à intensidade de acometimento, tamanho e forma dos depósitos nas diversas camadas corneanas.

  5. Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids.

    Science.gov (United States)

    Dutta, Devawati; Mandal, Chhabinath; Mandal, Chitra

    2017-12-01

    Glycosylation of proteins is the most common, multifaceted co- and post-translational modification responsible for many biological processes and cellular functions. Significant alterations and aberrations of these processes are related to various pathological conditions, and often turn out to be disease biomarkers. Conventional N-glycosylation occurs through the recognition of the consensus sequon, asparagine (Asn)-X-serine (Ser)/threonine (Thr), where X is any amino acid except for proline, with N-acetylglucosamine (GlcNAc) as the first glycosidic linkage. Usually, O-glycosylation adds a glycan to the hydroxyl group of Ser or Thr beginning with N-acetylgalactosamine (GalNAc). Protein glycosylation is further governed by additional diversifications in sequon and structure, which are yet to be fully explored. This review mainly focuses on the occurrence of N-glycosylation in non-consensus motifs, where Ser/Thr at the +2 position is substituted by other amino acids. Additionally, N-glycosylation is also observed in other amide/amine group-containing amino acids. Similarly, O-glycosylation occurs at hydroxyl group-containing amino acids other than serine/threonine. The neighbouring amino acids and local structural features around the potential glycosylation site also play a significant role in determining the extent of glycosylation. All of these phenomena that yield glycosylation at the atypical sites are reported in a variety of biological systems, including different pathological conditions. Therefore, the discovery of more novel sequence patterns for N- and O-glycosylation may help in understanding the functions of complex biological processes and cellular functions. Taken together, all these information provided in this review would be helpful for the biological readers. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model

    OpenAIRE

    Klemm, Matthias; Blum, Johannes; Link, Dietmar; Hammer, Martin; Haueisen, Jens; Schweitzer, Dietrich

    2016-01-01

    Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes d...

  7. Crystallization and preliminary crystallographic analysis of human glycosylated haemoglobin

    International Nuclear Information System (INIS)

    Syakhovich, Vitaly E.; Saraswathi, N. T.; Ruff, Marc; Bokut, Sergey B.; Moras, Dino

    2006-01-01

    Non enzymatic modification of haemoglobin by glucose plays an important role in diabetes pathogenesis. Here the purification, characterization and crystallization of human glycosylated haemoglobin are reported. Human glycosylated haemoglobin A 1C is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA 1C were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 Å at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 Å, α = 90.00, β = 125.40, γ = 90.00°. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (V M ) of 9.70 Å 3 Da −1 and a solvent content of 49%

  8. Crystallization and preliminary crystallographic analysis of human glycosylated haemoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Syakhovich, Vitaly E. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Saraswathi, N. T.; Ruff, Marc, E-mail: ruff@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Bokut, Sergey B. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Moras, Dino [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus)

    2006-02-01

    Non enzymatic modification of haemoglobin by glucose plays an important role in diabetes pathogenesis. Here the purification, characterization and crystallization of human glycosylated haemoglobin are reported. Human glycosylated haemoglobin A{sub 1C} is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA{sub 1C} were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 Å at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 Å, α = 90.00, β = 125.40, γ = 90.00°. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (V{sub M}) of 9.70 Å{sup 3} Da{sup −1} and a solvent content of 49%.

  9. Glycosylated yellow laccases of the basidiomycete Stropharia aeruginosa.

    Science.gov (United States)

    Daroch, Maurycy; Houghton, Catharine A; Moore, Jonathan K; Wilkinson, Mark C; Carnell, Andrew J; Bates, Andrew D; Iwanejko, Lesley A

    2014-05-10

    Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Demeclocycline as a contrast agent for detecting brain neoplasms using confocal microscopy

    Science.gov (United States)

    Wirth, Dennis; Smith, Thomas W.; Moser, Richard; Yaroslavsky, Anna N.

    2015-04-01

    Complete resection of brain tumors improves life expectancy and quality. Thus, there is a strong need for high-resolution detection and microscopically controlled removal of brain neoplasms. The goal of this study was to test demeclocycline as a contrast enhancer for the intraoperative detection of brain tumors. We have imaged benign and cancerous brain tumors using multimodal confocal microscopy. The tumors investigated included pituitary adenoma, meningiomas, glioblastomas, and metastatic brain cancers. Freshly excised brain tissues were stained in 0.75 mg ml-1 aqueous solution of demeclocyline. Reflectance images were acquired at 402 nm. Fluorescence signals were excited at 402 nm and registered between 500 and 540 nm. After imaging, histological sections were processed from the imaged specimens and compared to the optical images. Fluorescence images highlighted normal and cancerous brain cells, while reflectance images emphasized the morphology of connective tissue. The optical and histological images were in accordance with each other for all types of tumors investigated. Demeclocyline shows promise as a contrast agent for intraoperative detection of brain tumors.

