Laser scanning confocal microscope with programmable amplitude, phase, and polarization of the illumination beam.

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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.

Confocal microscope is able to detect calcium metabolic in neuronal infection by toxoplasma gondii

International Nuclear Information System (INIS)

Sensusiati, A D; Priya, T K S; Dachlan, Y P

2017-01-01

Calcium metabolism plays a very important role in neurons infected by Toxoplasma. Detection of change of calcium metabolism of neuron infected by Toxoplasma and Toxoplasma requires the calculation both quantitative and qualitative method. Confocal microscope has the ability to capture the wave of the fluorescent emission of the fluorescent dyes used in the measurement of cell calcium. The purpose of this study was to prove the difference in calcium changes between infected and uninfected neurons using confocal microscopy. Neuronal culture of human-skin-derived neural stem cell were divided into 6 groups, consisting 3 uninfected groups and 3 infected groups. Among the 3 groups were 2 hours, 24 hours and 48 hours. The neuron Toxoplasma gondii ratio was 1:5. Observation of intracellular calcium of neuron and tachyzoite, evidence of necrosis, apoptosis and the expression of Hsp 70 of neuron were examined by confocal microscope. The normality of the data was analysed by Kolmogorov-Smirnov Test, differentiation test was checked by t2 Test, and ANOVAs, for correlation test was done by Pearson Correlation Test. The calcium intensity of cytosolic neuron and T. gondii was significantly different from control groups (p<0.05). There was also significant correlation between calcium intensity with the evidence of necrosis and Hsp70 expression at 2 hours after infection. Apoptosis and necrosis were simultaneously shown with calcium contribution in this study. Confocal microscopy can be used to measure calcium changes in infected and uninfected neurons both in quantitatively and qualitatively. (paper)

Confocal microscope is able to detect calcium metabolic in neuronal infection by toxoplasma gondii

Science.gov (United States)

Sensusiati, A. D.; Priya, T. K. S.; Dachlan, Y. P.

2017-05-01

Calcium metabolism plays a very important role in neurons infected by Toxoplasma. Detection of change of calcium metabolism of neuron infected by Toxoplasma and Toxoplasma requires the calculation both quantitative and qualitative method. Confocal microscope has the ability to capture the wave of the fluorescent emission of the fluorescent dyes used in the measurement of cell calcium. The purpose of this study was to prove the difference in calcium changes between infected and uninfected neurons using confocal microscopy. Neuronal culture of human-skin-derived neural stem cell were divided into 6 groups, consisting 3 uninfected groups and 3 infected groups. Among the 3 groups were 2 hours, 24 hours and 48 hours. The neuron Toxoplasma gondii ratio was 1:5. Observation of intracellular calcium of neuron and tachyzoite, evidence of necrosis, apoptosis and the expression of Hsp 70 of neuron were examined by confocal microscope. The normality of the data was analysed by Kolmogorov-Smirnov Test, differentiation test was checked by t2 Test, and ANOVAs, for correlation test was done by Pearson Correlation Test. The calcium intensity of cytosolic neuron and T. gondii was significantly different from control groups (pneurons both in quantitatively and qualitatively.

Development of confocal laser microscope system for examination of microscopic characteristics of radiophotoluminescence glass dosemeters

International Nuclear Information System (INIS)

Maki, D.; Ishii, T.; Sato, F.; Kato, Y.; Yamamoto, T.; Iida, T.

2011-01-01

A confocal laser microscope system was developed for the measurement of radiophotoluminescence (RPL) photons emitted from a minute alpha-ray-irradiated area in an RPL glass dosemeter. The system was composed mainly of an inverted-type microscope, an ultraviolet laser, an XY movable stage and photon-counting circuits. The photon-counting circuits were effective in the reduction of the background noise level in the measurement of RPL photons. The performance of this microscope system was examined by the observation of standard RPL glass samples irradiated using 241 Am alpha rays. The spatial resolution of this system was ∼3 μm, and with regard to the sensitivity of this system, a hit of more than four to five alpha rays in unit area produced enough amount of RPL photons to construct the image. (authors)

Development of confocal laser microscope system for examination of microscopic characteristics of radiophotoluminescence glass dosemeters.

Science.gov (United States)

Maki, Daisuke; Ishii, Tetsuya; Sato, Fuminobu; Kato, Yushi; Yamamoto, Takayoshi; Iida, Toshiyuki

2011-03-01

A confocal laser microscope system was developed for the measurement of radiophotoluminescence (RPL) photons emitted from a minute alpha-ray-irradiated area in an RPL glass dosemeter. The system was composed mainly of an inverted-type microscope, an ultraviolet laser, an XY movable stage and photon-counting circuits. The photon-counting circuits were effective in the reduction of the background noise level in the measurement of RPL photons. The performance of this microscope system was examined by the observation of standard RPL glass samples irradiated using (241)Am alpha rays. The spatial resolution of this system was ∼ 3 μm, and with regard to the sensitivity of this system, a hit of more than four to five alpha rays in unit area produced enough amount of RPL photons to construct the image.

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

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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.

CCDiode: an optimal detector for laser confocal microscopes

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Pawley, James B.; Blouke, Morley M.; Janesick, James R.

1996-04-01

The laser confocal microscope (LCM) is now an established research tool in biology and materials science. In biological applications, it is usually employed to detect the location of fluorescent market molecules and, under these conditions, signal levels from bright areas are often digitizer. To maintain the desired +/- 3 e noise level at the relatively high data rate of 1 MHz, our new device utilizes 64 separate readout amplifier/digitizer systems, operating in sequence. The resulting detector is more compact, efficient and reliable than the PMT it replaces but as its sensitive area is smaller than that of a PMT, it will require auxiliary optics when used with any LCM having a large (mm) pinhole. As the signal light is parallel, a simple lens mounted axially and with the CCDiode at its focus would suffice. Future versions may use 3 X 3 or 5 X 5 arrays of sensors to `track' the confocal spot as it is deflected by inhomogeneities of the specimen, change its effective size or shape or detect system misalignment.

How the confocal laser scanning microscope entered biological research.

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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.

Efficacy of oral exfoliative cytology in diabetes mellitus patients: a light microscopic and confocal microscopic study.

Science.gov (United States)

Gopal, Deepika; Malathi, N; Reddy, B Thirupathi

2015-03-01

Diabetes mellitus (DM) has become a global problem. By monitoring the health status of these individuals, diabetic complications can be prevented. We aimed to analyze alterations in the morphology and cytomorphometry of buccal epithelial cells of type 2 DM patients using oral exfoliative cytology technique and determine its importance in public health screening, diagnosis and monitoring of diabetes mellitus. The study was carried out in 100 type 2 DM patients and 30 healthy individuals. Smears were taken from the right buccal mucosa and stained by the Papanicolaou technique. Staining with Acridine orange was carried out to view qualitative changes with confocal laser scanning microscope (LSM-510 Meta). The cytomorphometry was evaluated using IMAGE PRO PLUS 5.5 software with Evolution LC camera. All findings were statistically analyzed. The results showed that with increase in fasting plasma glucose levels, there is significant increase in nuclear area, decrease in cytoplasmic area, and increase in nuclear cytoplasmic ratio (p inclusion, candida and keratinization. In the present study, we found significant alterations in the cytomorphometry and cytomorphology of buccal epithelial cells of type 2 DM patients. This study supports and extends the view that these cellular changes can alert the clinician to the possibility of diabetes and aid in monitoring of diabetes throughout the lifetime of the patient.

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

Science.gov (United States)

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

2016-01-01

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

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.

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

Science.gov (United States)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

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

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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.

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.

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.

Nanoscale Energy-Filtered Scanning Confocal Electron Microscopy Using a Double-Aberration-Corrected Transmission Electron Microscope

International Nuclear Information System (INIS)

Wang Peng; Behan, Gavin; Kirkland, Angus I.; Nellist, Peter D.; Takeguchi, Masaki; Hashimoto, Ayako; Mitsuishi, Kazutaka; Shimojo, Masayuki

2010-01-01

We demonstrate that a transmission electron microscope fitted with two spherical-aberration correctors can be operated as an energy-filtered scanning confocal electron microscope. A method for establishing this mode is described and initial results showing 3D chemical mapping with nanoscale sensitivity to height and thickness changes in a carbon film are presented. Importantly, uncorrected chromatic aberration does not limit the depth resolution of this technique and moreover performs an energy-filtering role, which is explained in terms of a combined depth and energy-loss response function.

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.

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.

Automated Microscopy: Macro Language Controlling a Confocal Microscope and its External Illumination: Adaptation for Photosynthetic Organisms.

Science.gov (United States)

Steinbach, Gábor; Kaňa, Radek

2016-04-01

Photosynthesis research employs several biophysical methods, including the detection of fluorescence. Even though fluorescence is a key method to detect photosynthetic efficiency, it has not been applied/adapted to single-cell confocal microscopy measurements to examine photosynthetic microorganisms. Experiments with photosynthetic cells may require automation to perform a large number of measurements with different parameters, especially concerning light conditions. However, commercial microscopes support custom protocols (through Time Controller offered by Olympus or Experiment Designer offered by Zeiss) that are often unable to provide special set-ups and connection to external devices (e.g., for irradiation). Our new system combining an Arduino microcontroller with the Cell⊕Finder software was developed for controlling Olympus FV1000 and FV1200 confocal microscopes and the attached hardware modules. Our software/hardware solution offers (1) a text file-based macro language to control the imaging functions of the microscope; (2) programmable control of several external hardware devices (light sources, thermal controllers, actuators) during imaging via the Arduino microcontroller; (3) the Cell⊕Finder software with ergonomic user environment, a fast selection method for the biologically important cells and precise positioning feature that reduces unwanted bleaching of the cells by the scanning laser. Cell⊕Finder can be downloaded from http://www.alga.cz/cellfinder. The system was applied to study changes in fluorescence intensity in Synechocystis sp. PCC6803 cells under long-term illumination. Thus, we were able to describe the kinetics of phycobilisome decoupling. Microscopy data showed that phycobilisome decoupling appears slowly after long-term (>1 h) exposure to high light.

Living Matter Observations with a Novel Hyperspectral Supercontinuum Confocal Microscope for VIS to Near-IR Reflectance Spectroscopy

Directory of Open Access Journals (Sweden)

Francesca R. Bertani

2013-10-01

Full Text Available A broad range hyper-spectroscopic microscope fed by a supercontinuum laser source and equipped with an almost achromatic optical layout is illustrated with detailed explanations of the design, implementation and data. The real novelty of this instrument, a confocal spectroscopic microscope capable of recording high resolution reflectance data in the VIS-IR spectral range from about 500 nm to 2.5 μm wavelengths, is the possibility of acquiring spectral data at every physical point as defined by lateral coordinates, X and Y, as well as at a depth coordinate, Z, as obtained by the confocal optical sectioning advantage. With this apparatus we collect each single scanning point as a whole spectrum by combining two linear spectral detector arrays, one CCD for the visible range, and one InGaAs infrared array, simultaneously available at the sensor output channel of the home made instrument. This microscope has been developed for biomedical analysis of human skin and other similar applications. Results are shown illustrating the technical performances of the instrument and the capability in extracting information about the composition and the structure of different parts or compartments in biological samples as well as in solid statematter. A complete spectroscopic fingerprinting of samples at microscopic level is shown possible by using statistical analysis on raw data or analytical reflectance models based on Abelés matrix transfer methods.

A Clinical and Confocal Microscopic Comparison of Transepithelial PRK and LASEK for Myopia

Directory of Open Access Journals (Sweden)

Safak Korkmaz

2014-01-01

Full Text Available Purpose. To compare the clinical and confocal microscopic results of transepithelial PRK versus LASEK for correction of myopia. Materials and Methods. Twelve patients with myopia received transepithelial PRK in one eye and LASEK in the other. In transepithelial PRK-treated eyes, the corneal epithelium was removed with 40 microns of excimer laser ablation and in LASEK-treated eyes with 25-second application of 18% ethanol. Time to epithelial healing, ocular discomfort, uncorrected and best corrected visual acuities, manifest refraction, haze, greyscale value, and keratocyte apoptosis in confocal microscopy were recorded. Results. The mean time to epithelial healing was significantly longer after LASEK (4.00 ± 0.43 versus 3.17 ± 0.6 days. On day 1, ocular discomfort was significantly higher after transepithelial PRK. The grade of haze, keratocyte apoptosis, and greyscale value in confocal microscopy were significantly higher in transepithelial PRK-treated eyes at 1 month. All transepithelial PRK- and LASEK-treated eyes achieved 20/25 or better UCVA and were within ±1.00 D of emmetropia at final visits. Conclusions. Both transepithelial PRK and LASEK offer effective correction of myopia at 1 year. However, LASEK appeared to induce less discomfort and less intense wound healing in the early postoperative period.

A Clinical and Confocal Microscopic Comparison of Transepithelial PRK and LASEK for Myopia.

Science.gov (United States)

Korkmaz, Safak; Bilgihan, Kamil; Sul, Sabahattin; Hondur, Ahmet

2014-01-01

Purpose. To compare the clinical and confocal microscopic results of transepithelial PRK versus LASEK for correction of myopia. Materials and Methods. Twelve patients with myopia received transepithelial PRK in one eye and LASEK in the other. In transepithelial PRK-treated eyes, the corneal epithelium was removed with 40 microns of excimer laser ablation and in LASEK-treated eyes with 25-second application of 18% ethanol. Time to epithelial healing, ocular discomfort, uncorrected and best corrected visual acuities, manifest refraction, haze, greyscale value, and keratocyte apoptosis in confocal microscopy were recorded. Results. The mean time to epithelial healing was significantly longer after LASEK (4.00 ± 0.43 versus 3.17 ± 0.6 days). On day 1, ocular discomfort was significantly higher after transepithelial PRK. The grade of haze, keratocyte apoptosis, and greyscale value in confocal microscopy were significantly higher in transepithelial PRK-treated eyes at 1 month. All transepithelial PRK- and LASEK-treated eyes achieved 20/25 or better UCVA and were within ±1.00 D of emmetropia at final visits. Conclusions. Both transepithelial PRK and LASEK offer effective correction of myopia at 1 year. However, LASEK appeared to induce less discomfort and less intense wound healing in the early postoperative period.

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.

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...

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.

Optical depth sectioning in the aberration-corrected scanning transmission and scanning confocal electron microscope

International Nuclear Information System (INIS)

Behan, G; Nellist, P D

2008-01-01

The use of spherical aberration correctors in the scanning transmission electron microscope (STEM) has the effect of reducing the depth of field of the microscope, making three-dimensional imaging of a specimen possible by optical sectioning. Depth resolution can be improved further by placing aberration correctors and lenses pre and post specimen to achieve an imaging mode known as scanning confocal electron microscopy (SCEM). We present the calculated incoherent point spread functions (PSF) and optical transfer functions (OTF) of a STEM and SCEM. The OTF for a STEM is shown to have a missing cone region which results in severe blurring along the optic axis, which can be especially severe for extended objects. We also present strategies for reconstruction of experimental data, such as three-dimensional deconvolution of the point spread function.

Assessment of statistical agreement of three techniques for the study of cut marks: 3D digital microscope, laser scanning confocal microscopy and micro-photogrammetry.

Science.gov (United States)

Maté-González, Miguel Ángel; Aramendi, Julia; Yravedra, José; Blasco, Ruth; Rosell, Jordi; González-Aguilera, Diego; Domínguez-Rodrigo, Manuel

2017-09-01

In the last few years, the study of cut marks on bone surfaces has become fundamental for the interpretation of prehistoric butchery practices. Due to the difficulties in the correct identification of cut marks, many criteria for their description and classification have been suggested. Different techniques, such as three-dimensional digital microscope (3D DM), laser scanning confocal microscopy (LSCM) and micro-photogrammetry (M-PG) have been recently applied to the study of cut marks. Although the 3D DM and LSCM microscopic techniques are the most commonly used for the 3D identification of cut marks, M-PG has also proved to be very efficient and a low-cost method. M-PG is a noninvasive technique that allows the study of the cortical surface without any previous preparation of the samples, and that generates high-resolution models. Despite the current application of microscopic and micro-photogrammetric techniques to taphonomy, their reliability has never been tested. In this paper, we compare 3D DM, LSCM and M-PG in order to assess their resolution and results. In this study, we analyse 26 experimental cut marks generated with a metal knife. The quantitative and qualitative information registered is analysed by means of standard multivariate statistics and geometric morphometrics to assess the similarities and differences obtained with the different methodologies. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Confocal endomicroscopy for in vivo microscopic analysis of upper gastrointestinal tract premalignant and malignant lesions.

Science.gov (United States)

Gheorghe, Cristian; Iacob, Razvan; Becheanu, Gabriel; Dumbrav Abreve, Mona

2008-03-01

Confocal LASER endomicroscopy (CLE) is a new endoscopic technique which allows subsurface in vivo microscopic analysis during ongoing endoscopy, using systemically or topically administered fluorescent agents. It allows targeted biopsies to be taken, potentially improving the diagnostic rate in certain gastrointestinal diseases. Worldwide experience with CLE for upper gastrointestinal malignant and premalignant lesions is still reduced. Potential clinical applications are presented, including diagnosis of NERD, Barrett's esophagus, atrophic gatritis, gastric intestinal metaplasia and dysplasia, gastric adenomatous or hyperplastic polyps, gastric cancer.

Mode-mismatched confocal thermal-lens microscope with collimated probe beam

Energy Technology Data Exchange (ETDEWEB)

Cabrera, Humberto, E-mail: hcabrera@ictp.it [SPIE-ICTP Anchor Research Laboratory, International Centre for Theoretical Physics (ICTP), Strada Costiera 11, Trieste (Italy); Centro Multidisciplinartio de Ciencias, Instituto Venezolano de Investigaciones Científicas (IVIC), Mérida 5101 (Venezuela, Bolivarian Republic of); Korte, Dorota; Franko, Mladen [Laboratory for Environmental Research, University of Nova Gorica, Vipavska 13, 5000 Nova Gorica (Slovenia)

2015-05-15

We report a thermal lens microscope (TLM) based on an optimized mode-mismatched configuration. It takes advantage of the coaxial counter propagating tightly focused excitation and collimated probe beams, instead of both focused at the sample, as it is in currently known TLM setups. A simple mathematical model that takes into account the main features of the instrument is presented. The confocal detection scheme and the introduction of highly collimated probe beam allow enhancing the versatility, limit of detection (LOD), and sensitivity of the instrument. The theory is experimentally verified measuring ethanol’s absorption coefficient at 532.8 nm. Additionally, the presented technique is applied for detection of ultra-trace amounts of Cr(III) in liquid solution. The achieved LOD is 1.3 ppb, which represents 20-fold enhancement compared to transmission mode spectrometric techniques and a 7.5-fold improvement compared to previously reported methods for Cr(III) based on thermal lens effect.