  11. Demeclocycline as a contrast agent for detecting brain neoplasms using confocal microscopy

    International Nuclear Information System (INIS)

    Wirth, Dennis; Yaroslavsky, Anna N; Smith, Thomas W; Moser, Richard

    2015-01-01

    Complete resection of brain tumors improves life expectancy and quality. Thus, there is a strong need for high-resolution detection and microscopically controlled removal of brain neoplasms. The goal of this study was to test demeclocycline as a contrast enhancer for the intraoperative detection of brain tumors. We have imaged benign and cancerous brain tumors using multimodal confocal microscopy. The tumors investigated included pituitary adenoma, meningiomas, glioblastomas, and metastatic brain cancers. Freshly excised brain tissues were stained in 0.75 mg ml −1 aqueous solution of demeclocyline. Reflectance images were acquired at 402 nm. Fluorescence signals were excited at 402 nm and registered between 500 and 540 nm. After imaging, histological sections were processed from the imaged specimens and compared to the optical images. Fluorescence images highlighted normal and cancerous brain cells, while reflectance images emphasized the morphology of connective tissue. The optical and histological images were in accordance with each other for all types of tumors investigated. Demeclocyline shows promise as a contrast agent for intraoperative detection of brain tumors. (paper)

  12. Femtosecond laser subsurface scleral treatment in cadaver human sclera and evaluation using two-photon and confocal microscopy

    Science.gov (United States)

    Sun, Hui; Fan, Zhongwei; Yan, Ying; Lian, Fuqiang; Kurtz, Ron; Juhasz, Tibor

    2016-03-01

    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial-thickness drainage channels can be created with femtosecond laser in the translucent sclera for the potential treatment of glaucoma. We demonstrate the creation of partial-thickness subsurface drainage channels with the femtosecond laser in the cadaver human eyeballs and describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. A femtosecond laser operating at a wavelength of 1700 nm was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of cadaver human eyes. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such partial thickness subsurface scleral channels. Our studies suggest that the confocal and two-photon microscopy can be used to investigate femtosecond-laser created partial-thickness drainage channels in the sclera of cadaver human eyes.

  13. Real-Time Live Confocal Fluorescence Microscopy as a New Tool for Assessing Platelet Vitality.

    Science.gov (United States)

    Hermann, Martin; Nussbaumer, Oliver; Knöfler, Ralf; Hengster, Paul; Nussbaumer, Walter; Streif, Werner

    2010-01-01

    BACKGROUND: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. METHODS: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. RESULTS: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2-positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. CONCLUSION: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders.

  14. 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.

  15. Quantification of Cardiomyocyte Alignment from Three-Dimensional (3D) Confocal Microscopy of Engineered Tissue.

    Science.gov (United States)

    Kowalski, William J; Yuan, Fangping; Nakane, Takeichiro; Masumoto, Hidetoshi; Dwenger, Marc; Ye, Fei; Tinney, Joseph P; Keller, Bradley B

    2017-08-01

    Biological tissues have complex, three-dimensional (3D) organizations of cells and matrix factors that provide the architecture necessary to meet morphogenic and functional demands. Disordered cell alignment is associated with congenital heart disease, cardiomyopathy, and neurodegenerative diseases and repairing or replacing these tissues using engineered constructs may improve regenerative capacity. However, optimizing cell alignment within engineered tissues requires quantitative 3D data on cell orientations and both efficient and validated processing algorithms. We developed an automated method to measure local 3D orientations based on structure tensor analysis and incorporated an adaptive subregion size to account for multiple scales. Our method calculates the statistical concentration parameter, κ, to quantify alignment, as well as the traditional orientational order parameter. We validated our method using synthetic images and accurately measured principal axis and concentration. We then applied our method to confocal stacks of cleared, whole-mount engineered cardiac tissues generated from human-induced pluripotent stem cells or embryonic chick cardiac cells and quantified cardiomyocyte alignment. We found significant differences in alignment based on cellular composition and tissue geometry. These results from our synthetic images and confocal data demonstrate the efficiency and accuracy of our method to measure alignment in 3D tissues.