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

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

Laser confocal microscope for analysis of 3013 inner container closure weld region

Energy Technology Data Exchange (ETDEWEB)

Martinez-Rodriguez, M. J. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

2017-10-26

As part of the protocol to investigate the corrosion in the inner container closure weld region (ICCWR) a laser confocal microscope (LCM) was used to perform close visual examination of the surface and measurements of corrosion features on the surface. However, initial analysis of selected destructively evaluated (DE) containers using the LCM revealed several challenges for acquiring, processing and interpreting the data. These challenges include topography of the ICCWR sample, surface features, and the amount of surface area for collecting data at high magnification conditions. In FY17, the LCM parameters were investigated to identify the appropriate parameter values for data acquisition and identification of regions of interest. Using these parameter values, selected DE containers were analyzed to determine the extent of the ICCWR to be examined.

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.

Confocal microscopic observation of structural changes in glass-ionomer cements and tooth interfaces.

Science.gov (United States)

Watson, T F; Pagliari, D; Sidhu, S K; Naasan, M A

1998-03-01

This study aimed to develop techniques to allow dynamic imaging of a cavity before, during and after placement of glass-ionomer restorative materials. Cavities were cut in recently extracted third molars and the teeth longitudinally sectioned. Each hemisected tooth surface was placed in green modelling compound at 90 to the optical axis of the microscope. The cavity surface was imaged using a video rate confocal microscope in conjunction with an internally focusable microscope objective. The sample on the stage was pushed up to the objective lens which 'clamped' the cover glass onto it. Water, glycerine or oil was placed below the coverglass, with oil above. Internal tooth structures were imaged by changing the internal focus of the objective. The restorative material was then placed into the cavity. Video images were stored either onto video tape or digitally, using a frame grabber, computer and mass memory storage. Software controls produced time-lapse recordings of the interface over time. Preliminary experiments have examined the placement and early maturation of conventional glass-ionomer cements and a syringeable resin-modified glass-ionomer cement. Initial contact of the cement matrix and glass particles was visible as the plastic material rolled past the enamel and dentine, before making a bond. Evidence for water movement from the dentine into the cement has also been seen. After curing, the early dimensional changes in the cements due to water flux were apparent using the time-lapse facility. This new technique enables examination of developing tooth/restoration interfaces and the tracking of movement in materials.

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.

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...

Orbital single particle tracking on a commercial confocal microscope using piezoelectric stage feedback

International Nuclear Information System (INIS)

Lanzanò, L; Gratton, E

2014-01-01

Single Particle Tracking (SPT) is a technique used to locate fluorescent particles with nanometer precision. In the orbital tracking method the position of a particle is obtained analyzing the distribution of intensity along a circular orbit scanned around the particle. In combination with an active feedback this method allows tracking of particles in 2D and 3D with millisecond temporal resolution. Here we describe a SPT setup based on a feedback approach implemented with minimal modification of a commercially available confocal laser scanning microscope, the Zeiss LSM 510, in combination with an external piezoelectric stage scanner. The commercial microscope offers the advantage of a user-friendly software interface and pre-calibrated hardware components. The use of an external piezo-scanner allows the addition of feedback into the system but also represents a limitation in terms of its mechanical response. We describe in detail this implementation of the orbital tracking method and discuss advantages and limitations. As an example of application to live cell experiments we perform the 3D tracking of acidic vesicles in live polarized epithelial cells. (paper)

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

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.

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

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.

Scanning differential polarization microscope: Its use to image linear and circular differential scattering

International Nuclear Information System (INIS)

Mickols, W.; Maestre, M.F.

1988-01-01

A differential polarization microscope that couples the sensitivity of single-beam measurement of circular dichroism and circular differential scattering with the simultaneous measurement of linear dichroism and linear differential scattering has been developed. The microscope uses a scanning microscope stage and single-point illumination to give the very shallow depth of field found in confocal microscopy. This microscope can operate in the confocal mode as well as in the near confocal condition that can allow one to program the coherence and spatial resolution of the microscope. This microscope has been used to study the change in the structure of chromatin during the development of sperm in Drosophila

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...

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.

Line-scanning tomographic optical microscope with isotropic transfer function

International Nuclear Information System (INIS)

Gajdátsy, Gábor; Dudás, László; Erdélyi, Miklós; Szabó, Gábor

2010-01-01

An imaging method and optical system, referred to as a line-scanning tomographic optical microscope (LSTOM) using a combination of line-scanning technique and CT reconstruction principle, is proposed and studied theoretically and experimentally. In our implementation a narrow focus line is scanned over the sample and the reflected light is measured in a confocal arrangement. One such scan is equivalent to a transverse projection in tomography. Repeating the scanning procedure in several directions, a number of transverse projections are recorded from which the image can be obtained using conventional CT reconstruction algorithms. The resolution of the image is independent of the spatial dimensions and structure of the applied detector; furthermore, the transfer function of the system is isotropic. The imaging performance of the implemented confocal LSTOM was compared with a point-scanning confocal microscope, based on recorded images. These images demonstrate that the resolution of the confocal LSTOM exceeds (by 15%) the resolution limit of a point-scanning confocal microscope

Avances en el estudio fractográfico de fibras cerámicas de circonaerbia mediante microscopía óptica confocal

Directory of Open Access Journals (Sweden)

López-Cepero, J. M.

2005-10-01

Full Text Available Laser scanning confocal microscopy (LSCM is a microscopic technique based on an optical construction which allows the microscope to discard the light coming from unfocused zones of the sample. Whereas LSCM is extensively used in Natural Sciences (Biology, Medicine..., its use in Materials Science is almost unexplored and, in particular, there are essentially no fractographical studies using LSCM. However, its characteristics (gathering of 3D information, better than micron resolution and simple sample preparation make LSCM an ideal tool in a wide selection of fractographical problems, owing to the fast adquisition of valuable information and to the excellent sinergy with scanning electron microscopy (SEM. In the present work, the authors study an interesting system (ZrO2-5% mol Er2O3 fibers, submitted to tensile strength in hightemperature conditions in detail with LSCM. In addition to showcasing the usefulness of LSCM in fractographical studies and revealing the characteristic texture of the fracture surface in such fibers, said texture is found to closely resemble the nanometric precipitate structure which is unique to this material.

La microscopía óptica confocal (LSCM, Laser Scanning Confocal Microscopy es una técnica microscópica basada en una construcción óptica que permite eliminar la luz procedente de zonas no enfocadas de la muestra. Mientras que es de amplio uso en Ciencias de la Vida, la aplicación de LSCM a la Ciencia de Materiales no ha sido apenas explorada, siendo prácticamente inexistentes los estudios fractográficos que se apoyen en LSCM. A pesar de ello, sus características (obtención de información tridimensional, resolución por debajo de la micra y sencilla preparación de muestras la convierten en una herramienta idónea para una multitud de problemas fractográficos, debido a la obtención rápida de valiosa información y a su buena coordinación con la microscopía electrónica de barrido (SEM. En este

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.

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

Poly(diacetylene) Monolayers Studied with a Fluorescence Scanning Near-Field Optical Microscope

NARCIS (Netherlands)

Moers, Marco H.P.; Moers, M.H.P.; Gaub, Hermann E.; van Hulst, N.F.

1994-01-01

A novel and powerful method to study the optical properties of thin lipid films which a resolution superior to confocal microscopy is presented. With a scanning near-field optical microscope, fluorescence images of a Langmuir-Blodgett film of diethylene glycol diamine pentacosadiynoic amide are

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

the characteristics of the corneal tissue in patients undergoing this type of transplantation through in vivo confocal microscopy. METHODS: A cross-sectional descriptive study was carried out in 40 eyes from 40 patients operated with penetrating keratoplasty in the Corneal Service of "Ramón Pando Ferrer" Cuban Institute of Ophthalmology from March 2006 to March 2007. A clinical ophthalmologic history was drafted, and they were all subjected to confocal microscopic examination of the corneal graft using confocal microscope CONFOSCAN 4. RESULTS: Pseuphakic bullosa keratopathy was the most common disease prior to surgery and was present in 77.5 % of patients. Corneal thickness decreased in 72.5 % of the operated patients. The epithelium was altered in 62.5 %. All of them presented with problems in the endotelial cellular form and size. Nervous plexuses were not observed in 82.5 % of patients. CONCLUSIONS: Confocal microscopy as a new science in the field of ophthalmology favors the follow-up of penetrating keratoplasty and allows preventing possible complications and assuring the success of surgery and the restoration of the refractive function of the cornea.

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.

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...

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.

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.

Analysis of mitochondrial mechanical dynamics using a confocal fluorescence microscope with a bent optical fibre.

Science.gov (United States)

Li, Yongbo; Honda, Satoshi; Iwami, Kentaro; Ohta, Yoshihiro; Umeda, Norihiro

2015-11-01

The cells in the cardiovascular system are constantly subjected to mechanical forces created by blood flow and the beating heart. The effect of forces on cells has been extensively investigated, but their effect on cellular organelles such as mitochondria remains unclear. We examined the impact of nano-Newton forces on mitochondria using a bent optical fibre (BOF) with a flat-ended tip (diameter exceeding 2 μm) and a confocal fluorescence microscope. By indenting a single mitochondrion with the BOF tip, we found that the mitochondrial elastic modulus was proportional to the (-1/2) power of the mitochondrial radius in the 9.6-115 kPa range. We stained the mitochondria with a potential-metric dye (TMRE) and measured the changes in TMRE fluorescence intensity. We confirmed that more active mitochondria exhibit a higher frequency of repetitive transient depolarization. The same trend was observed at forces lower than 50 nN. We further showed that the depolarization frequency of mitochondria decreases under an extremely large force (nearly 100 nN). We conclude that mitochondrial function is affected by physical environmental factors, such as external forces at the nano-Newton level. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

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.

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.

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.

Inverted follicular keratosis: dermoscopic and reflectance confocal microscopic features.

Science.gov (United States)

Armengot-Carbo, M; Abrego, A; Gonzalez, T; Alarcon, I; Alos, L; Carrera, C; Malvehy, J; Puig, S

2013-01-01

Inverted follicular keratosis (IFK) is a rare benign tumor which usually appears as a firm papule on the face. The diagnosis is generally made by histopathology because the clinical appearance is difficult to differentiate from other lesions. Dermoscopic features of IFK have not been established to date. Herein we describe the dermoscopic findings of 4 cases of IFK. Radial peripheral hairpin vessels surrounded by a whitish halo arranged around a central white-yellowish amorphous area were observed in 3 cases, and glomerular vessels were present in the central area of one of them. The fourth case also presented a central white amorphous area but showed arborizing vessels. Reflectance confocal microscopy (available in 1 case) revealed a broadened honeycomb pattern, epidermal projections and hairpin and glomerular vessels. To our knowledge this is the first case series describing the dermoscopic features of inverted follicular keratosis and the first confocal microscopy description of this entity.

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

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.

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.

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....

Polarization-preserving confocal microscope for optical experiments in a dilution refrigerator with high magnetic field.

Science.gov (United States)

Sladkov, Maksym; Bakker, M P; Chaubal, A U; Reuter, D; Wieck, A D; van der Wal, C H

2011-04-01

We present the design and operation of a fiber-based cryogenic confocal microscope. It is designed as a compact cold-finger that fits inside the bore of a superconducting magnet, and which is a modular unit that can be easily swapped between use in a dilution refrigerator and other cryostats. We aimed at application in quantum optical experiments with electron spins in semiconductors and the design has been optimized for driving with and detection of optical fields with well-defined polarizations. This was implemented with optical access via a polarization maintaining fiber together with Voigt geometry at the cold finger, which circumvents Faraday rotations in the optical components in high magnetic fields. Our unit is versatile for use in experiments that measure photoluminescence, reflection, or transmission, as we demonstrate with a quantum optical experiment with an ensemble of donor-bound electrons in a thin GaAs film. © 2011 American Institute of Physics

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

Directory of Open Access Journals (Sweden)

A. Beaudet

1998-11-01

Full Text Available This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

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...

Adipocyte size and cellular expression of caveolar proteins analyzed by confocal microscopy

DEFF Research Database (Denmark)

Hulstrøm, Veronica; Prats Gavalda, Clara; Vinten, Jørgen

2013-01-01

Caveolae are abundant in adipocytes and are involved in the regulation of lipid accumulation, which is the main volume determinant of these cells. We have developed and applied a confocal microscopic technique for measuring individual cellular expression of the caveolar proteins cavin-1 and caveo......Caveolae are abundant in adipocytes and are involved in the regulation of lipid accumulation, which is the main volume determinant of these cells. We have developed and applied a confocal microscopic technique for measuring individual cellular expression of the caveolar proteins cavin-1...

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 ...

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.

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

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

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.

Scanning Color Laser Microscope

Science.gov (United States)

Awamura, D.; Ode, T.; Yonezawa, M.

1988-01-01

A confocal color laser microscope which utilizes a three color laser light source (Red: He-Ne, Green: Ar, Blue: Ar) has been developed and is finding useful applications in the semiconductor field. The color laser microscope, when compared to a conventional microscope, offers superior color separation, higher resolution, and sharper contrast. Recently some new functions including a Focus Scan Memory, a Surface Profile Measurement System, a Critical Dimension Measurement system (CD) and an Optical Beam Induced Current Function (OBIC) have been developed for the color laser microscope. This paper will discuss these new features.

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.

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)

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

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

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.

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

Quantitative detection of caffeine in human skin by confocal Raman spectroscopy--A systematic in vitro validation study.

Science.gov (United States)

Franzen, Lutz; Anderski, Juliane; Windbergs, Maike

2015-09-01

For rational development and evaluation of dermal drug delivery, the knowledge of rate and extent of substance penetration into the human skin is essential. However, current analytical procedures are destructive, labor intense and lack a defined spatial resolution. In this context, confocal Raman microscopy bares the potential to overcome current limitations in drug depth profiling. Confocal Raman microscopy already proved its suitability for the acquisition of qualitative penetration profiles, but a comprehensive investigation regarding its suitability for quantitative measurements inside the human skin is still missing. In this work, we present a systematic validation study to deploy confocal Raman microscopy for quantitative drug depth profiling in human skin. After we validated our Raman microscopic setup, we successfully established an experimental procedure that allows correlating the Raman signal of a model drug with its controlled concentration in human skin. To overcome current drawbacks in drug depth profiling, we evaluated different modes of peak correlation for quantitative Raman measurements and offer a suitable operating procedure for quantitative drug depth profiling in human skin. In conclusion, we successfully demonstrate the potential of confocal Raman microscopy for quantitative drug depth profiling in human skin as valuable alternative to destructive state-of-the-art techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Development of confocal X-ray fluorescence (XRF) microscopy at the Cornell high energy synchrotron source

International Nuclear Information System (INIS)

Woll, A.R.; Huang, R.; Mass, J.; Bisulca, C.; Bilderback, D.H.; Gruner, S.; Gao, N.

2006-01-01

A confocal X-ray fluorescence microscope was built at the Cornell High Energy Synchrotron Source (CHESS) to obtain compositional depth profiles of historic paintings. The microscope consists of a single-bounce, borosilicate monocapillary optic to focus the incident beam onto the painting and a commercial borosilicate polycapillary lens to collect the fluorescent X-rays. The resolution of the microscope was measured by scanning a variety of thin metal films through this confocal volume while monitoring the fluorescence signal. The capabilities of the technique were then probed using test paint microstructures with up to four distinct layers, each having a thickness in the range of 10-80 microns. Results from confocal XRF were compared with those from stand-alone XRF and visible light microscopy of the paint cross-sections. A large area, high-resolution scanner is currently being built to perform 3D scans on moderately sized paintings. (orig.)

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

Directory of Open Access Journals (Sweden)

Kobayashi A

2014-02-01

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

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.

Análisis fractográfico de fibras de circona y de zafiro mediante microscopía óptica confocal

Directory of Open Access Journals (Sweden)

López-Cepero, J. M.

2005-08-01

Full Text Available Fractography is a very useful tool to learn about the mechanisms controlling the fracture process. In this work, the fractographical uses of laser scanning confocal microscopy (LSCM are shown. LSCM is a widely used technique in Biology and similar disciplines, but its use in Materials Science is not yet as explored. However, it is an ideal technique for fractographical studies, since by using it the three-dimensional profile of the fracture surface can be obtained. From this profile, it is possible to extract information, like the roughness of the fracture surface, which would be very difficult to obtain from other studies. In this paper, LSCM is applied to the study of the fracture surface in ceramic fibers submitted to tensile stress, making the interest of the technique evident due to features such as easy sample preparation, gathering of real 3D information, and good SEM-LSCM synergy.

La fractografía en materiales resulta de gran utilidad para la caracterización de los mecanismos que dominan la rotura. En el presente artículo se investigan las aplicaciones fractográficas de la microscopía óptica confocal o LSCM (Laser Scanning Confocal Microscopy. Dicha técnica es de amplio uso en Biología y disciplinas afines, pero su empleo en Ciencia de Materiales está poco explorado. Sin embargo, resulta ideal para el estudio fractográfico, ya que permite obtener reconstrucciones del perfil tridimensional de la superficie de fractura. De este perfil tridimensional puede extraerse información —la rugosidad de la superficie, por ejemplo— muy difícil de obtener a partir de otro tipo de estudios. En este trabajo se aplica LSCM a la caracterización de superficies de fractura en fibras cerámicas sometidas a tracción y se pone de manifiesto el interés de la técnica, debido a características tales como la preparación sencilla de muestras, la obtención de información tridimensional real y la buena coordinación SEM-LSCM.

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.

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

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...

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.

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.

Caustic meso-optical confocal microscope for vertical particle tracks. Proposal

International Nuclear Information System (INIS)

Soroko, L.M.

1995-01-01

The principal of the proposed caustic meso-optical microscope for vertical particle tracks in the nuclear photoemulsion is explained. The results of the experiments performed to illustrate the main features of this new meso-optical microscope are given. The proposed caustic meso-optical microscope for vertical particle tracks in the nuclear photoemulsion can be effectively used in the experimental investigation of such rare processes as ν μ - ν τ oscillations and of the Pb-Pb interactions. 2 refs., 7 figs

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.

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...

Evaluation of Enterococcus faecalis adhesion, penetration, and method to prevent the penetration of Enterococcus faecalis into root cementum: Confocal laser scanning microscope and scanning electron microscope analysis.