  16. Intraoperative diagnosis of nonpigmented nail tumours with ex vivo fluorescence confocal microscopy: 10 cases.

    Science.gov (United States)

    Debarbieux, S; Gaspar, R; Depaepe, L; Dalle, S; Balme, B; Thomas, L

    2015-04-01

    Ex vivo fluorescence confocal microscopy (FCM) permits real-time imaging of freshly excised skin tissues. Its usefulness as a time-sparing alternative to frozen sections in Mohs surgery of basal cell carcinoma is well documented. The purpose of this study was to describe the ex vivo FCM features of a series of benign and malignant nonpigmented tumours of the nail unit, and to correlate them with conventional histopathology. Nail apparatus tumours from 10 patients were imaged during surgical exploration using ex vivo FCM after immersion in acridine orange. Confocal mosaics of the freshly performed biopsies were evaluated in real time and retrospectively compared with haematoxylin and eosin sections. Our series included two invasive epithelial tumours (Group 1), four in situ or minimally invasive squamous cell carcinomas (SCC) (Group 2), three benign epithelial tumours (Group 3) and one nodular melanoma (Group 4). The correlation was excellent for malignant epithelial tumours exhibiting marked cytological and architectural atypias (Bowen disease, invasive SCC and onycholemmal carcinoma). Onychomatricomas exhibited a very peculiar aspect with densely cellular papillae. The correlation was less favourable for minimally invasive well-differentiated SCCs with slight cytological atypias. The correlation was poor for our case of amelanotic invasive subungual melanoma. Ex vivo FCM could be a useful tool to shorten management of nonpigmented nail tumours: in the case of a malignant tumour, it could indeed lead to performing wide excision during the same surgical procedure and possibly assessing the surgical margins. © 2014 British Association of Dermatologists.

  17. Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy.

    Science.gov (United States)

    Bondt, Albert; Wuhrer, Manfred; Kuijper, T Martijn; Hazes, Johanna M W; Dolhain, Radboud J E M

    2016-11-25

    Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation. The overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab. In contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.

  18. Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

    NARCIS (Netherlands)

    A. Bondt (Albert); M. Wuhrer (Manfred); T.M. Kuijper (Martijn); J.M.W. Hazes (Mieke); R.J.E.M. Dolhain (Radboud)

    2016-01-01

    textabstractBackground: Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. Methods:

  19. Model-based analysis of N-glycosylation in Chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Krambeck, Frederick J.; Bennun, Sandra V; Andersen, Mikael Rørdam

    2017-01-01

    The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower...

  20. O-GLYCOBASE version 4.0: a revised database of O-glycosylated proteins

    DEFF Research Database (Denmark)

    Gupta, Ramneek; Birch, Hanne; Rapacki, Krzysztof

    1999-01-01

    O-GLYCBASE is a database of glycoproteins with O-linked glycosylation sites. Entries with at least one experimentally verified O-glycosylation site have been complied from protein sequence databases and literature. Each entry contains information about the glycan involved, the species, sequence, ...

  1. A novel cerebello-ocular syndrome with abnormal glycosylation due to abnormalities in dolichol metabolism.

    NARCIS (Netherlands)

    Morava, E.; Wevers, R.A.; Cantagrel, V.; Hoefsloot, L.H.; Al-Gazali, L.; Schoots, J.; Rooij, A. van; Huijben, K.; Ravenswaaij-Arts, C.M.A. van; Jongmans, M.C.J.; Sykut-Cegielska, J.; Hoffmann, G.F.; Bluemel, P.; Adamowicz, M.; Reeuwijk, J. van; Ng, B.G.; Bergman, J.E.; Bokhoven, J.H.L.M. van; Korner, C.; Babovic-Vuksanovic, D.; Willemsen, M.A.A.P.; Gleeson, J.G.; Lehle, L.; Brouwer, A.P.M. de; Lefeber, D.J.

    2010-01-01

    Cerebellar hypoplasia and slowly progressive ophthalmological symptoms are common features in patients with congenital disorders of glycosylation type I. In a group of patients with congenital disorders of glycosylation type I with unknown aetiology, we have previously described a distinct phenotype

  2. Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

    DEFF Research Database (Denmark)

    Pedersen, Nis Borbye; Wang, Shengjun; Narimatsu, Yoshiki

    2014-01-01

    , which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide Gal...

  3. O-GLYCBASE version 3.0: a revised database of O-glycosylated proteins

    DEFF Research Database (Denmark)

    Hansen, Jan; Lund, Ole; Nilsson, Jette

    1998-01-01

    O-GLYCBASE is a revised database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the sequence databases. Entries include informations about species, sequence, glycosylation sites and glycan type and is fully cr...

  4. Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

    DEFF Research Database (Denmark)

    Julenius, Karin; Mølgaard, Anne; Gupta, Ramneek

    2005-01-01

    could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc....

  5. Oxytocin analogues with O-glycosylated serine and threonine in position 4

    Czech Academy of Sciences Publication Activity Database

    Marcinkowska, A.; Borovičková, Lenka; Slaninová, Jiřina; Grzonka, Z.

    2007-01-01

    Roč. 81, č. 7 (2007), s. 1335-1344 ISSN 0137- 5083 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z90210515 Keywords : oxytocin * glycosylated serin * glycosylated threonin * position 4 Subject RIV: CE - Biochemistry Impact factor: 0.483, year: 2007

  6. Evaluation and purchase of confocal microscopes: numerous factors to consider.

    Science.gov (United States)

    Zucker, Robert M; Chua, Michael

    2010-10-01

    The purchase of a confocal microscope is a difficult decision. Many factors need to be considered, which include hardware, software, company, support, service, and price. These issues are discussed to help guide the purchasing process. © 2010 by John Wiley & Sons, Inc.

  7. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY AND FOUNDATIONS FOR QUANTITATION

    Science.gov (United States)

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The reliability of the CLSM to obtain specific measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. For man...

  8. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR CALIBRATION, QUANTITATION AND SPECTROSCOPY

    Science.gov (United States)

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

  9. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY

    Science.gov (United States)

    The confocal laser-scanning microscopy (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of the emitted signal. The accuracy of these measurements demands t...

  10. TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation

    NARCIS (Netherlands)

    Jansen, Jos C.; Timal, Sharita; van Scherpenzeel, Monique; Michelakakis, Helen; Vicogne, Dorothée; Ashikov, Angel; Moraitou, Marina; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; van den Boogert, Marjolein A. W.; Porta, Francesco; Calvo, Pier Luigi; Mavrikou, Mersyni; Cenacchi, Giovanna; van den Bogaart, Geert; Salomon, Jody; Holleboom, Adriaan G.; Rodenburg, Richard J.; Drenth, Joost P. H.; Huynen, Martijn A.; Wevers, Ron A.; Morava, Eva; Foulquier, François; Veltman, Joris A.; Lefeber, Dirk J.

    2016-01-01

    Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously

  11. Glycosylation of the N-terminal potential N-glycosylation sites in the human α1,3-fucosyltransferase V and -VI (hFucTV and -VI)

    DEFF Research Database (Denmark)

    Christensen, Lise Lotte; Bross, Peter Gerd; Ørntoft, Torben Falck

    2000-01-01

    Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and h......FucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins...... in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced...

  12. '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.

  13. The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

    Science.gov (United States)

    URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

    2015-01-01

    The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

  14. [Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins].

    Science.gov (United States)

    Avanov, A Ia; Lipkind, G M

    1990-03-01

    Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.

  15. N-Glycosylation optimization of recombinant antibodies in CHO cell through process and metabolic engineering

    DEFF Research Database (Denmark)

    Fan, Yuzhou

    , analysis, control and optimization of N-glycosylation were thoroughly reviewed. In particular, how to control and optimize N-glycosylation in CHO cells was exclusively studied. The main focus of this PhD project is to find effective approaches of modulating N-glycosylation of CHO-derived recombinant...... galactose as feed additives, changing process parameters such as seeding density and cultivation duration are all demonstrated to be effective. The causal explanation of their impact on glycosylation can be various, including product, metabolism, proteome and physiology-associated mechanism. In the middle...... part of the thesis, both literature reviews and experimental applications were provided to demonstrate how to use omics data and implement systems biology to understand biological activities, especially N-glycosylation in CHO cells. In the last part of the thesis, the second strategy that apply genetic...