Science.gov (United States)

Halkai, Rahul S; Hegde, Mithra N; Halkai, Kiran R

2016-01-01

To ascertain the role of Enterococcus faecalis in persistent infection and a possible method to prevent the penetration of E. faecalis into root cementum. One hundred and twenty human single-rooted extracted teeth divided into five groups. Group I (control): intact teeth, Group II: no apical treatment done, Group III divided into two subgroups. In Groups IIIa and IIIb, root apex treated with lactic acid of acidic and neutral pH, respectively. Group IV: apical root cementum exposed to lactic acid and roughened to mimic the apical resorption. Group V: apical treatment done same as Group IV and root-end filling done using mineral trioxide aggregate (MTA). Apical one-third of all samples immersed in E. faecalis broth for 8 weeks followed by bone morphogenetic protein and obturation and again immersed into broth for 8 weeks. Teeth split into two halves and observed under confocal laser scanning microscope and scanning electron microscope, organism identified by culture and polymerase chain reaction techniques. Adhesion and penetration was observed in Group IIIa and Group IV. Only adhesion in Group II and IIIB and no adhesion and penetration in Group I and V. Adhesion and penetration of E. faecalis into root cementum providing a long-term nidus for subsequent infection are the possible reason for persistent infection and root-end filling with MTA prevents the adhesion and penetration.

Surface profile measurement by using the integrated Linnik WLSI and confocal microscope system

Science.gov (United States)

Wang, Wei-Chung; Shen, Ming-Hsing; Hwang, Chi-Hung; Yu, Yun-Ting; Wang, Tzu-Fong

2017-06-01

The white-light scanning interferometer (WLSI) and confocal microscope (CM) are the two major optical inspection systems for measuring three-dimensional (3D) surface profile (SP) of micro specimens. Nevertheless, in practical applications, WLSI is more suitable for measuring smooth and low-slope surfaces. On the other hand, CM is more suitable for measuring uneven-reflective and low-reflective surfaces. As for aspect of surface profiles to be measured, the characteristics of WLSI and CM are also different. WLSI is generally used in semiconductor industry while CM is more popular in printed circuit board industry. In this paper, a self-assembled multi-function optical system was integrated to perform Linnik white-light scanning interferometer (Linnik WLSI) and CM. A connecting part composed of tubes, lenses and interferometer was used to conjunct finite and infinite optical systems for Linnik WLSI and CM in the self-assembled optical system. By adopting the flexibility of tubes and lenses, switching to perform two different optical measurements can be easily achieved. Furthermore, based on the shape from focus method with energy of Laplacian filter, the CM was developed to enhance the on focal information of each pixel so that the CM can provide all-in-focus image for performing the 3D SP measurement and analysis simultaneously. As for Linnik WLSI, eleven-step phase shifting algorithm was used to analyze vertical scanning signals and determine the 3D SP.

(LMRG): Microscope Resolution, Objective Quality, Spectral Accuracy and Spectral Un-mixing

Science.gov (United States)

Bayles, Carol J.; Cole, Richard W.; Eason, Brady; Girard, Anne-Marie; Jinadasa, Tushare; Martin, Karen; McNamara, George; Opansky, Cynthia; Schulz, Katherine; Thibault, Marc; Brown, Claire M.

2012-01-01

The second study by the LMRG focuses on measuring confocal laser scanning microscope (CLSM) resolution, objective lens quality, spectral imaging accuracy and spectral un-mixing. Affordable test samples for each aspect of the study were designed, prepared and sent to 116 labs from 23 countries across the globe. Detailed protocols were designed for the three tests and customized for most of the major confocal instruments being used by the study participants. One protocol developed for measuring resolution and objective quality was recently published in Nature Protocols (Cole, R. W., T. Jinadasa, et al. (2011). Nature Protocols 6(12): 1929–1941). The first study involved 3D imaging of sub-resolution fluorescent microspheres to determine the microscope point spread function. Results of the resolution studies as well as point spread function quality (i.e. objective lens quality) from 140 different objective lenses will be presented. The second study of spectral accuracy looked at the reflection of the laser excitation lines into the spectral detection in order to determine the accuracy of these systems to report back the accurate laser emission wavelengths. Results will be presented from 42 different spectral confocal systems. Finally, samples with double orange beads (orange core and orange coating) were imaged spectrally and the imaging software was used to un-mix fluorescence signals from the two orange dyes. Results from 26 different confocal systems will be summarized. Time will be left to discuss possibilities for the next LMRG study.

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.

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.

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

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

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.

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.

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. ...

In-situ Crystallization of Highly Volatile Commercial Mold Flux Using an Isolated Observation System in the Confocal Laser Scanning Microscope

Science.gov (United States)

Park, Jun-Yong; Ryu, Jae Wook; Sohn, Il

2014-08-01

The in situ crystallization behavior of highly volatile commercial mold fluxes for medium carbon steels was investigated using the confocal laser scanning microscope (CLSM) equipped with an optimized isolated observation system. The highly volatile compounds of the mold flux were suppressed during heating allowing direct observation in the CLSM. Cooling rates of 25, 50, 100, 400, and 800 K/min were incorporated and continuous cooling transformation (CCT) diagrams of 4 different commercial mold fluxes for medium carbon steels were developed. Identification of the crystalline phase was conducted with XRD and SEM-EDS analysis. A cuspidine crystalline was observed in all samples at various cooling rates. With higher basicity, CaF2, and NaF, the crystallization of the fluxes was enhanced according to the CCT diagram. As the slag structure becomes depolymerized, the diffusion rate of the cathodic ions seems to increase.

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.

Upgrading the GSI beamline microscope with a confocal fluorescence lifetime scanner to monitor charged particle induced chromatin decondensation in living cells

Energy Technology Data Exchange (ETDEWEB)

Abdollahi, Elham; Taucher-Scholz, Gisela [Department of Biophysics, GSI Helmholtz Center for Heavy Ion Research, Planckstrasse 1, 64291 Darmstadt (Germany); Durante, Marco [Department of Biophysics, GSI Helmholtz Center for Heavy Ion Research, Planckstrasse 1, 64291 Darmstadt (Germany); Institute for Condensed Matter Physics, Darmstadt University of Technology, 64289 Darmstadt (Germany); Jakob, Burkhard, E-mail: B.Jakob@gsi.de [Department of Biophysics, GSI Helmholtz Center for Heavy Ion Research, Planckstrasse 1, 64291 Darmstadt (Germany)

2015-12-15

We report the upgrade of the GSI beamline microscope coupled to the linear accelerator UNILAC by a confocal FLIM scanner utilizing time correlated single photon counting technique (TCSPC). The system can now be used to address the radiation induced chromatin decondensation in more detail and with higher sensitivity compared to intensity based methods. This decondensation of heterochromatic areas is one of the early DNA damage responses observed after charged particle irradiation and might facilitate the further processing of the induced lesions. We describe here the establishment of different DNA dyes as chromatin compaction probes usable for quantification of the DNA condensation status in living cells utilizing lifetime imaging. In addition, we find an evidence of heterochromatic chromatin decondensation in ion irradiated murine chromocenters detected after subsequent fixation using FLIM measurements.

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.

In vivo cellular imaging with microscopes enabled by MEMS scanners

Science.gov (United States)

Ra, Hyejun

High-resolution optical imaging plays an important role in medical diagnosis and biomedical research. Confocal microscopy is a widely used imaging method for obtaining cellular and sub-cellular images of biological tissue in reflectance and fluorescence modes. Its characteristic optical sectioning capability also enables three-dimensional (3-D) image reconstruction. However, its use has mostly been limited to excised tissues due to the requirement of high numerical aperture (NA) lenses for cellular resolution. Microscope miniaturization can enable in vivo imaging to make possible early cancer diagnosis and biological studies in the innate environment. In this dissertation, microscope miniaturization for in vivo cellular imaging is presented. The dual-axes confocal (DAC) architecture overcomes limitations of the conventional single-axis confocal (SAC) architecture to allow for miniaturization with high resolution. A microelectromechanical systems (MEMS) scanner is the central imaging component that is key in miniaturization of the DAC architecture. The design, fabrication, and characterization of the two-dimensional (2-D) MEMS scanner are presented. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer and is actuated by self-aligned vertical electrostatic combdrives. The imaging performance of the MEMS scanner in a DAC configuration is shown in a breadboard microscope setup, where reflectance and fluorescence imaging is demonstrated. Then, the MEMS scanner is integrated into a miniature DAC microscope. The whole imaging system is integrated into a portable unit for research in small animal models of human biology and disease. In vivo 3-D imaging is demonstrated on mouse skin models showing gene transfer and siRNA silencing. The siRNA silencing process is sequentially imaged in one mouse over time.

In vivo detection of basal cell carcinoma: comparison of a reflectance confocal microscope and a multiphoton tomograph

Science.gov (United States)

Ulrich, Martina; Klemp, Marisa; Darvin, Maxim E.; König, Karsten; Lademann, Jürgen; Meinke, Martina C.

2013-06-01

The standard diagnostic procedure for basal cell carcinoma (BCC) is invasive tissue biopsy with time-consuming histological examination. To reduce the number of biopsies, noninvasive optical methods have been developed providing high-resolution skin examination. We present direct comparison of a reflectance confocal microscope (RLSM) and a multiphoton tomograph (MPT) for BCC diagnosis. Both systems are applied to nine patients prior to surgery, and the results are analyzed, including histological results. Both systems prove suitable for detecting typical characteristics of BCC in various stages. The RLSM allows large horizontal overview images to be obtained, enabling the investigator to find the regions of interest quickly, e.g., BCC nests. Elongated cells and palisading structures are easily recognized using both methods. Due to the higher resolution, changes in nucleus diameter or cytoplasm could be visualized with the MPT. Therefore, the nucleus diameter, nucleus/cytoplasm ratio, and cell density are estimated for normal and BCC cells using the MPT. The nucleus of elongated BCC cells is significantly longer than other measured normal skin cells, whereas the cell density and nucleus/cytoplasm ratio of BCC cannot be significantly distinguished from granular cells.

[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.

Two-Photon Fluorescence Microscope for Microgravity Research

Science.gov (United States)

Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

2005-01-01

A two-photon fluorescence microscope has been developed for the study of biophysical phenomena. Two-photon microscopy is a novel form of laser-based scanning microscopy that enables three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon optical microscopy, two-photon microscopy utilizes the simultaneous nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption, so an ultra-fast pulsed laser source is typically employed. On the other hand, the critical energy threshold for two-photon absorption leads to fluorophore excitation that is intrinsically localized to the focal volume. Consequently, two-photon microscopy enables optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction (relative to one-photon optical microscopy) in photon-induced damage because of the longer excitation wavelength. This reduction is especially advantageous for in vivo studies. Relative to confocal microscopy, there is also a reduction in background fluorescence, and, because of a reduction in Rayleigh scattering, there is a 4 increase of penetration depth. The prohibitive cost of a commercial two-photon fluorescence-microscope system, as well as a need for modularity, has led to the construction of a custom-built system (see Figure 1). This system includes a coherent mode-locked titanium: sapphire laser emitting 120-fs-duration pulses at a repetition rate of 80 MHz. The pulsed laser has an average output power of 800 mW and a wavelength tuning range of 700 to 980 nm, enabling the excitation of a variety of targeted fluorophores. The output from the laser is attenuated, spatially filtered, and then directed into a confocal scanning head that has been modified to provide for side entry of the laser beam. The laser output coupler has been replaced with a dichroic filter that reflects the

Chondrocytes provide a model for in-situ confocal microscopy and 3D reconstructions

Science.gov (United States)

Hirsch, Michelle S.; Svoboda, Kathy K. H.

1994-04-01

Hyaline cartilage is composed of chondrocytes that reside in lacunae surrounded by extracellular matrix molecules. Microscopic and histochemical features of cartilage have been studied with many techniques. Many of these techniques can be time consuming and may alter natural cartilage characteristics. In addition, the orientation and order of sectioned tissue must be maintained to create 3D reconstructions. We show that confocal laser scanning microscopy may replace traditional methods for studying cartilage.

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

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...

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.

Fluorescence Confocal Microscopy for Ex Vivo Diagnosis of Conjunctival Tumors: A Pilot Study.

Science.gov (United States)

Iovieno, Alfonso; Longo, Caterina; De Luca, Mariacarla; Piana, Simonetta; Fontana, Luigi; Ragazzi, Moira

2016-08-01

To evaluate the potential use of fluorescence confocal microscopy (FCM) for ex vivo diagnosis and excision margin assessment of conjunctival neoplasms. Validity study. setting: Single institution. Consecutive patients with clinically suspicious conjunctival lesions. Conjunctival lesions were excised in toto using a standard "no-touch technique" by a single surgeon (A.I.). Collected specimens were examined with a commercially available laser scanning fluorescence confocal microscope after immersion in a 0.6 mM solution of acridine orange dye for 10-20 seconds. Specimens were subsequently processed with standard histologic analysis. FCM diagnosis of the nature and extension of conjunctival lesions. Sixteen consecutive patients were included in the study (11 male, 5 female; mean age 58.1 ± 26.1 years, range 10-90 years). The median time needed to process and analyze a sample with FCM was 15 minutes. Eleven of 16 lesions were identified by FCM as squamous (2 benign papillomas, 2 grade 2 conjunctival intraepithelial neoplasias, 7 in situ squamous carcinomas) and 5 as nonsquamous (1 pingueculum, 1 dermolipoma, 2 melanocytic nevi, 1 melanoma). In all cases FCM was able to detect horizontal and vertical extension of the lesion. All FCM findings were confirmed by corresponding subsequent histologic examination. FCM provides a fast ex vivo preliminary diagnosis of suspicious conjunctival lesions with good histologic details and margin assessment, and may represent a novel tool for intraoperative and postsurgical management of conjunctival tumors. This is the first study to investigate ex vivo FCM application in ophthalmology. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Diving under a microscope--a new simple and versatile in vitro diving device for fluorescence and confocal microscopy allowing the controls of hydrostatic pressure, gas pressures, and kinetics of gas saturation.

Science.gov (United States)

Wang, Qiong; Belhomme, Marc; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Theron, Michaël

2013-06-01

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

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.

Numerical study of a confocal ultrasonic setup for creation of cavitation

Energy Technology Data Exchange (ETDEWEB)

Lafond, Maxime, E-mail: maxime.lafond@inserm.fr; Chavrier, Françoise; Prieur, Fabrice [Inserm, U1032, LabTau, Lyon, F-69003 (France); Université de Lyon, Lyon, F-69003 (France); Université Lyon 1, Lyon, F-69003 (France); Mestas, Jean-Louis; Lafon, Cyril [Inserm, U1032, LabTau, Lyon, F-69003 (France); Université de Lyon, Lyon, F-69003 (France); Université Lyon 1, Lyon, F-69003 (France); Caviskills SAS, Vaulx-En-Velin, F-69120 (France)

2015-10-28

Acoustic cavitation is used for various therapeutic applications such as local enhancement of drug delivery, histotripsy or hyperthermia. One of the utmost important parameter for cavitation creation is the rarefaction pressure. The typical magnitude of the rarefaction pressure required to initiate cavitation from gas dissolved in tissue is beyond the range of the megapascal. Because nonlinear effects need to be taken into account, a numerical simulator based on the Westervelt equation was used to study the pressure waveform and the acoustic field generated by a setup for creation of cavitation consisting of two high intensity focused ultrasound transducers mounted confocally. At constant acoustic power, simulations with only one and both transducers from the confocal setup showed that the distortion of the pressure waveform due to the combined effects of nonlinearity and diffraction is less pronounced when both confocal transducers are used. Consequently, the confocal setup generates a greater peak negative pressure at focus which is more favorable for cavitation initiation. Comparison between the confocal setup and a single transducer with the same total emitting surface puts in evidence the role of the spatial separation of the two beams. Furthermore, it has been previously shown that the location of the peak negative pressure created by a single transducer shifts from focus towards the transducers in the presence of nonlinear effects. The simulator was used to study a configuration where the acoustical axes of transducers intersect on the peak negative pressure instead of the geometrical focus. For a representative confocal setup, namely moderate nonlinear effects, a 2% increase of the peak negative pressure and 8% decrease of the peak positive pressure resulted from this configuration. These differences tend to increase by increasing nonlinear effects. Although the optimal position of the transducers varies with the nonlinear regimen, the intersection point

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.

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.

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)

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)

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.

Ex vivo confocal microscopy: a new diagnostic technique for mucormycosis.

Science.gov (United States)

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

2016-05-01

Skin-dedicated ex vivo confocal microscopy (EVCM) has so far mainly been employed to identify cutaneous tumours on freshly excised samples. We present two cases where EVCM has been used to diagnose cutaneous mucormycosis. The skin biopsies were evaluated by the skin-dedicated ex vivo confocal microscope VivaScope 2500(®) (MAVIG GmbH, Munich Germany) under both reflectance and fluorescence mode. Conventional direct optical examination on skin scraping and histological examination were later performed. Mucormycetes observed by EVCM presented as hyper-reflective elongated 20 μm in diameter structures with perpendicular ramifications. Fungi were found both under reflectance and fluorescence mode and were better visible with acridine orange under fluorescence EVCM. Conventional direct optical examination on skin scraping and histological examination found the same elongated and branching structures confirming the presence of Mucormycetes. Ex vivo confocal microscopy has both the advantages of being fast as the direct optical examination, and to be able to show the localisation of the fungi in the tissue like the histological examination. In our cases, EVCM allowed to rapidly confirm the clinical diagnosis of mucormycosis, which is essential for the treatment of this fungal infection. Further studies are needed to compare the performance of EVCM with the findings of conventional histological and mycological examinations. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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)

Superresolution confocal technology for displacement measurements based on total internal reflection

International Nuclear Information System (INIS)

Kuang Cuifang; Hao Xiang; Wang Tingting; Liu Xu; Ali, M. Yakut

2010-01-01

In order to achieve a higher axial resolution for displacement measurement, a novel method is proposed based on total internal reflection filter and confocal microscope principle. A theoretical analysis of the basic measurement principles is presented. The analysis reveals that the proposed confocal detection scheme is effective in enhancing the resolution of nonlinearity of the reflectance curve greatly. In addition, a simple prototype system has been developed based on the theoretical analysis and a series of experiments have been performed under laboratory conditions to verify the system feasibility, accuracy, and stability. The experimental results demonstrate that the axial resolution in displacement measurements is better than 1 nm in a range of 200 nm which is threefold better than that can be achieved using the plane reflector.

Superresolution confocal technology for displacement measurements based on total internal reflection.

Science.gov (United States)

Kuang, Cuifang; Ali, M Yakut; Hao, Xiang; Wang, Tingting; Liu, Xu

2010-10-01

In order to achieve a higher axial resolution for displacement measurement, a novel method is proposed based on total internal reflection filter and confocal microscope principle. A theoretical analysis of the basic measurement principles is presented. The analysis reveals that the proposed confocal detection scheme is effective in enhancing the resolution of nonlinearity of the reflectance curve greatly. In addition, a simple prototype system has been developed based on the theoretical analysis and a series of experiments have been performed under laboratory conditions to verify the system feasibility, accuracy, and stability. The experimental results demonstrate that the axial resolution in displacement measurements is better than 1 nm in a range of 200 nm which is threefold better than that can be achieved using the plane reflector.

Confocal nanoscanning, bead picking (CONA): PickoScreen microscopes for automated and quantitative screening of one-bead one-compound libraries.

Science.gov (United States)

Hintersteiner, Martin; Buehler, Christof; Uhl, Volker; Schmied, Mario; Müller, Jürgen; Kottig, Karsten; Auer, Manfred

2009-01-01

Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead

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.

Transient gels in colloid-polymer mixtures studied with fluorescence confocal scanning laser microscopy

NARCIS (Netherlands)

Verhaegh, N.A.M.; Asnaghi, D.; Lekkerkerker, H.N.W.

1999-01-01

We study the structure and the time evolution of transient gels formed in colloid-polymer mixtures, by means of uorescence Confocal Scanning Laser Microscopy (CSLM). This technique is used in conjunction with novel colloidal silica particles containing a uorescent core. The confocal micrographs

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...

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.

Digital Position Encoding Of Galvanometer Scanner In A Laser Microscope

Science.gov (United States)

Liljeborg, Anders

1988-09-01

An account is given of a realization of a feedback method to digitize the analog position signal from a moving iron galvanometer. It is employed in a confocal scanning laser microscope for generating digital images. The photometric sampling has to be closely coupled to the position of a mirror that scans a focused laser beam across a microscope specimen. Pictures with low geometric distortion are obtained up to the size 1024 x 1024 pixels.

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.

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.

Microscope Raman scattering and X-ray diffraction study of near-stoichiometric Ti:LiNbO3 waveguides

International Nuclear Information System (INIS)

Zhang, De-Long; Siu, G.G.; Pun, E.Y.B.

2005-01-01

The crystalline phase within guiding layers of near-stoichiometric strip and planar Ti:LiNbO 3 wave-guides, prepared by the method of simultaneous work of vapour transport equilibration (VTE) treatment and indiffusion of Ti film, was studied by combined confocal microscope Raman scattering and X-ray powder diffraction. The results show that the strip and planar waveguide layers still retain the LiNbO 3 phase and no other non-LiNbO 3 phases can be identified within the guiding layer. Li/Nb ratios inside and outside the strip and planar waveguide layers were determined from the microscope Raman scattering results and compared to those obtained from the measured optical absorption edge. It is shown that the Li/Nb ratios are homogeneous within the waveguide layer and are close inside and outside the waveguide layer. (copyright 2005 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

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.

A confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium

Directory of Open Access Journals (Sweden)

Sparrow Malcolm P

2005-10-01

Full Text Available Abstract Background Pulmonary neuroendocrine cells (PNEC are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy. Methods Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table 1 undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5, sensory nerves (calcitonin gene related peptide, CGRP, and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP. The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop. Results PNEC were abundant but not homogenously distributed within the epithelium, with densities ranging from 65/mm2 to denser patches of 250/mm2, depending on the individual wholemount. Rotation of 3-D images revealed a complex morphology; flask-like with the cell body near the basement membrane and a thick stem extending to the lumen. Long processes issued laterally from its base, some lumenal and others with feet-like processes. Calcitonin gene-related peptide (CGRP was present in about 20% of PNEC, mainly in the processes. CGRP-positive nerves were sparse, with some associated with the apical part of the PNEC. Conclusion Our 3D-data demonstrates that PNEC are numerous and exhibit a heterogeneous peptide content suggesting an active and diverse PNEC population.

Probing thermal evanescent waves with a scattering-type near-field microscope

International Nuclear Information System (INIS)

Kajihara, Y; Kosaka, K; Komiyama, S

2011-01-01

Long wavelength infrared (LWIR) waves contain many important spectra of matters like molecular motions. Thus, probing spontaneous LWIR radiation without external illumination would reveal detailed mesoscopic phenomena that cannot be probed by any other measurement methods. Here we developed a scattering-type scanning near-field optical microscope (s-SNOM) and demonstrated passive near-field microscopy at 14.5 µm wavelength. Our s-SNOM consists of an atomic force microscope and a confocal microscope equipped with a highly sensitive LWIR detector, called a charge-sensitive infrared phototransistor (CSIP). In our s-SNOM, photons scattered by a tungsten probe are collected by an objective of the confocal LWIR microscope and are finally detected by the CSIP. To suppress the far-field background, we vertically modulated the probe and demodulated the signal with a lock-in amplifier. With the s-SNOM, a clear passive image of 3 µm pitch Au/SiC gratings was successfully obtained and the spatial resolution was estimated to be 60 nm (λ/240). The radiation from Au and GaAs was suggested to be due to thermally excited charge/current fluctuations and surface phonons, respectively. This s-SNOM has the potential to observe mesoscopic phenomena such as molecular motions, biomolecular protein interactions and semiconductor conditions in the future

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

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.

Adaptive optics plug-and-play setup for high-resolution microscopes with multi-actuator adaptive lens

Science.gov (United States)

Quintavalla, M.; Pozzi, P.; Verhaegen, Michelle; Bijlsma, Hielke; Verstraete, Hans; Bonora, S.

2018-02-01

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.

Towards modeling of cardiac micro-structure with catheter-based confocal microscopy: a novel approach for dye delivery and tissue characterization.

Science.gov (United States)

Lasher, Richard A; Hitchcock, Robert W; Sachse, Frank B

2009-08-01

This work presents a methodology for modeling of cardiac tissue micro-structure. The approach is based on catheter-based confocal imaging systems, which are emerging as tools for diagnosis in various clinical disciplines. A limitation of these systems is that a fluorescent marker must be available in sufficient concentration in the imaged region. We introduce a novel method for the local delivery of fluorescent markers to cardiac tissue based on a hydro-gel carrier brought into contact with the tissue surface. The method was tested with living rabbit cardiac tissue and applied to acquire three-dimensional image stacks with a standard inverted confocal microscope and two-dimensional images with a catheter-based confocal microscope. We processed these image stacks to obtain spatial models and quantitative data on tissue microstructure. Volumes of atrial and ventricular myocytes were 4901 +/- 1713 and 10 299 +/-3598 mum (3) (mean+/-sd), respectively. Atrial and ventricular myocyte volume fractions were 72.4 +/-4.7% and 79.7 +/- 2.9% (mean +/-sd), respectively. Atrial and ventricular myocyte density was 165 571 +/- 55 836 and 86 957 +/- 32 280 cells/mm (3) (mean+/-sd), respectively. These statistical data and spatial descriptions of tissue microstructure provide important input for modeling studies of cardiac tissue function. We propose that the described methodology can also be used to characterize diseased tissue and allows for personalized modeling of cardiac tissue.

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.

'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.

Estudio comparativo entre microscopía confocal y microscopía especular en la valoración del endotelio en córneas con distrofia de Fuchs

OpenAIRE

Charafeddin, Wissam

2011-01-01

La distròfia endotelial de Fuchs es caracteritza per la formació de guttas endotelials i en estadis avançats pot induir edema corneal i pèrdua d'agudesa visual. A diferència del microscopi especular, el microscopi confocal té un disseny que permet evitar la llum aberrant causada per l'edema corneal o opacitats estromals. Comparant ambdues probes en la valoració de l'endoteli corneal en pacients amb distròfia de Fuchs, s'observa millor qualitat d'imatge per microscòpia confocal tot i que no es...

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.

Improving axial resolution in confocal microscopy with new high refractive index mounting media.

Science.gov (United States)

Fouquet, Coralie; Gilles, Jean-François; Heck, Nicolas; Dos Santos, Marc; Schwartzmann, Richard; Cannaya, Vidjeacoumary; Morel, Marie-Pierre; Davidson, Robert Stephen; Trembleau, Alain; Bolte, Susanne

2015-01-01

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.

Improving axial resolution in confocal microscopy with new high refractive index mounting media.

Directory of Open Access Journals (Sweden)

Coralie Fouquet

Full Text Available Resolution, high signal intensity and elevated signal to noise ratio (SNR are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF, a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.

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...

Visualizing 3-D microscopic specimens

Science.gov (United States)

Forsgren, Per-Ola; Majlof, Lars L.

1992-06-01

The confocal microscope can be used in a vast number of fields and applications to gather more information than is possible with a regular light microscope, in particular about depth. Compared to other three-dimensional imaging devices such as CAT, NMR, and PET, the variations of the objects studied are larger and not known from macroscopic dissections. It is therefore important to have several complementary ways of displaying the gathered information. We present a system where the user can choose display techniques such as extended focus, depth coding, solid surface modeling, maximum intensity and other techniques, some of which may be combined. A graphical user interface provides easy and direct control of all input parameters. Motion and stereo are available options. Many three- dimensional imaging devices give recordings where one dimension has different resolution and sampling than the other two which requires interpolation to obtain correct geometry. We have evaluated algorithms with interpolation in object space and in projection space. There are many ways to simplify the geometrical transformations to gain performance. We present results of some ways to simplify the calculations.

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

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.

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)

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

Confocal microscopy and spectroscopy of nanocrystals on a high-Q microsphere resonator

International Nuclear Information System (INIS)

Goetzinger, S; Menezes, L de S; Benson, O; Talapin, D V; Gaponik, N; Weller, H; Rogach, A L; Sandoghdar, V

2004-01-01

We report on experiments where we used a home-made confocal microscope to excite single nanocrystals on a high-Q microsphere resonator. In that way spectra of an individual quantum emitter could be recorded. The Q factor of the microspheres coated with nanocrystals was still up to 10 9 . We also demonstrate the use of a prism coupler as a well-defined output port to collect the fluorescence of an ensemble of nanocrystals coupled to whispering-gallery modes

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

International Nuclear Information System (INIS)

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

2008-01-01

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

Proper alignment of the microscope.

Science.gov (United States)

Rottenfusser, Rudi

2013-01-01

The light microscope is merely the first element of an imaging system in a research facility. Such a system may include high-speed and/or high-resolution image acquisition capabilities, confocal technologies, and super-resolution methods of various types. Yet more than ever, the proverb "garbage in-garbage out" remains a fact. Image manipulations may be used to conceal a suboptimal microscope setup, but an artifact-free image can only be obtained when the microscope is optimally aligned, both mechanically and optically. Something else is often overlooked in the quest to get the best image out of the microscope: Proper sample preparation! The microscope optics can only do its job when its design criteria are matched to the specimen or vice versa. The specimen itself, the mounting medium, the cover slip, and the type of immersion medium (if applicable) are all part of the total optical makeup. To get the best results out of a microscope, understanding the functions of all of its variable components is important. Only then one knows how to optimize these components for the intended application. Different approaches might be chosen to discuss all of the microscope's components. We decided to follow the light path which starts with the light source and ends at the camera or the eyepieces. To add more transparency to this sequence, the section up to the microscope stage was called the "Illuminating Section", to be followed by the "Imaging Section" which starts with the microscope objective. After understanding the various components, we can start "working with the microscope." To get the best resolution and contrast from the microscope, the practice of "Koehler Illumination" should be understood and followed by every serious microscopist. Step-by-step instructions as well as illustrations of the beam path in an upright and inverted microscope are included in this chapter. A few practical considerations are listed in Section 3. Copyright © 2013 Elsevier Inc. All rights

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.)

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

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...

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.

Quantification of whey in fluid milk using confocal Raman microscopy and artificial neural network.

Science.gov (United States)

Alves da Rocha, Roney; Paiva, Igor Moura; Anjos, Virgílio; Furtado, Marco Antônio Moreira; Bell, Maria José Valenzuela

2015-06-01

In this work, we assessed the use of confocal Raman microscopy and artificial neural network as a practical method to assess and quantify adulteration of fluid milk by addition of whey. Milk samples with added whey (from 0 to 100%) were prepared, simulating different levels of fraudulent adulteration. All analyses were carried out by direct inspection at the light microscope after depositing drops from each sample on a microscope slide and drying them at room temperature. No pre- or posttreatment (e.g., sample preparation or spectral correction) was required in the analyses. Quantitative determination of adulteration was performed through a feed-forward artificial neural network (ANN). Different ANN configurations were evaluated based on their coefficient of determination (R2) and root mean square error values, which were criteria for selecting the best predictor model. In the selected model, we observed that data from both training and validation subsets presented R2>99.99%, indicating that the combination of confocal Raman microscopy and ANN is a rapid, simple, and efficient method to quantify milk adulteration by whey. Because sample preparation and postprocessing of spectra were not required, the method has potential applications in health surveillance and food quality monitoring. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

In vivo confocal Raman spectroscopy of the human cornea.

Science.gov (United States)

Bauer, N J; Hendrikse, F; March, W F

1999-07-01

To investigate the feasibility of a confocal Raman spectroscopic technique for the noninvasive assessment of corneal hydration in vivo in two legally blind subjects. A laser beam (632.8 nm; 15 mJ) was maintained on the cornea by using a microscope objective lens (x25 magnification, NA = 0.5, f = 10 mm) both for focusing the incident light as well as collecting the Raman backscattered light, in a 180 degrees backscatter configuration. An optical fiber, acting as the confocal pinhole for elimination of light from out-of-focus places, was coupled to a spectrometer that dispersed the collected light onto a sensitive array detector for rapid spectral data acquisition over a range from 2,890 to 3,590/cm(-1). Raman spectra were recorded from the anterior 100-150 microm of the cornea over a period before and after topical application of a mild dehydrating solution. The ratio between the amplitudes of the signals at 3,400/cm(-1) (OH-vibrational mode of water) and 2,940/cm(-1) (CH-vibrational mode of proteins) was used as a measure for corneal hydration. High signal-to-noise ratio (SNR = 25) Raman spectra were obtained from the human corneas by using 15 mJ of laser light energy. Qualitative changes in the hydration of the anteriormost part of the corneas could be observed as a result of the dehydrating agent. With adequate improvements in system safety, confocal Raman spectroscopy could potentially be applied clinically as a noninvasive tool for the assessment of corneal hydration in vivo.

Intracellular photoinduced oxidative stress by zinc phthalocyanine photosensitization: a study of the early events in real time using confocal microscopy

Science.gov (United States)

Alexandratou, Eleni; Yova, Dido; Handris, Panagiotis; Kletsas, Dimitris; Loukas, Spyros

2003-10-01

Oxidative stress has been implicated in several biological and pathological aspects. Reactive oxygen species (ROS) have been proposed to act as signal transduction molecules activating reactions leading to cell rescue or to cell apoptosis/necrosis. In the present study, oxidative stress was induced by photosensitization of zinc phthalocyanine (ZnPc) in human fibroblasts using a photodynamic dose that did not lead to apoptosis or necrosis. The induction of oxidative stress was performed at the microscope stage in preassigned time. The cascade of phenomena evoked was studied in real time and at the single cell level using confocal laser scanning microscopy. Using specific vital fluorescent probes, alterations induced by oxidative stress in mitochondria membrane potential, in intracellular pH and in calcium concentration were recorded. Image processing and analysis techniques were used to quantify the observed changes. Subcellular localization of the photosensitizer was studied in order to determine the primary and immediate ROS target. It was found that ZnPc is mainly localized in the mitochondria region.

[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.

Automated Microscopy: Macro Language Controlling a Confocal Microscope and its External Illumination: Adaptation for Photosynthetic Organisms

Czech Academy of Sciences Publication Activity Database

Steinbach, Gabor; Kaňa, Radek

2016-01-01

Roč. 22, č. 2 (2016), s. 258-263 ISSN 1431-9276 R&D Projects: GA ČR GAP501/12/0304; GA MŠk EE2.3.30.0059; GA MŠk ED2.1.00/03.0110; GA MŠk(CZ) LO1416 Institutional support: RVO:61388971 Keywords : automated microscopy * remote controlled microscopy * confocal microscopy Subject RIV: BH - Optics, Masers, Lasers Impact factor: 1.891, year: 2016

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.

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.

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.

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.

Ex Vivo Confocal Spectroscopy of Autofluorescence in Age-Related Macular Degeneration.

Directory of Open Access Journals (Sweden)

Joel Kaluzny

Full Text Available We investigated the autofluorescence (AF signature of the microscopic features of retina with age-related macular degeneration (AMD using 488 nm excitation.The globes of four donors with AMD and four age-matched controls were embedded in paraffin and sectioned through the macula. Sections were excited using a 488 nm argon laser, and the AF emission was captured using a laser scanning confocal microscope (496-610 nm, 6 nm resolution. The data cubes were then analyzed to compare peak emission spectra between the AMD and the controls. Microscopic features, including individual lipofuscin and melanolipofuscin granules, Bruch's Membrane, as well macroscopic features, were considered.Overall, the AMD eyes showed a trend of blue-shifted emission peaks compared with the controls. These differences were statistically significant when considering the emission of the combined RPE/Bruch's Membrane across all the tissue cross-sections (p = 0.02.The AF signatures of ex vivo AMD RPE/BrM show blue-shifted emission spectra (488 nm excitation compared with the control tissue. The magnitude of these differences is small (~4 nm and highlights the potential challenges of detecting these subtle spectral differences in vivo.

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

Directory of Open Access Journals (Sweden)

Attila Bokros

2013-08-01

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

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

Directory of Open Access Journals (Sweden)

Zulema Gómez Castillo

2012-06-01

disease notified from May 2007 to May 2008 were considered. Results: The 100 studied eyes came from apparently healthy patients, and the pachymetry values of 64 of them were above the average. Since they were healthy corneas, the 3 types of epithelial cells as well as the keratocytes were present in almost all the patients and at different depths of the corneal stroma, respectively. Most of the studied eyes had endothelial cell counts above 2 500, a figure within the normal range, and there were nerve fibers in each of their layers. Conclusion: Confocal microscope is a new tool that allows in vivo observation of corneal histology and thus supplements conventional biomicroscopy observations. It contributes to better understanding of corneal pathology, with a view to prophylactically and therapeutically acting upon corneal pathologies during their follow-up and evolution.

Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvatochromism in the rubber-producing plant, Eucommia ulmoides Oliver.

Science.gov (United States)

Nakazawa, Yoshihisa; Takeda, Tsuyoshi; Suzuki, Nobuaki; Hayashi, Tatsushi; Harada, Yoko; Bamba, Takeshi; Kobayashi, Akio

2013-09-01

A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry.

Minimizing inter-microscope variability in dental microwear texture analysis

Science.gov (United States)

Arman, Samuel D.; Ungar, Peter S.; Brown, Christopher A.; DeSantis, Larisa R. G.; Schmidt, Christopher; Prideaux, Gavin J.

2016-06-01

A common approach to dental microwear texture analysis (DMTA) uses confocal profilometry in concert with scale-sensitive fractal analysis to help understand the diets of extinct mammals. One of the main benefits of DMTA over other methods is the repeatable, objective manner of data collection. This repeatability, however, is threatened by variation in results of DMTA of the same dental surfaces yielded by different microscopes. Here we compare DMTA data of five species of kangaroos measured on seven profilers of varying specifications. Comparison between microscopes confirms that inter-microscope differences are present, but we show that deployment of a number of automated treatments to remove measurement noise can help minimize inter-microscope differences. Applying these same treatments to a published hominin DMTA dataset shows that they alter some significant differences between dietary groups. Minimising microscope variability while maintaining interspecific dietary differences requires then that these factors are balanced in determining appropriate treatments. The process outlined here offers a solution for allowing comparison of data between microscopes, which is essential for ongoing DMTA research. In addition, the process undertaken, including considerations of other elements of DMTA protocols also promises to streamline methodology, remove measurement noise and in doing so, optimize recovery of a reliable dietary signature.