  16. Distribution of N-glycosylation sequons in proteins: how apart are they?

    DEFF Research Database (Denmark)

    Rao, Shyama Prasad; Buus, Ole Thomsen; Wollenweber, Bernd

    2011-01-01

    of experimentally confirmed eukaryotic N-glycoproteins we analyzed the relative position and distribution of sequons. N-Glycosylation probability was found to be lower in the termini of protein sequences compared to the mid region. N-glycosylated sequons were found much farther from C terminus compared to the N......N-glycosylation is a common protein modification process, which affects a number of properties of proteins. Little is known about the distribution of N-glycosylation sequons, for example, the distance between glycosylated sites and their position in the protein primary sequence. Using a large set......-terminus of the protein sequence and this effect was more pronounced for NXS sequons. The distribution of sequons, modeled based on balls-in-boxes classical occupancy, showed a near-maximum probability. Considerable proportion of sequons was found within a distance of ten amino acids, indicating that the steric hindrance...

  17. Demons registration for in vivo and deformable laser scanning confocal endomicroscopy

    Science.gov (United States)

    Chiew, Wei Ming; Lin, Feng; Seah, Hock Soon

    2017-09-01

    A critical effect found in noninvasive in vivo endomicroscopic imaging modalities is image distortions due to sporadic movement exhibited by living organisms. In three-dimensional confocal imaging, this effect results in a dataset that is tilted across deeper slices. Apart from that, the sequential flow of the imaging-processing pipeline restricts real-time adjustments due to the unavailability of information obtainable only from subsequent stages. To solve these problems, we propose an approach to render Demons-registered datasets as they are being captured, focusing on the coupling between registration and visualization. To improve the acquisition process, we also propose a real-time visual analytics tool, which complements the imaging pipeline and the Demons registration pipeline with useful visual indicators to provide real-time feedback for immediate adjustments. We highlight the problem of deformation within the visualization pipeline for object-ordered and image-ordered rendering. Visualizations of critical information including registration forces and partial renderings of the captured data are also presented in the analytics system. We demonstrate the advantages of the algorithmic design through experimental results with both synthetically deformed datasets and actual in vivo, time-lapse tissue datasets expressing natural deformations. Remarkably, this algorithm design is for embedded implementation in intelligent biomedical imaging instrumentation with customizable circuitry.

  18. The value of in vivo reflectance confocal microscopy in the diagnosis and monitoring of inflammatory and infectious skin diseases: a systematic review

    NARCIS (Netherlands)

    Hoogedoorn, L.; Peppelman, M.; Kerkhof, P.C.M. van de; Erp, P.E.J. van; Gerritsen, M.J.P.

    2015-01-01

    In vivo examination of the skin by reflectance confocal microscopy (RCM) has been performed for about 20 years, leading to a broad spectrum of imaged infectious and inflammatory skin diseases (ISD) with many described RCM features. We systematically reviewed all available literature concerning ISD

  19. Confocal laser scanning microscopy to estimate nanoparticles’ human skin penetration in vitro

    Directory of Open Access Journals (Sweden)

    Zou Y

    2017-10-01

    Full Text Available Ying Zou,1,2,* Anna Celli,2,3,* Hanjiang Zhu,2,* Akram Elmahdy,2 Yachao Cao,2 Xiaoying Hui,2 Howard Maibach2 1Skin & Cosmetic Research Department, Shanghai Skin Disease Hospital, Shanghai, People’s Republic of China; 2Department of Dermatology, School of Medicine, University of California San Francisco, San Francisco, CA, USA; 3San Francisco Veterans Medical Center, San Francisco, CA, USA *These authors contributed equally to this work Objective: With rapid development of nanotechnology, there is increasing interest in nanoparticle (NP application and its safety and efficacy on human skin. In this study, we utilized confocal laser scanning microscopy to estimate NP skin penetration.Methods: Three different-sized polystyrene NPs marked with red fluorescence were applied to human skin, and Calcium Green 5N was used as a counterstain. Dimethyl sulfoxide (DMSO and ethanol were used as alternative vehicles for NPs. Tape stripping was utilized as a barrier-damaged skin model. Skin biopsies dosed with NPs were incubated at 4°C or 37°C for 24 hours and imaged using confocal laser scanning microscopy.Results: NPs were localized in the stratum corneum (SC and hair follicles without penetrating the epidermis/dermis. Barrier alteration with tape stripping and change in incubation temperature did not induce deeper penetration. DMSO enhanced NP SC penetration but ethanol did not.Conclusion: Except with DMSO vehicle, these hydrolyzed polystyrene NPs did not penetrate intact or barrier-damaged human “viable” epidermis. For further clinical relevance, in vivo human skin studies and more sensitive analytic chemical methodology are suggested. Keywords: nanoparticles, skin penetration, stratum corneum, confocal laser scanning microscopy, tape stripping

  20. 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.