Imaging the Microscopic Structure of Shear Thinning and Thickening Colloidal Suspensions

KAUST Repository

Cheng, X.

2011-09-01

The viscosity of colloidal suspensions varies with shear rate, an important effect encountered in many natural and industrial processes. Although this non-Newtonian behavior is believed to arise from the arrangement of suspended particles and their mutual interactions, microscopic particle dynamics are difficult to measure. By combining fast confocal microscopy with simultaneous force measurements, we systematically investigate a suspension\\'s structure as it transitions through regimes of different flow signatures. Our measurements of the microscopic single-particle dynamics show that shear thinning results from the decreased relative contribution of entropic forces and that shear thickening arises from particle clustering induced by hydrodynamic lubrication forces. This combination of techniques illustrates an approach that complements current methods for determining the microscopic origins of non-Newtonian flow behavior in complex fluids.

Confocal microscopy of corneal stroma and endothelium after LASIK and PRK.

Science.gov (United States)

Amoozadeh, Javad; Aliakbari, Soheil; Behesht-Nejad, Amir-Houshang; Seyedian, Mohammad-Amin; Rezvan, Bijan; Hashemi, Hassan

2009-10-01

To compare with confocal microscopy the changes in stromal keratocyte density and endothelial cell count due to photorefractive keratectomy (PRK) and LASIK. In this prospective study, 32 eyes (16 myopic patients) were examined with the NIDEK Confoscan 3 confocal microscope before and 6 months after PRK and LASIK. The preoperative mean myopia was -2.85+/-0.99 diopters (D) (range: -1.00 to -4.00 D) in 24 eyes that underwent PRK and -2.94+/-0.96 D (range: -2.00 to -4.25 D) in 8 eyes that underwent LASIK. Keratocyte density in the anterior and posterior stroma and the endothelial cell count were measured. Statistically significant changes were assessed using the t test. PPRK group. Postoperatively, the percentages were 52.96+/-7.55 and 53.34+/-10.2, respectively. Six months postoperatively, keratocyte density changed by 367.12+/-103.35 cells/mm(2) (34.7% reduction) in the anterior stroma (P.05) for the LASIK group. In the PRK group, these values were 319.71+/-83.45 cells/mm(2) (31.13% reduction) in the anterior stroma (P.05). The changes in keratocyte densities were not statistically significant between groups (P>.05). The mean number of keratocytes decreased by 37.2% in the retroablation zone of the LASIK group (PPRK groups (P>.05). Copyright 2009, SLACK Incorporated.

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

Directory of Open Access Journals (Sweden)

KA. Al-Salihi

2009-12-01

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

Efficacy of 4 Irrigation Protocols in Killing Bacteria Colonized in Dentinal Tubules Examined by a Novel Confocal Laser Scanning Microscope Analysis.

Science.gov (United States)

Azim, Adham A; Aksel, Hacer; Zhuang, Tingting; Mashtare, Terry; Babu, Jegdish P; Huang, George T-J

2016-06-01

The aim of this study was to determine the efficiency of 4 irrigation systems in eliminating bacteria in root canals, particularly in dentinal tubules. Roots of human teeth were prepared to 25/04, autoclaved, and inoculated with Enterococcus faecalis for 3 weeks. Canals were then disinfected by (1) standard needle irrigation, (2) sonically agitating with EndoActivator, (3) XP Endo finisher, or (4) erbium:yttrium aluminum garnet laser (PIPS) (15 roots/group). The bacterial reduction in the canal was determined by MTT assays. For measuring live versus dead bacteria in the dentinal tubules (4 teeth/group), teeth were split open and stained with LIVE/DEAD BackLight. Coronal, middle, and apical thirds of the canal dentin were scanned by using a confocal laser scanning microscope (CLSM) to determine the ratio of dead/total bacteria in the dentinal tubules at various depths. All 4 irrigation protocols significantly eliminated bacteria in the canal, ranging from 89.6% to 98.2% reduction (P bacteria in the coronal, middle, and apical segments at 50-μm depth. On the other hand, PIPS had the greatest bacterial killing efficiency at the 150-μm depth in all 3 root segments. XP Endo appears to be more efficient than other 3 techniques in disinfecting the main canal space and up to 50 μm deep into the dentinal tubules. PIPS appears to be most effective in killing the bacteria deep in the dentinal tubules. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout.

Science.gov (United States)

McConnell, Gail; Trägårdh, Johanna; Amor, Rumelo; Dempster, John; Reid, Es; Amos, William Bradshaw

2016-09-23

Current optical microscope objectives of low magnification have low numerical aperture and therefore have too little depth resolution and discrimination to perform well in confocal and nonlinear microscopy. This is a serious limitation in important areas, including the phenotypic screening of human genes in transgenic mice by study of embryos undergoing advanced organogenesis. We have built an optical lens system for 3D imaging of objects up to 6 mm wide and 3 mm thick with depth resolution of only a few microns instead of the tens of microns currently attained, allowing sub-cellular detail to be resolved throughout the volume. We present this lens, called the Mesolens, with performance data and images from biological specimens including confocal images of whole fixed and intact fluorescently-stained 12.5-day old mouse embryos.

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.

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...

Comparison of skin responses from macroscopic and microscopic UV challenges

Science.gov (United States)

Seo, InSeok; Bargo, Paulo R.; Chu, Melissa; Ruvolo, Eduardo; Kollias, Nikiforos

2011-03-01

The minimal erythema dose induced by solar-simulated radiation is a useful measure of UV sensitivity of skin. Most skin phototests have been conducted by projecting a flat field of UV radiation onto the skin in an area greater than 15 cm × 15 cm with an increment of radiation doses. In this study, we investigated the responses of human skin to solar-simulated radiation of different field sizes. Twelve human subjects of skin phototype I-IV were exposed to solar-simulated radiation (SSR) on their upper inner arm or on their lower back with a series of doses in increments of 20% in order to determine the threshold dose to induce a minimal perceptible erythema response (MED). Each dose was delivered with a liquid light guide (8 mm diameter on the back or 6 mm on the upper inner arm) and with quartz optical fibers of 200 μm diameter. The resulting skin responses were evaluated visually and investigated with a reflectance confocal microscope and imaging. The erythema response to the microscopic challenge was always diffuse with no clear boundaries extending to several times the exposed site diameter at doses greater than 2 MED. The skin returned to normal appearance from the microscopic challenge after two weeks of exposure while change in appearance for the larger areas persisted for several weeks to months. This new modality of testing provides the possibility to study skin at the microscopic level with a rapid recovery following challenge.

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.

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.

Development of HiLo Microscope and its use in In-Vivo Applications

Science.gov (United States)

Patel, Shreyas J.

The functionality of achieving optical sectioning in biomedical research is invaluable as it allows for visualization of a biological sample at different depths while being free of background scattering. Most current microscopy techniques that offer optical sectioning, unfortunately, require complex instrumentation and thus are generally costly. HiLo microscopy, on the other hand, offers the same functionality and advantage at a relatively low cost. Hence, the work described in this thesis involves the design, build, and application of a HiLo microscope. More specifically, a standalone HiLo microscope was built in addition to implementing HiLo microscopy on a standard fluorescence microscope. In HiLo microscopy, optical sectioning is achieved by acquiring two different types of images per focal plane. One image is acquired under uniform illumination and the other is acquired under speckle illumination. These images are processed using an algorithm that extracts in-focus information and removes features and glare that occur as a result of background fluorescence. To show the benefits of the HiLo microscopy, several imaging experiments on various samples were performed under a HiLo microscope and compared against a traditional fluorescence microscope and a confocal microscope, which is considered the gold standard in optical imaging. In-vitro and ex-vivo imaging was performed on a set of pollen grains, and optically cleared mouse brain and heart slices. Each of these experiments showed great reduction in background scattering at different depths under HiLo microscopy. More importantly, HiLo imaging of optically cleared heart slice demonstrated emergence of different vasculature at different depths. Reduction of out-of-focus light increased the spatial resolution and allowed better visualization of capillary vessels. Furthermore, HiLo imaging was tested in an in-vivo model of a rodent dorsal window chamber model. When imaging the same sample under confocal microscope

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

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

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

Directory of Open Access Journals (Sweden)

Elif Demirkilinc Biler

2015-01-01

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

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

Color Laser Microscope

Science.gov (United States)

Awamura, D.; Ode, T.; Yonezawa, M.

1987-04-01

A color laser microscope utilizing a new color laser imaging system has been developed for the visual inspection of semiconductors. The light source, produced by three lasers (Red; He-Ne, Green; Ar, Blue; He-Cd), is deflected horizontally by an AOD (Acoustic Optical Deflector) and vertically by a vibration mirror. The laser beam is focused in a small spot which is scanned over the sample at high speed. The light reflected back from the sample is reformed to contain linear information by returning to the original vibration mirror. The linear light is guided to the CCD image sensor where it is converted into a video signal. Individual CCD image sensors are used for each of the three R, G, or B color image signals. The confocal optical system with its laser light source yields a color TV monitor image with no flaring and a much sharper resolution than that of the conventional optical microscope. The AOD makes possible a high speed laser scan and a NTSC or PAL TV video signal is produced in real time without any video memory. Since the light source is composed of R, G, and B laser beams, color separation superior to that of white light illumination is achieved. Because of the photometric linearity of the image detector, the R, G, and B outputs of the system are most suitably used for hue analysis. The CCD linear image sensors in the optical system produce no geometrical distortion, and good color registration is available principally. The output signal can be used for high accuracy line width measuring. The many features of the color laser microscope make it ideally suited for the visual inspection of semiconductor processing. A number of these systems have already been installed in such a capacity. The Color Laser Microscope can also be a very useful tool for the fields of material engineering and biotechnology.

Influence of operating microscope in the sealing of cervical perforations.

Science.gov (United States)

Schmidt, Bruna Schwingel; Zaccara, Ivana Maria; Reis Só, Marcus Vinícius; Kuga, Milton Carlos; Palma-Dibb, Regina Guenka; Kopper, Patrícia Maria Poli

2016-01-01

Accidental root canal perforations are among the main complications of endodontic treatment. This study evaluated the influence of operating microscope (OM) in the marginal adaptation of mineral trioxide aggregate (MTA) (Angelus(®)) and glass ionomer (Vitremer) inserted into cervical perforations. Perforations were made in the cervical third of the buccal wall of the root canal in mandibular incisors. Next, the teeth were divided into four groups (N = 10): MG - MTA without OM; VG - Vitremer without OM; MOMG - MTA with OM; VOMG - Vitremer with OM. The perforations were sealed according to the group and the teeth were prepared for analysis by confocal laser scanning microscope. Images of perforation region (1,024×) were made and the gap presented by the materials was measured using the Image J program. LEXT OLS4100 three dimensional (3D) measuring laser microscope measured the volumetric misfit. Data of gap were analyzed by Kruskal-Wallis and Dunn's tests. Analysis of variance (ANOVA) and Tukey's tests compared the volumetric misfits. The results showed lower volume and gap in the interface dentin/material in VOMG compared to the other groups (P < 0.05). The use of OM improved the quality of cervical perforations sealed with Vitremer, being indicated in clinical situations of iatrogenic cervical perforations.

InGaP - CREATE portal | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available : Fixed mouse brain sections were fluorescent-labeled and observed by confocal laser scanning microscope usi...ted GFP-fusion KIAA/mKIAA in HEK293 cells were observed by fluorescent microscope...ope (neocortex 1) Observation by Confocal laser scanning microscope (neocortex 1) C...onfocal laser scanning microscope (neocortex 2) Observation by Confocal laser scanning microscope (neocortex... 2) Confocal laser scanning microscope (hippocampus) Observation by Confocal laser scanning microscope

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.

A confocal laser scanning microscopic study on thermoresponsive

Indian Academy of Sciences (India)

Monodisperse poly(N -isopropylacrylamide) (PNIPAM) particles loaded with cadmium telluride (CdTe) quantum dots (QDs) of two different sizes (4.7 nm and 5.6 nm) were synthesized in aqueous medium by bonding the capping agent on the quantum dots to the amide groups of PNIPAM and incubating the samples at 45° ...

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.

Confocal fluorescence microscopy to evaluate changes in adipocytes in the tumor microenvironment associated with invasive ductal carcinoma and ductal carcinoma in situ.

Science.gov (United States)

Dobbs, Jessica L; Shin, Dongsuk; Krishnamurthy, Savitri; Kuerer, Henry; Yang, Wei; Richards-Kortum, Rebecca

2016-09-01

Adipose tissue is a dynamic organ that provides endocrine, inflammatory and angiogenic factors, which can assist breast carcinoma cells with invasion and metastasis. Previous studies have shown that adipocytes adjacent to carcinoma, known as cancer-associated adipocytes, undergo extensive changes that correspond to an "activated phenotype," such as reduced size relative to adipocytes in non-neoplastic breast tissue. Optical imaging provides a tool that can be used to characterize adipocyte morphology and other features of the tumor microenvironment. In this study, we used confocal fluorescence microscopy to acquire images of freshly excised breast tissue stained topically with proflavine. We developed a computerized algorithm to identify and quantitatively measure phenotypic properties of adipocytes located adjacent to and far from normal collagen, ductal carcinoma in situ and invasive ductal carcinoma. Adipocytes were measured in confocal fluorescence images of fresh breast tissue collected from 22 patients. Results show that adipocytes adjacent to neoplastic tissue margins have significantly smaller area compared to adipocytes far from the margins of neoplastic lesions and compared to adipocytes adjacent to non-neoplastic collagenous stroma. These findings suggest that confocal microscopic images can be utilized to evaluate phenotypic properties of adipocytes in breast stroma which may be useful in defining alterations in microenvironment that may aid in the development and progression of neoplastic lesions. © 2016 UICC.

Comparative study of image contrast in scanning electron microscope and helium ion microscope.

Science.gov (United States)

O'Connell, R; Chen, Y; Zhang, H; Zhou, Y; Fox, D; Maguire, P; Wang, J J; Rodenburg, C

2017-12-01

Images of Ga + -implanted amorphous silicon layers in a 110 n-type silicon substrate have been collected by a range of detectors in a scanning electron microscope and a helium ion microscope. The effects of the implantation dose and imaging parameters (beam energy, dwell time, etc.) on the image contrast were investigated. We demonstrate a similar relationship for both the helium ion microscope Everhart-Thornley and scanning electron microscope Inlens detectors between the contrast of the images and the Ga + density and imaging parameters. These results also show that dynamic charging effects have a significant impact on the quantification of the helium ion microscope and scanning electron microscope contrast. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Electron microscope studies

International Nuclear Information System (INIS)

Crewe, A.V.; Kapp, O.H.

1992-01-01

This is a report covering the research performed in the Crewe laboratory between 1964 and 1992. Because of limitations of space we have provided relatively brief summaries of the major research directions of the facility during these years. A complete bibliography has been included and we have referenced groups of pertinent publications at the beginning of each section. This report summarizes our efforts to develop better electron microscopes and chronicles many of the experimental programs, in materials science and biology, that acted both as a stimulus to better microscope design and also as a testing ground for many instrumental innovations

Electron microscope studies

Energy Technology Data Exchange (ETDEWEB)

Crewe, A.V.; Kapp, O.H.

1992-07-01

This is a report covering the research performed in the Crewe laboratory between 1964 and 1992. Because of limitations of space we have provided relatively brief summaries of the major research directions of the facility during these years. A complete bibliography has been included and we have referenced groups of pertinent publications at the beginning of each section. This report summarizes our efforts to develop better electron microscopes and chronicles many of the experimental programs, in materials science and biology, that acted both as a stimulus to better microscope design and also as a testing ground for many instrumental innovations.

Analysis of polymer grafted inside the porous hydrogel using confocal laser scanning microscopy

Directory of Open Access Journals (Sweden)

2007-04-01

Full Text Available Graft polymerization of glycidyl methacrylate onto the pore surface of polyacrylamide macroporous gel was implemented in DMSO-aqueous solution using diperiodatocuprate(III complexes as an initiator. The grafting densities up to 410% were achieved. The graft polymerization was confirmed by gravimetrical methods and FTIR. The graft polymerization of polymer inside the pores of the macroporous gel resulted in increased flow resistance through the gel matrix. The distribution of grafted polymer on the gel pore surface material was studied by scanning electron microscopy (SEM and confocal laser scanning microscopy (CLSM. CLSM is an alternative method for studying morphology of gel surface with grafted polymer having the advantages over the SEM allowing to investigate the distribution of grafted polymer inside the hydrogel in a native hydrated state. The microscopic techniques demonstrated uneven distribution of the grafted polymer inside the gel pores as a result of initiating the graft polymerization by insoluble initiator deposited on the pore surface.

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.

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.

A multiphoton laser scanning microscope setup for transcranial in vivo brain imaging on mice

Science.gov (United States)

Nase, Gabriele; Helm, P. Johannes; Reppen, Trond; Ottersen, Ole Petter

2005-12-01

We describe a multiphoton laser scanning microscope setup for transcranial in vivo brain imaging in mice. The modular system is based on a modified industrial standard Confocal Scanning Laser Microscope (CSLM) and is assembled mainly from commercially available components. A special multifunctional stage, which is optimized for both laser scanning microscopic observation and preparative animal surgery, has been developed and built. The detection unit includes a highly efficient photomultiplier tube installed in a Peltier-cooled thermal box shielding the detector from changes in room temperature and from distortions caused by external electromagnetic fields. The images are recorded using a 12-bit analog-to-digital converter. Depending on the characteristics of the staining, individual nerve cells can be imaged down to at least 100μm below the intact cranium and down to at least 200μm below the opened cranium.

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

Degeneration process of fungiform taste buds after severing the human chorda tympani nerve--observation by confocal laser scanning microscopy.

Science.gov (United States)

Saito, Takehisa; Ito, Tetsufumi; Ito, Yumi; Kato, Yuji; Manabe, Yasuhiro; Narita, Norihiko

2015-03-01

To elucidate the degeneration process of fungiform taste buds after severing the chorda tympani nerve (CTN) by confocal laser scanning microscopy in vivo. Prospective study. University hospital. Seven consecutive patients whose CTN was severed during tympanoplasty for middle ear cholesteatoma. Diagnostic. Preoperative and postoperative gustatory functions were assessed by electrogustometry (EGM). An average of 10 fungiform papillae (FP) in the midlateral region of the tongue were periodically observed, and the number of taste buds was counted using a confocal laser microscope. Among them, 2 to 3 reference FPs were selected based on the typical form of the FP or characteristic arrangements of taste pores. Observation was performed before surgery, 1 or 2 days after surgery, 2 or 3 times a week until 2 weeks after surgery, once a week between 2 and 4 weeks, and every 2 to 4 weeks thereafter until all taste buds had disappeared. EGM thresholds showed no response within 1 month after surgery in all patients. The initial change in the degeneration process was the disappearance of taste pores. The surface of taste buds became covered with epithelium. Finally, taste buds themselves atrofied and disappeared. The time course of degeneration differed depending upon individuals, each FP, and each taste bud. By employing the generalized linear mixed model under the Poisson distribution, it was calculated that all taste buds would disappear at around 50 days after surgery. Confocal laser scanning microscopy was useful for clarifying the degeneration process of fungiform taste buds.

Optical Computed-Tomographic Microscope for Three-Dimensional Quantitative Histology

Directory of Open Access Journals (Sweden)

Ravil Chamgoulov

2004-01-01

Full Text Available A novel optical computed‐tomographic microscope has been developed allowing quantitative three‐dimensional (3D imaging and analysis of fixed pathological material. Rather than a conventional two‐dimensional (2D image, the instrument produces a 3D representation of fixed absorption‐stained material, from which quantitative histopathological features can be measured more accurately. The accurate quantification of these features is critically important in disease diagnosis and the clinical classification of cancer. The system consists of two high NA objective lenses, a light source, a digital spatial light modulator (DMD, by Texas Instrument, an x–y stage, and a CCD detector. The DMD, positioned at the back pupil‐plane of the illumination objective, is employed to illuminate the specimen with parallel rays at any desired angle. The system uses a modification of the convolution backprojection algorithm for reconstruction. In contrast to fluorescent images acquired by a confocal microscope, this instrument produces 3D images of absorption stained material. Microscopic 3D volume reconstructions of absorption‐stained cells have been demonstrated. Reconstructed 3D images of individual cells and tissue can be cut virtually with the distance between the axial slices less than 0.5 μm.

Electric field and energy of a point electric charge between confocal hyperbolaidal electrodes

Energy Technology Data Exchange (ETDEWEB)

Ley-Koo, E. [Universidad Nacional Autonoma de Mexico, Mexico, D. F. (Mexico)

2001-06-01

The electric potential and intensity field, as well as the energy of a point electric charge between confocal hyperboloidal electrodes is evaluated as a superposition of prolate spheroidal harmonics using the Green-function technique. This study is motivated by the need to model the electric field between the tip and the sample in a scanning tunnelling microscope, and it can also be applied to a conductor-insulator-conductor junction. [Spanish] Los campos de potencial y de intensidad electrica, asi como la energia de una carga electrica puntual entre electrodos hiperboloidales confocales se evaluan como superposiciones de armonicos esferoidales prolatos usando la tecnica de la funcion de Green. Este estudio ha sido motivado por la necesidad de modelar el campo electrico entre la punta y la muestra de un microscopio de tunelamiento y barrido, y se puede aplicar tambien a una union de conductor-aislante-conductor.

Embryological study of Herminium monorchis (Orchidaceae) using confocal scanning laser microscopy

International Nuclear Information System (INIS)

Fredrikson, M.

1990-01-01

The embryology of Herminium monorchis (Orchidaceae) was studied using confocal scanning laser microscopy (CSLM), a new technique for embryological studies. This technique may contribute new information to plant embryology. Herminium monorchis has a monosporic embryo sac development. The mature embryo sac is 8-nucleate. Two integuments, both 2-layered, are formed, but only the inner takes part in formation of the micropyle. Double fertilization takes place. The primary endosperm nucleus does not divide, but remains alive at least at the 3-celled stage of embryo development. The three antipodals do not show any sign of degeneration at this stage. (author)

Attempt of correlative observation of morphological synaptic connectivity by combining confocal laser-scanning microscope and FIB-SEM for immunohistochemical staining technique.

Science.gov (United States)

Sonomura, Takahiro; Furuta, Takahiro; Nakatani, Ikuko; Yamamoto, Yo; Honma, Satoru; Kaneko, Takeshi

2014-11-01

Ten years have passed since a serial block-face scanning electron microscopy (SBF-SEM) method was developed [1]. In this innovative method, samples were automatically sectioned with an ultramicrotome placed inside a scanning electron microscope column, and the block surfaces were imaged one after another by SEM to capture back-scattered electrons. The contrast-inverted images obtained by the SBF-SEM were very similar to those acquired using conventional TEM. SFB-SEM has made easy to acquire image stacks of the transmission electron microscopy (TEM) in the mesoscale, which is taken with the confocal laser-scanning microcopy(CF-LSM).Furthermore, serial-section SEM has been combined with the focused ion beam (FIB) milling method [2]. FIB-incorporated SEM (FIB-SEM) has enabled the acquisition of three-dimensional images with a higher z-axis resolution com- pared to ultramicrotome-equipped SEM.We tried immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in CF-LSM. Dendrites of neurons in the rat neostriatum were visualized using a recombinant viral vector. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively.We showed that conventional immuno-cytochemical staining for TEM was applicable to FIB-SEM. Furthermore, several synaptic contacts, which were thought to exist on the basis of CF-LSM findings, were confirmed with FIB-SEM, revealing the usefulness of the combined method of CF-LSM and FIB-SEM. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluation of the therapeutic results of actinic keratosis treated with topical 5% fluorouracil by reflectance confocal laser microscopy: preliminary study*

Science.gov (United States)

Ishioka, Priscila; Maia, Marcus; Rodrigues, Sarita Bartholomei; Marta, Alessandra Cristina; Hirata, Sérgio Henrique

2015-01-01

Topical treatment for actinic keratosis with 5% fluorouracil has a recurrence rate of 54% in 12 months of follow-up. This study analyzed thirteen actinic keratoses on the upper limbs through confocal microscopy, at the time of clinical diagnosis and after 4 weeks of treatment with fluorouracil. After the treatment was established and evidence of clinical cure was achieved, in two of the nine actinic keratoses, confocal microscopy enabled visualization of focal areas of atypical honeycomb pattern in the epidermis indicating therapeutic failure. Preliminary data suggest the use of confocal microscopy as a tool for diagnosis and therapeutic control of actinic keratosis. PMID:26131881

Observation of regenerated fungiform taste buds after severing the chorda tympani nerve using confocal laser scanning microscopy in vivo.

Science.gov (United States)

Saito, Takehisa; Ito, Tetsufumi; Kato, Yuji; Yamada, Takechiyo; Manabe, Yasuhiro; Narita, Norihiko

2014-03-01

To evaluate whether regenerated fungiform taste buds after severing the chorda tympani nerve can be detected by confocal laser scanning microscopy in vivo. Retrospective study. University hospital. Six patients with a normal gustatory function (Group 1), 9 patients with taste function recovery after severing the CTN (Group 2), and 5 patients without taste function recovery (Group 3) were included. In Groups 2 and 3, canal wall up (closed) tympanoplasty or canal wall down with canal reconstruction tympanoplasty was performed in all patients. Diagnostic. The severed nerves were readapted or approximated on the temporalis muscle fascia used to reconstruct the eardrum during surgery. Preoperative and postoperative gustatory functions were assessed using electrogustometry. Twelve to 260 months after severing the CTN, the surface of the midlateral region of the tongue was observed with a confocal laser microscope. EGM thresholds showed no response 1 month after surgery in all patients of Groups 2 and 3. In Group 2, EGM thresholds showed recovery 1 to 2 years after surgery and before confocal microscopy (-1.3 ± 6.5 dB). There was a significant difference between Group 1 (-5.7 ± 2.0 dB; p taste buds were observed in each FP, and 55 (79.7%) of 69 FP contained at least 1 taste bud. The mean number of taste bud per papilla was 3.7 ± 3.6. In patients with a recovered taste function (Group 2), 0 to 8 taste buds were observed in each FP. In this group, 54 (56.2%) of 94 FP contained at least 1 taste bud. The mean number of taste bud per papilla was 2.0 ± 2.2 (p taste bud was observed. Regenerated fungiform taste bud could be observed in vivo using confocal laser scanning microscopy, indicating that regenerated taste bud can be detected without biopsy.

Confocal examination of subsurface cracking in ceramic materials.

Science.gov (United States)

Etman, Maged K

2009-10-01

The original ceramic surface finish and its microstructure may have an effect on crack propagation. The purpose of this study was to investigate the relation between crack propagation and ceramic microstructure following cyclic fatigue loading, and to qualitatively evaluate and quantitatively measure the surface and subsurface crack depths of three types of ceramic restorations with different microstructures using a Confocal Laser Scanning Microscope (CLSM) and Scanning Electron Microscope (SEM). Twenty (8 x 4 x 2 mm(3)) blocks of AllCeram (AC), experimental ceramic (EC, IPS e.max Press), and Sensation SL (SSL) were prepared, ten glazed and ten polished of each material. Sixty antagonist enamel specimens were made from the labial surfaces of permanent incisors. The ceramic abraders were attached to a wear machine, so that each enamel specimen presented at 45 degrees to the vertical movement of the abraders, and immersed in artificial saliva. Wear was induced for 80K cycles at 60 cycles/min with a load of 40 N and 2-mm horizontal deflection. The specimens were examined for cracks at baseline, 5K, 10K, 20K, 40K, and 80K cycles. Twenty- to 30-microm deep subsurface cracking appeared in SSL, with 8 to 10 microm in AC, and 7 microm close to the margin of the wear facets in glazed EC after 5K cycles. The EC showed no cracks with increasing wear cycles. Seventy-microm deep subsurface cracks were detected in SSL and 45 microm in AC after 80K cycles. Statistically, there was significant difference among the three materials (p 0.05) in crack depth within the same ceramic material with different surface finishes. The ceramic materials with different microstructures showed different patterns of subsurface cracking.

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.

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.

Microscopic Study of Surface Microtopographic Characteristics of Dental Implants

Science.gov (United States)

Sezin, M.; Croharé, L.; Ibañez, J.C.

2016-01-01

Objective: To determine and compare the micro topographic characteristics of dental implants submitted to different surface treatments, using scanning electron microscopy (SEM). Materials and Methods: Implants were divided into 7 groups of 3 specimens each, according to the surface treatment used: group 1: Osseotite, BIOMET 3i; group 2: SLA surface, Institut Straumann AG; group 3: Oxalife surface, Tree-Oss implant; group 4: B&W implant surface; group 5: Q-implant surface; group 6: ML implant surface; group 7: RBM surface, Rosterdent implant. The surfaces were examined under SEM (Carl Zeiss FE-SEM-SIGMA). Image Proplus software was used to determine the number and mean diameter of pores per area unit (mm). The data obtained were analyzed with the Mann-Whitney test. A confocal laser microscope (LEXT-OLS4100 Olympus) was used to conduct the comparative study of surface roughness (Ra). Data were analyzed using Tukey's HSD test. Results: The largest average pore diameter calculated in microns was found in group 5 (3.45 µm+/-1.91) while the smallest in group 7 (1.47µm+/-1.29). Significant differences were observed among each one of the groups studied (p<0.05). The largest number of pores/mm2 was found in group 2 (229343) and the smallest number in group 4 (10937). Group 2 showed significant differences regarding the other groups (p<0.05). The greatest roughness (Ra) was observed in group 2 (0.975µm+/-0.115) and the smallest in group 4 (0.304µm+/-0.063). Group 2 was significantly different from the other groups (p<0.05). Conclusion: The micro topography observed in the different groups presented dissimilar and specific features, depending on the chemical treatment used for the surfaces.. PMID:27335615

Molecular confocal laser endomicroscopy: a novel technique for in vivo cellular characterization of gastrointestinal lesions.

Science.gov (United States)

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

2014-06-28

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 endoscope or via a needle guided by endoscopic ultrasound. The second system has a confocal microscope integrated into the distal part of an endoscope. By adding molecular probes like fluorescein conjugated antibodies or fluorescent peptides to this procedure (either topically or systemically administered 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 the diagnostic accuracy of endoscopic procedures by identifying otherwise invisible mucosal lesions. Furthermore, studies have shown that fluorescein labelled drugs can be used to estimate the affinity of the drug to a target organ, which probably can be correlated to the efficacy of the drug. However, several of the studies in this research field have been conducted in animal facilities or in vitro, while only a limited number of trials have actually been carried out in vivo. Therefore, safety issues still needs further evaluations. This review will present an overview of the implications and pitfalls, as well as future challenges of molecular CLE in gastrointestinal diseases.

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

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.

Microscopic hydrodynamics study with nuclear track membrane

International Nuclear Information System (INIS)

Shilun Guo; Yuhua Zhao; Yulan Wang; Hiuhong Hao; Brandt, R.; Vater, P.

1988-01-01

Microscopic hydrodynamics has been studied using different liquids and nuclear track membranes with pores perpendicularly piercing through them. The flow rate of water and alcohol has been studied with polycarbonate track membranes with pore diameters 1.48 micrometres and 1.08 micrometres. It has been shown that the flow rate both for water and alcohol on a microscopic scale can be determined by the Poiseuille law which characterizes macroscopic laminar flow. The Reynolds number used in macroscopic fluid flow has been calculated from the flow rate and parameters of the liquids and the geometry of the pores. It has been shown that this Reynolds number can also be used to characterize microscopic flow. Based on the above results, the filtration capacity (or limit) of polycarbonate track microfilters for water had been calculated. Some possible limits on the application of the calculation are pointed out and discussed. (author)

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)

Application of function-oriented roughness parameters using confocal microscopy

Directory of Open Access Journals (Sweden)

K. Klauer

2018-06-01

Full Text Available Optical measuring instruments are widely used for the functional characterization of surface topography. However, due to the interaction of the surface with the incident light, effects occur that can influence the measured topography height values and the obtained surface texture parameters. Therefore, we describe a systematic investigation of the influences of optical surface topography measurement on the acquisition of function-oriented roughness parameters. The same evaluation areas of varying cylinder liners which represent a typical application of function-oriented roughness parameters were measured with a confocal microscope and a stylus instrument. Functional surface texture parameters as given in the standards ISO 13565–2, ISO 13565–3 and ISO 25178–2 were evaluated for both measurement methods and compared. The transmission of specific surface features was described and a correlation analysis for the surface topographies obtained with the different measurement methods and their resulting functional roughness parameters was carried out. Keywords: Functional surface characterization, Optical metrology, Topography measurement, Roughness

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

Science.gov (United States)

Attota, Ravi Kiran; Park, Haesung

2017-06-15

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

Visual and confocal microscopic interpretation of patch tests to benzethonium chloride and benzalkonium chloride.

Science.gov (United States)

Benjamin, Bohaty; Chris, Fricker; Salvador, González; Melissa, Gill; Susan, Nedorost

2012-08-01

Quaternary ammonium compounds (Quats), such as benzalkonium chloride (BAC) and benzethonium chloride (BEC), are widely used as antibacterial active ingredients and preservatives in personal care products, disinfectants, and ophthalmic preparations. BAC is known to be a marginal irritant when patch tested at 0.15% aq. Data on BEC are limited. To differentiate irritant from allergic patch test reactions to quaternary ammonium compounds. Eight subjects who were considered likely to react based on history of rash after exposure to disinfectants or a history of prior positive patch test to BAC were recruited, as well as two patients undergoing routine patch testing. BAC (0.15% aq), BAC (0.15% pet), BEC (0.05% aq), BEC (0.15% pet), BEC (0.15% aq), BEC (0.5% aq), sodium lauryl sulfate (2.0%), and deionized water were applied under Finn chambers for 48 h. Four days and 7 days after application, the sites were examined visually and then by in vivo reflectance confocal microscopy (RCM) which was interpreted by blinded experts. Two patients with definite allergic reactions according to visual patch test reads and RCM were clinically relevant. Cross-reaction between BEC and BAC was demonstrated in one patient. RCM imaging correlated well with clinical scoring and interpretation of patch test reactions in terms of irritancy vs. allergy for BEC and BAC. Relevant allergic reactions to quats occur in humans. Possible cross-reaction was noted to occur between BAC and BEC. RCM appears to be a useful tool in distinguishing between irritancy and sensitization during patch testing to BAC and BEC. Further study of prevalence and best test concentration and vehicle is needed. © 2011 John Wiley & Sons A/S.

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.

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.

Virtual reality microscope versus conventional microscope regarding time to diagnosis: an experimental study.

Science.gov (United States)

Randell, Rebecca; Ruddle, Roy A; Mello-Thoms, Claudia; Thomas, Rhys G; Quirke, Phil; Treanor, Darren

2013-01-01

  To create and evaluate a virtual reality (VR) microscope that is as efficient as the conventional microscope, seeking to support the introduction of digital slides into routine practice.   A VR microscope was designed and implemented by combining ultra-high-resolution displays with VR technology, techniques for fast interaction, and high usability. It was evaluated using a mixed factorial experimental design with technology and task as within-participant variables and grade of histopathologist as a between-participant variable. Time to diagnosis was similar for the conventional and VR microscopes. However, there was a significant difference in the mean magnification used between the two technologies, with participants working at a higher level of magnification on the VR microscope.   The results suggest that, with the right technology, efficient use of digital pathology for routine practice is a realistic possibility. Further work is required to explore what magnification is required on the VR microscope for histopathologists to identify diagnostic features, and the effect on this of the digital slide production process. © 2012 Blackwell Publishing Limited.

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.

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.

Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

Science.gov (United States)

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

2010-01-01

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

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

Science.gov (United States)

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

2009-11-26

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

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,.

A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy

Science.gov (United States)

Li, Hao; Yang, Haw

2018-03-01

This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy.

Science.gov (United States)

Li, Hao; Yang, Haw

2018-03-28

This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

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.

Quantitative analysis of microbicide concentrations in fluids, gels and tissues using confocal Raman spectroscopy.

Directory of Open Access Journals (Sweden)

Oranat Chuchuen

Full Text Available Topical vaginal anti-HIV microbicides are an important focus in female-based strategies to prevent the sexual transmission of HIV. Understanding microbicide pharmacokinetics is essential to development, characterization and implementation of efficacious microbicide drug delivery formulations. Current methods to measure drug concentrations in tissue (e.g., LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry are highly sensitive, but destructive and complex. This project explored the use of confocal Raman spectroscopy to detect microbicide drugs and to measure their local concentrations in fluids, drug delivery gels, and tissues. We evaluated three candidate microbicide drugs: tenofovir, Dapivirine and IQP-0528. Measurements were performed in freshly excised porcine buccal tissue specimens, gel vehicles and fluids using two Horiba Raman microscopes, one of which is confocal. Characteristic spectral peak calibrations for each drug were obtained using serial dilutions in the three matrices. These specific Raman bands demonstrated strong linear concentration dependences in the matrices and were characterized with respect to their unique vibrational signatures. At least one specific Raman feature was identified for each drug as a marker band for detection in tissue. Sensitivity of detection was evaluated in the three matrices. A specific peak was also identified for tenofovir diphosphate, the anti-HIV bioactive product of tenofovir after phosphorylation in host cells. Z-scans of drug concentrations vs. depth in excised tissue specimens, incubated under layers of tenofovir solution in a Transwell assay, showed decreasing concentration with depth from the surface into the tissue. Time-dependent concentration profiles were obtained from tissue samples incubated in the Transwell assay, for times ranging 30 minutes - 6 hours. Calibrations and measurements from tissue permeation studies for tenofovir showed good correlation with gold

Quantitative Analysis of Microbicide Concentrations in Fluids, Gels and Tissues Using Confocal Raman Spectroscopy

Science.gov (United States)

Chuchuen, Oranat; Henderson, Marcus H.; Sykes, Craig; Kim, Min Sung; Kashuba, Angela D. M.; Katz, David F.

2013-01-01

Topical vaginal anti-HIV microbicides are an important focus in female-based strategies to prevent the sexual transmission of HIV. Understanding microbicide pharmacokinetics is essential to development, characterization and implementation of efficacious microbicide drug delivery formulations. Current methods to measure drug concentrations in tissue (e.g., LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry) are highly sensitive, but destructive and complex. This project explored the use of confocal Raman spectroscopy to detect microbicide drugs and to measure their local concentrations in fluids, drug delivery gels, and tissues. We evaluated three candidate microbicide drugs: tenofovir, Dapivirine and IQP-0528. Measurements were performed in freshly excised porcine buccal tissue specimens, gel vehicles and fluids using two Horiba Raman microscopes, one of which is confocal. Characteristic spectral peak calibrations for each drug were obtained using serial dilutions in the three matrices. These specific Raman bands demonstrated strong linear concentration dependences in the matrices and were characterized with respect to their unique vibrational signatures. At least one specific Raman feature was identified for each drug as a marker band for detection in tissue. Sensitivity of detection was evaluated in the three matrices. A specific peak was also identified for tenofovir diphosphate, the anti-HIV bioactive product of tenofovir after phosphorylation in host cells. Z-scans of drug concentrations vs. depth in excised tissue specimens, incubated under layers of tenofovir solution in a Transwell assay, showed decreasing concentration with depth from the surface into the tissue. Time-dependent concentration profiles were obtained from tissue samples incubated in the Transwell assay, for times ranging 30 minutes - 6 hours. Calibrations and measurements from tissue permeation studies for tenofovir showed good correlation with gold standard LC-MS/MS data

Laser-induced cartilage damage: an ex-vivo model using confocal microscopy

Science.gov (United States)

Frenz, Martin; Zueger, Benno J.; Monin, D.; Weiler, C.; Mainil-Varlet, P. M.; Weber, Heinz P.; Schaffner, Thomas

1999-06-01

Although there is an increasing popularity of lasers in orthopedic surgery, there is a growing concern about negative side effects of this therapy e.g. prolonged restitution time, radiation damage to adjacent cartilage or depth effects like bone necrosis. Despite case reports and experimental investigations over the last few years little is known about the extent of acute cartilage damage induced by different lasers types and energies. Histological examination offers only limited insights in cell viability and metabolism. Ho:YAG and Er:YAG lasers emitting at 2.1 micrometer and 2.94 micrometer, respectively, are ideally suited for tissue treatment because these wavelengths are strongly absorbed in water. The Purpose of the present study is to evaluate the effect of laser type and energy on chondrocyte viability in an ex vivo model. Free running Er:YAG (E equals 100 and 150 mJ) and Ho:YAG (E equals 500 and 800 mJ) lasers were used at different energy levels using a fixed pulse length of 400 microseconds. The energy was delivered at 8 Hz through optical fibers. Fresh bovine hyaline cartilage samples were mounted in a water bath at room temperature and the fiber was positioned at 30 degree and 180 degree angles relative to the tissue surface. After laser irradiation the samples were assessed by a life-dead cell viability test using a confocal microscope and by standard histology. Thermal damage was much deeper with Ho:YAG (up to 1800 micrometer) than with the Er:YAG laser (up to 70 micrometer). The cell viability test revealed a damage zone about twice the one determined by standard histology. Confocal microscopy is a powerful tool for assessing changes in tissue structure after laser treatment. In addition this technique allows to quantify these alterations without necessitating time consuming and expensive animal experiments.

Conversion efficiency of implanted ions by confocal micro-luminescence mapping

International Nuclear Information System (INIS)

Deshko, Y.; Huang, Mengbing; Gorokhovsky, A.A.

2013-01-01

We report on the further development of the statistical approach to determine the conversion efficiency of implanted ions into emitting centers and present the measurement method based on the confocal micro-luminescence mapping. It involves the micro-luminescence mapping with a narrow-open confocal aperture, followed by the statistical analysis of the photoluminescence signal from an ensemble of emitting centers. The confocal mapping method has two important advantages compared to the recently discussed aperture-free method (J. Lumin. 131 (2011) 489): it is less sensitive to errors in the laser spot size and has a well defined useful area. The confocal mapping has been applied to the Xe center in diamond. The conversion efficiency has been found to be about 0.28, which is in good agreement with the results of the aperture-free method. - Highlights: ► Conversion efficiency of implanted ions into emitting centers – statistical approach. ► Micro-luminescence mapping with open and narrow confocal aperture – comparison. ► Advantages of the confocal micro-luminescence mapping. ► Confocal micro-luminescence mapping has been applied to the Xe center in diamond. ► The conversion efficiency has been found to be about 0.28.

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.

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)

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.

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.

Quantification of Multilayer Samples by Confocal μXRF

International Nuclear Information System (INIS)

Perez, R. Daniel; Sanchez, H. J.; Rubio, M.; Perez, C. A.

2009-01-01

The confocal setup consists of x-ray lenses in the excitation as well as in the detection channel. In this configuration, a micro volume defined by the overlap of the foci of both x-ray lenses is analyzed. Scanning this micro volume through the sample, 1-3 dimensional studies can be performed. For intermediate thin homogeneous layers a scanning in the normal direction to the surface sample provides information of its thickness and elemental composition. For multilayer samples it also provides the order of each layer in the stratified structure. For the confocal setup, we used a glass monocapillary in the excitation channel and a monolithic half polycapillary in the detection channel. The experiment was carried out at the D09B beamline of the LNLS using white beam. In the present work, a new algorithm was applied to analyze in detail by confocal μXRF a sample of three paint layers on a glass substrate. Using the proposed algorithm, information about thickness and elemental densities was obtained for each layer of these samples.

Microscopic studies of RIB target materials and ion induced nanostructures

International Nuclear Information System (INIS)

Karmakar, Prasanta; Bhattacharya, Shampa; Roy, Tapatee Kundu; Bhowmick, Debasis; Chakrabarti, Alok

2010-01-01

The invention of electron microscope and scanning probe microscope has empowered us to visualize the tiny world that has explored many fundamental laws of natures. Further technological advancements have made these tools capable to probe micron size structures to individual atom. These microscopes are used to image and study micron size fibers or grain structures used for high yield radioactive products, to few nanometer size ripple, dot and hole structures produced by ion irradiation. Electron Microscope has also been used to characterize the ion beam synthesized dilute magnetic systems

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.

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...

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...

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

Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures

Science.gov (United States)

Tatini, Francesca; Rossi, Francesca; Coppi, Elisabetta; Magni, Giada; Fusco, Irene; Menabuoni, Luca; Pedata, Felicita; Pugliese, Anna Maria; Pini, Roberto

2016-04-01

The characterization of the ion channels in corneal endothelial cells and the elucidation of their involvement in corneal pathologies would lead to the identification of new molecular target for pharmacological treatments and to the clarification of corneal physiology. The corneal endothelium is an amitotic cell monolayer with a major role in preserving corneal transparency and in regulating the water and solute flux across the posterior surface of the cornea. Although endothelial cells are non-excitable, they express a range of ion channels, such as voltage-dependent Na+ channels and K+ channels, L-type Ca2 channels and many others. Interestingly, purinergic receptors have been linked to a variety of conditions within the eye but their presence in the endothelium and their role in its pathophysiology is still uncertain. In this study, we were able to extract endothelial cells from single human corneas, thus obtaining primary cultures that represent the peculiarity of each donor. Corneas were from tissues not suitable for transplant in patients. We characterized the endothelial cells by confocal microscopy, both within the intact cornea and in the primary endothelial cells cultures. We also studied the functional role of the purinergic system (adenosine, ATP and their receptors) by means of electrophysiological recordings. The experiments were performed by patch clamp recordings and confocal time-lapse microscopy and our results indicate that the application of purinergic compounds modulates the amplitude of outward currents in the isolated endothelial cells. These findings may lead to the proposal of new therapies for endothelium-related corneal diseases.

Cheese Matrix Microstructure Studied by Advanced Microscopic Techniques

Czech Academy of Sciences Publication Activity Database

Burdiková, Z.; Hickey, C.; Auty, M. A. E.; Pala, J.; Švindrych, Z.; Steinmetz, I.; Krzyžánek, Vladislav; Hrubanová, Kamila; Sheehan, J. J.

2014-01-01

Roč. 20, S3 (2014), s. 1336-1337 ISSN 1431-9276 R&D Projects: GA MŠk(CZ) LO1212 Institutional support: RVO:68081731 Keywords : cheese matrix * cryo-SEM * confocal laser scanning microscopy Subject RIV: EA - Cell Biology Impact factor: 1.877, year: 2014

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.

Photon event distribution sampling: an image formation technique for scanning microscopes that permits tracking of sub-diffraction particles with high spatial and temporal resolutions.

Science.gov (United States)

Larkin, J D; Publicover, N G; Sutko, J L

2011-01-01

In photon event distribution sampling, an image formation technique for scanning microscopes, the maximum likelihood position of origin of each detected photon is acquired as a data set rather than binning photons in pixels. Subsequently, an intensity-related probability density function describing the uncertainty associated with the photon position measurement is applied to each position and individual photon intensity distributions are summed to form an image. Compared to pixel-based images, photon event distribution sampling images exhibit increased signal-to-noise and comparable spatial resolution. Photon event distribution sampling is superior to pixel-based image formation in recognizing the presence of structured (non-random) photon distributions at low photon counts and permits use of non-raster scanning patterns. A photon event distribution sampling based method for localizing single particles derived from a multi-variate normal distribution is more precise than statistical (Gaussian) fitting to pixel-based images. Using the multi-variate normal distribution method, non-raster scanning and a typical confocal microscope, localizations with 8 nm precision were achieved at 10 ms sampling rates with acquisition of ~200 photons per frame. Single nanometre precision was obtained with a greater number of photons per frame. In summary, photon event distribution sampling provides an efficient way to form images when low numbers of photons are involved and permits particle tracking with confocal point-scanning microscopes with nanometre precision deep within specimens. © 2010 The Authors Journal of Microscopy © 2010 The Royal Microscopical Society.

Latest developments and opportunities for 3D analysis of biological samples by confocal μ-XRF

International Nuclear Information System (INIS)

Perez, Roberto D.; Sanchez, Hector J.; Perez, Carlos A.; Rubio, Marcelo

2010-01-01

X-ray fluorescence analysis performed with a primary radiation focused in the micrometer range is known as micro-X-ray fluorescence (μ-XRF). It is characterized by a penetration depth higher than other micro-analytical methods, reaching hundreds of micrometers in biological samples. This characteristic of the X-ray beam can be employed in 3D analysis. An innovative method to perform 3D analysis by μ-XRF is the so-called confocal setup. The confocal setup consists of X-ray lenses in the excitation as well as in the detection channel. In this configuration, a micro-volume defined by the overlap of the foci of both X-ray lenses is analyzed. Scanning this micro-volume through the sample can be used to perform a study in three dimensions. At present, X-ray lenses used in confocal μ-XRF experiments are mainly glass capillaries and polycapillaries. Glass capillaries are used in the excitation channel with sources of high photon flux like synchrotron radiation. Half polycapillaries or conical polycapillary concentrators are used almost exclusively in the detection channel. Spatial resolution of the confocal μ-XRF depends on the dimensions of the foci of both X-ray lenses. The overlap of these foci forms an ellipsoid which is the probing volume of the confocal setup. The axis length of the probing volume reported in confocal μ-XRF experiments are of order of few tens of micrometer. In our confocal setup, we used a commercial glass monocapillary in the excitation channel and a monolithic half polycapillary in the detection channel. The polycapillary was home-made by means of drawing of multibundles of glass capillaries in a heating furnace. The experiment was carried out at the beamline D09B-XRF of the Synchrotron Light National Laboratory (Laboratorio Nacional de Luz Sincrotron, LNLS) using white beam. A model for the theoretical description of X-ray fluorescence intensity registered by confocal μ-XRF was introduced by Malzer and Kanngieβer [2005. A model for the

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.

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

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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.

Systematic study of alginate-based microcapsules by micropipette aspiration and confocal fluorescence microscopy.

Science.gov (United States)

Kleinberger, Rachelle M; Burke, Nicholas A D; Dalnoki-Veress, Kari; Stöver, Harald D H

2013-10-01

Micropipette aspiration and confocal fluorescence microscopy were used to study the structure and mechanical properties of calcium alginate hydrogel beads (A beads), as well as A beads that were additionally coated with poly-L-lysine (P) and sodium alginate (A) to form, respectively, AP and APA hydrogels. A beads were found to continue curing for up to 500 h during storage in saline, due to residual calcium chloride carried over from the gelling bath. In subsequent saline washes, micropipette aspiration proved to be a sensitive indicator of gel weakening and calcium loss. Aspiration tests were used to compare capsule stiffness before and after citrate extraction of calcium. They showed that the initial gel strength is largely due to the calcium alginate gel cores, while the long term strength is solely due to the poly-L-lysine-alginate polyelectrolyte complex (PEC) shells. Confocal fluorescence microscopy showed that calcium chloride exposure after PLL deposition led to PLL redistribution into the hydrogel bead, resulting in thicker but more diffuse and weaker PEC shells. Adding a final alginate coating to form APA capsules did not significantly change the PEC membrane thickness and stiffness, but did speed the loss of calcium from the bead core. © 2013.

A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

Directory of Open Access Journals (Sweden)

Lim Tit-Meng

2010-10-01

Full Text Available Abstract Background Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy. Results Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®. Conclusions The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic

Detecting plant silica fibres in animal tissue by confocal fluorescence microscopy.

Science.gov (United States)

Hodson, M J; Smith, R J; van Blaaderen, A; Crafton, T; O'Neill, C H

1994-04-01

Silica fibres from the inflorescence bracts of the grass Phalaris canariensis L. cause dermatitis, and have been implicated in the aetiology of oesophageal cancer in northeastern Iran. Here we describe a method for labelling these fibres so that they can be located in mammalian tissue. Fluorescein was covalently linked to isolated, purified fibres with the silane coupling agent 3-aminopropyl triethoxysilane. The labelled hairs were then rubbed into the backs of mice. These were later killed and their skin fixed, stained and sliced at a thickness of 250 microns. A confocal laser scanning microscope gave brilliant images of the fibres at any depth up to 100 microns or more beneath the surface of the slice. Fibres penetrated deeply into the dermis. Several cubic millimetres of tissue could be surveyed in 1 h. The number of fibres present was approximately 2 mm-3 initially, falling to 0.1 mm-3 after 7 days.

Spontaneous confocal Raman microscopy--a tool to study the uptake of nanoparticles and carbon nanotubes into cells

Science.gov (United States)

Romero, Gabriela; Rojas, Elena; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio Enrique

2011-06-01

Confocal Raman microscopy as a label-free technique was applied to study the uptake and internalization of poly(lactide- co-glycolide) (PLGA) nanoparticles (NPs) and carbon nanotubes (CNTs) into hepatocarcinoma human HepG2 cells. Spontaneous confocal Raman spectra was recorded from the cells exposed to oxidized CNTs and to PLGA NPs. The Raman spectra showed bands arising from the cellular environment: lipids, proteins, nucleic acids, as well as bands characteristic for either PLGA NPs or CNTs. The simultaneous generation of Raman bands from the cell and nanomaterials from the same spot proves internalization, and also indicates the cellular region, where the nanomaterial is located. For PLGA NPs, it was found that they preferentially co-localized with lipid bodies, while the oxidized CNTs are located in the cytoplasm.

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.

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.

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

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.

Microscopic and spectroscopic evaluation of novel PLGA-chitosan Nanoplexes as an ocular delivery system.

Science.gov (United States)

Jain, Gaurav K; Pathan, Shadab A; Akhter, Sohail; Jayabalan, Nirmal; Talegaonkar, Sushma; Khar, Roop K; Ahmad, Farhan J

2011-02-01

The interaction of PLGA-chitosan Nanoplexes with ocular mucosa was investigated ex vivo and in vivo to assess their potential as ocular delivery system. Fluorescent Rhodamine Nanoplexes (Rd-Nanoplexes) were prepared by ionotropic gelation method. The size and morphology of Nanoplexes was investigated by TEM, SEM and PCS. The corneal retention, uptake and penetration of Nanoplexes were analyzed by spectrofluorimetry and confocal microscopy. Corneas from Rd-Nanoplexes-treated rabbits were evaluated for the in vivo uptake and ocular tolerance. The Nanoplexes prepared were round with a mean diameter of 115.6±17nm and the encapsulation efficiency of Rd was 59.4±2.5%. Data from ex vivo and in vivo studies showed that the amounts of Rd in the cornea were significantly higher for Nanoplexes than for a control Rd solution, these amounts being fairly constant for up to 24h. Confocal microscopy of the corneas revealed paracellular and transcellular uptake of the Nanoplexes. The uptake mechanism postulated was adsorptive-mediated endocytosis and opening of the tight junctions between epithelial cells. No alteration was microscopically observed after ocular surface exposure to Nanoplexes. Taken together, these data demonstrate that Nanoplexes are potentially useful as ocular drug carriers. Copyright © 2010 Elsevier B.V. All rights reserved.

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.

The HVAC Challenges of Upgrading an Old Lab for High-end Light Microscopes

Science.gov (United States)

Richard, R.; Martone, P.; Callahan, L.M.

2014-01-01

The University of Rochester Medical Center forms the centerpiece of the University of Rochester's health research, teaching, patient care, and community outreach missions. Within this large facility of over 5 million square feet, demolition and remodeling of existing spaces is a constant activity. With more than $145 million in federal research funding, lab space is frequently repurposed and renovated to support this work. The URMC Medical Center Facilities Organization supporting small to medium space renovations is constantly challenged and constrained by the existing mechanical infrastructure and budgets to deliver a renovated space that functions within the equipment environmental parameters. One recent project, sponsored by the URMC Shared Resources Laboratory, demonstrates these points. The URMC Light Microscopy Shared Resource Laboratory requested renovation of a 121 sq. ft. room in a 40 year old building which would enable placement of a laser capture microdissection microscope and a Pascal 5 laser scanning confocal microscope with the instruments separated by a blackout curtain. This poster discusses the engineering approach implemented to bring an older lab into the environmental specifications needed for the proper operation of the high-end light microscopes.

Near-infrared-excited confocal Raman spectroscopy advances in vivo diagnosis of cervical precancer.

Science.gov (United States)

Duraipandian, Shiyamala; Zheng, Wei; Ng, Joseph; Low, Jeffrey J H; Ilancheran, Arunachalam; Huang, Zhiwei

2013-06-01

Raman spectroscopy is a unique optical technique that can probe the changes of vibrational modes of biomolecules associated with tissue premalignant transformation. This study evaluates the clinical utility of confocal Raman spectroscopy over near-infrared (NIR) autofluorescence (AF) spectroscopy and composite NIR AF/Raman spectroscopy for improving early diagnosis of cervical precancer in vivo at colposcopy. A rapid NIR Raman system coupled with a ball-lens fiber-optic confocal Raman probe was utilized for in vivo NIR AF/Raman spectral measurements of the cervix. A total of 1240 in vivo Raman spectra [normal (n=993), dysplasia (n=247)] were acquired from 84 cervical patients. Principal components analysis (PCA) and linear discriminant analysis (LDA) together with a leave-one-patient-out, cross-validation method were used to extract the diagnostic information associated with distinctive spectroscopic modalities. The diagnostic ability of confocal Raman spectroscopy was evaluated using the PCA-LDA model developed from the significant principal components (PCs) [i.e., PC4, 0.0023%; PC5, 0.00095%; PC8, 0.00022%, (p<0.05)], representing the primary tissue Raman features (e.g., 854, 937, 1095, 1253, 1311, 1445, and 1654 cm(-1)). Confocal Raman spectroscopy coupled with PCA-LDA modeling yielded the diagnostic accuracy of 84.1% (a sensitivity of 81.0% and a specificity of 87.1%) for in vivo discrimination of dysplastic cervix. The receiver operating characteristic curves further confirmed that the best classification was achieved using confocal Raman spectroscopy compared to the composite NIR AF/Raman spectroscopy or NIR AF spectroscopy alone. This study illustrates that confocal Raman spectroscopy has great potential to improve early diagnosis of cervical precancer in vivo during clinical colposcopy.

Real-time monitoring of magnetic drug targeting using fibered confocal fluorescence microscopy.

Science.gov (United States)

Bai, Jie; Wang, Julie Tzu-Wen; Mei, Kuo-Ching; Al-Jamal, Wafa T; Al-Jamal, Khuloud T

2016-12-28

Magnetic drug targeting has been proposed as means of concentrating therapeutic agents at a target site and the success of this approach has been demonstrated in a number of studies. However, the behavior of magnetic carriers in blood vessels and tumor microcirculation still remains unclear. In this work, we utilized polymeric magnetic nanocapsules (m-NCs) for magnetic targeting in tumors and dynamically visualized them within blood vessels and tumor tissues before, during and after magnetic field exposure using fibered confocal fluorescence microscopy (FCFM). Our results suggested that the distribution of m-NCs within tumor vasculature changed dramatically, but in a reversible way, upon application and removal of a magnetic field. The m-NCs were concentrated and stayed as clusters near a blood vessel wall when tumors were exposed to a magnetic field but without rupturing the blood vessel. The obtained FCFM images provided in vivo in situ microvascular observations of m-NCs upon magnetic targeting with high spatial resolution but minimally invasive surgical procedures. This proof-of-concept descriptive study in mice is envisaged to track and quantify nanoparticles in vivo in a non-invasive manner at microscopic resolution. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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.

3D widefield light microscope image reconstruction without dyes

Science.gov (United States)

Larkin, S.; Larson, J.; Holmes, C.; Vaicik, M.; Turturro, M.; Jurkevich, A.; Sinha, S.; Ezashi, T.; Papavasiliou, G.; Brey, E.; Holmes, T.

2015-03-01

3D image reconstruction using light microscope modalities without exogenous contrast agents is proposed and investigated as an approach to produce 3D images of biological samples for live imaging applications. Multimodality and multispectral imaging, used in concert with this 3D optical sectioning approach is also proposed as a way to further produce contrast that could be specific to components in the sample. The methods avoid usage of contrast agents. Contrast agents, such as fluorescent or absorbing dyes, can be toxic to cells or alter cell behavior. Current modes of producing 3D image sets from a light microscope, such as 3D deconvolution algorithms and confocal microscopy generally require contrast agents. Zernike phase contrast (ZPC), transmitted light brightfield (TLB), darkfield microscopy and others can produce contrast without dyes. Some of these modalities have not previously benefitted from 3D image reconstruction algorithms, however. The 3D image reconstruction algorithm is based on an underlying physical model of scattering potential, expressed as the sample's 3D absorption and phase quantities. The algorithm is based upon optimizing an objective function - the I-divergence - while solving for the 3D absorption and phase quantities. Unlike typical deconvolution algorithms, each microscope modality, such as ZPC or TLB, produces two output image sets instead of one. Contrast in the displayed image and 3D renderings is further enabled by treating the multispectral/multimodal data as a feature set in a mathematical formulation that uses the principal component method of statistics.

Ocular surface alterations and in vivo confocal microscopic characteristics of corneas in patients with myasthenia gravis.

Science.gov (United States)

Erkan Turan, Kadriye; Kocabeyoglu, Sibel; Bekircan-Kurt, Can Ebru; Bezci, Figen; Erdem-Ozdamar, Sevim; Irkec, Murat

2018-03-01

To evaluate ocular surface alterations and characteristics of corneal basal epithelium and subbasal nerves in patients with myasthenia gravis. Myasthenia gravis patients (n = 21) and healthy controls (n = 20) were enrolled. All participants underwent ocular surface testing in the following order: tear break-up time, lissamine green staining, Schirmer I test with anesthesia, and Ocular Surface Disease Index questionnaire. The Cochet-Bonnet esthesiometer was used to measure corneal sensitivity. Basal epithelial cells and subbasal nerves were evaluated using in vivo confocal microscopy. Myasthenia gravis patients had higher Ocular Surface Disease Index score (13.9 ± 15.0 vs 1.4 ± 2.2, p myasthenia gravis had lower basal epithelial cell density (3775.7 ± 938.1 vs 4983.1 ± 608.5, p myasthenia gravis and the number of corneal nerves (rho = -0.497, p = 0.022). Significant alterations of basal epithelial cells and subbasal nerves were demonstrated in myasthenia gravis patients although there was no difference of corneal sensitivity between myasthenia gravis patients and healthy controls. Thus, it should be borne in mind that myasthenia gravis patients deserve further evaluation with regard to ocular surface disease.

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning.

Science.gov (United States)

Perner, Birgit; Schnerwitzki, Danny; Graf, Michael; Englert, Christoph

2016-04-07

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development. The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed. In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance. The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

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....

Microscopia confocal de la córnea en facoemulsificación Confocal microscopy of the cornea on phacoemulsification

Directory of Open Access Journals (Sweden)

Juan Raúl Hernández Silva

2011-12-01

Full Text Available Objetivo: Determinar los cambios estructurales de la córnea en la cirugía de catarata por facoemulsificación sin complicaciones. Métodos: Se realizó un estudio prospectivo de pacientes operados de catarata por facoemulsificación coaxial por la técnica de pre chop sin complicaciones. A estos se les realizó microscopia confocal de la córnea con el CONFOSCAN 4 (Nidek Technologies con el objetivo de 40x y adaptador Z-Ring. Se realizó el estudio en el preoperatorio y en el posoperatorio (a las 24 horas, después de una semana, de un mes y a los tres meses. Resultados: Se demostraron cambios estructurales en la córnea como células epiteliales con núcleos hiperreflectivos alargadas en ocasiones y áreas de hiperreflectividad anómala a las 24 horas del posoperatorio. Persistieron queratocitos activados y la disminución de la hiperreflectividad de la matriz extracelular que desapareció al mes. Conclusiones: Aunque por biomicroscopia no se observen alteraciones corneales en el posoperatorio de la cirugía de catarata por facoemulsificación, sí se pueden demostrar por microscopia confocal de la córnea. Estas variaciones no influyen en la recuperación visual óptima de los pacientes.Objective: To determine the structural changes in the cornea in the cataract surgery using phacoemulsification without complications. Methods: A prospective study of patients operated on from cataract using the coaxial phacoemulsification (Pre Chop technique without complications was carried out. These patients also underwent confocal microscopy of the cornea with Confoscan4 (Nidek Technologies with 40x target and Z - Ring adapter. The study was performed in the preoperative period and postoperative period for 24 hours, one week, one month and three months after surgery. Results: Structural changes were observed in the cornea such as epithelial cells with hypereflectivity nucleus, occasionally elongated, , areas of anomalous hypereflectivity 24 hours after

Confocal fluorescence microscopy investigation of visible emitting defects induced by electron beam lithography in LIF films

International Nuclear Information System (INIS)

Montereali, R. M.; Bigotta, S.; Pace, A.; Piccinini, M.; Burattini, E.; Grilli, A.; Raco, A.; Giammatteo, M.; L'Aquila Univ., L'Aquila; Picozzi, P.; Santucci, S.; L'Aquila Univ., L'Aquila

2000-01-01

Low energy electron irradiation of lithium fluoride (LiF), in the form of bulk crystals and films, gives rise to the stable formation of primary F defects and aggregated color centers in a thin layer located at the surface of the investigated material. For the first time a confocal light scanning microscope (CLSM) in fluorescence mode was used to reconstruct the depth distribution of efficiently emitting laser active color centers in a stripe-like region induced by 12 and 16 keV electrons on LiF films thermally evaporated on glass. The formation of the F3+ and F2 aggregated defects appears restricted to the electron penetration and proportional to their energy depth profile, as obtained from Monte Carlo simulations [it

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

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.

Theoretical investigation of confocal microscopy using an elliptically polarized cylindrical vector laser beam: Visualization of quantum emitters near interfaces

Science.gov (United States)

Boichenko, Stepan

2018-04-01

We theoretically study laser-scanning confocal fluorescence microscopy using elliptically polarized cylindrical vector excitation light as a tool for visualization of arbitrarily oriented single quantum dipole emitters located (1) near planar surfaces enhancing fluorescence, (2) in a thin supported polymer film, (3) in a freestanding polymer film, and (4) in a dielectric planar microcavity. It is shown analytically that by using a tightly focused azimuthally polarized beam, it is possible to exclude completely the orientational dependence of the image intensity maximum of a quantum emitter that absorbs light as a pair of incoherent independent linear dipoles. For linear dipole quantum emitters, the orientational independence degree higher than 0.9 can normally be achieved (this quantity equal to 1 corresponds to completely excluded orientational dependence) if the collection efficiency of the microscope objective and the emitter's total quantum yield are not strongly orientationally dependent. Thus, the visualization of arbitrarily oriented single quantum emitters by means of the studied technique can be performed quite efficiently.

Apoptosis study of the macrophage via near-field scanning optical microscope

International Nuclear Information System (INIS)

Wang, D-C; Chen, K-Y; Chen, G-Y; Chen, S-H; Wun, S-J

2008-01-01

The cell apoptosis phenomenon was studied by traditional optical microscope with much lower resolution and also observed by Atomic Force Microscope (AFM) with nano-resolution recently. They both detect the cell apoptosis through the change of cell topography. In this study, the cell apoptosis was investigated via Near-Field Scanning Optical Microscope (NSOM). The cell topography, with nano-scaled resolution, and its optical characteristics were observed by NSOM at the same measurement scanning. The macrophage was chosen as the cell investigated. To understand the cell apoptosis process is the goal set for the research. The apoptosis process was related to the variations of the optical characteristics of the cell

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.

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

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

2011-05-01

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

Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome, Picrosirius red and confocal microscopy techniques.

Science.gov (United States)

Marcos-Garcés, V; Harvat, M; Molina Aguilar, P; Ferrández Izquierdo, A; Ruiz-Saurí, A

2017-08-01

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong

Relationship between gustatory function and average number of taste buds per fungiform papilla measured by confocal laser scanning microscopy in humans.

Science.gov (United States)

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

2017-02-01

The aim of this study was to elucidate the relationship between the gustatory function and average number of taste buds per fungiform papilla (FP) in humans. Systemically healthy volunteers (n = 211), pre-operative patients with chronic otitis media (n = 79), and postoperative patients, with or without a chorda tympani nerve (CTN) severed during middle ear surgery (n = 63), were included. Confocal laser scanning microscopy was employed to observe fungiform taste buds because it allows many FP to be observed non-invasively in a short period of time. Taste buds in an average of 10 FP in the midlateral region of the tongue were counted. In total, 3,849 FP were observed in 353 subjects. The gustatory function was measured by electrogustometry (EGM). An inverse relationship was found between the gustatory function and average number of fungiform taste buds per papilla. The healthy volunteers showed a lower EGM threshold (better gustatory function) and had more taste buds than did the patients with otitis media, and the patients with otitis media showed a lower EGM threshold and had more taste buds than did postoperative patients, reflecting the severity of damage to the CTN. It was concluded that the confocal laser scanning microscope is a very useful tool for using to observe a large number of taste buds non-invasively. © 2017 Eur J Oral Sci.

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.

Cement paste surface roughness analysis using coherence scanning interferometry and confocal microscopy

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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.

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

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.

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.

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

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.

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.

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.

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)

Study of Scanning Tunneling Microscope control electronics

International Nuclear Information System (INIS)

Oliva, A.J.; Pancarobo, M.; Denisenko, N.; Aguilar, M.; Rejon, V.; Pena, J.L.

1994-01-01

A theoretical study of Scanning Tunneling Microscope control electronics is made. The knowledge of its behaviour allows us to determine accurately the region where the unstable operation could effect the measurements, and also to set the optimal working parameters. Each feedback circuitry compound is discussed as well as their mutual interaction. Different working conditions analysis and results are presented. (Author) 12 refs

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.

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

Light Microscopy Module: International Space Station Premier Automated Microscope

Science.gov (United States)

Sicker, Ronald J.; Foster, William M.; Motil, Brian J.; Meyer, William V.; Chiaramonte, Francis P.; Abbott-Hearn, Amber; Atherton, Arthur; Beltram, Alexander; Bodzioney, Christopher; Brinkman, John;

21 CFR 884.6190 - Assisted reproductive microscopes and microscope accessories.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Assisted reproductive microscopes and microscope... Devices § 884.6190 Assisted reproductive microscopes and microscope accessories. (a) Identification. Assisted reproduction microscopes and microscope accessories (excluding microscope stage warmers, which are...

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.

Study of skin of an Egyptian mummy using a scanning electron microscope

Directory of Open Access Journals (Sweden)

Mańkowska-Pliszka Hanna

2017-06-01

Full Text Available The first study of modified human remains using an electron microscope was carried out at the end of the 1950 and in 1979 the first result of the study involving a scanning electron microscope (SEM was published for the first time. The study was mainly focused on the structure of tissues and cells. With the help of this technique cell and tissue elements, viruses and bacterial endospores as well as the structure of epithelium and the collagen contents of dermis were identified and described. In the above-mentioned case the object of the study using a SEM was a free part of the right hand (forearm with the dorsal and palmar parts of hand of unknown origin, with signs of mummification revealed during microscopic analysis. Our study was aimed at finding the answer to the question if the mummification of the studied limb was natural or intentional, and if the study using a SEM could link the anonymous remains with ancient Egypt.

In vitro studies of Rickettsia-host cell interactions: Confocal laser scanning microscopy of Rickettsia helvetica-infected eukaryotic cell lines.

Science.gov (United States)

Speck, Stephanie; Kern, Tanja; Aistleitner, Karin; Dilcher, Meik; Dobler, Gerhard; Essbauer, Sandra

2018-02-01

Rickettsia (R.) helvetica is the most prevalent rickettsia found in Ixodes ricinus ticks in Germany. Several studies reported antibodies against R. helvetica up to 12.5% in humans investigated, however, fulminant clinical cases are rare indicating a rather low pathogenicity compared to other rickettsiae. We investigated growth characteristics of R. helvetica isolate AS819 in two different eukaryotic cell lines with focus on ultra-structural changes of host cells during infection determined by confocal laser scanning microscopy. Further investigations included partially sequencing of rickA, sca4 and sca2 genes, which have been reported to encode proteins involved in cell-to-cell spread and virulence in some rickettsiae. R. helvetica grew constantly but slowly in both cell lines used. Confocal laser scanning microscopy revealed that the dissemination of R. helvetica AS819 in both cell lines was rather mediated by cell break-down and bacterial release than cell-to-cell spread. The cytoskeleton of both investigated eukaryotic cell lines was not altered. R. helvetica possesses rickA, but its expression is not sufficient to promote actin-based motility as demonstrated by confocal laser scanning microscopy. Hypothetical Sca2 and Sca4 proteins were deduced from nucleotide gene sequences but the predicted amino acid sequences were disrupted or truncated compared to other rickettsiae most likely resulting in non-functional proteins. Taken together, these results might give a first hint to the underlying causes of the reduced virulence and pathogenicity of R. helvetica.

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.

Spatially resolved analyses of uranium species using a coupled system made up of confocal laser-scanning microscopy (CLSM) and laser induced fluorescence spectroscopy (LIFS)

International Nuclear Information System (INIS)

Brockmann, S.; Grossmann, K.; Arnold, T.

2014-01-01

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

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.

Characterization of titanyl phthalocyanine (TiOPc) thin films by microscopic and spectroscopic method

Science.gov (United States)

Skonieczny, R.; Makowiecki, J.; Bursa, B.; Krzykowski, A.; Szybowicz, M.

2018-02-01

The titanyl phthalocyanine (TiOPc) thin film deposited on glass, silicon and gold substrate have been studied using Raman spectroscopy, atomic force microscopy (AFM), absorption and profilometry measurements. The TiOPc thin layers have been deposited at room temperature by the quasi-molecular beam evaporation technique. The Raman spectra have been recorded using micro Raman system equipped with a confocal microscope. Using surface Raman mapping techni que with polarized Raman spectra the polymorphic forms of the TiOPc thin films distribution have been obtained. The AFM height and phase image were examined in order to find surface features and morphology of the thin films. Additionally to compare experimental results, structure optimization and vibrational spectra calculation of single TiOPc molecule were performed using DFT calculations. The received results showed that the parameters like polymorphic form, grain size, roughness of the surface in TiOPc thin films can well characterize the obtained organic thin films structures in terms of their use in optoelectronics and photovoltaics devices.

Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

Science.gov (United States)

Surmacki, Jakub M.; Quirós Gonzalez, Isabel; Bohndiek, Sarah E.

2018-02-01

Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (pmicro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

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

Science.gov (United States)

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

2016-03-01

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

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.

The application of confocal technology based on polycapillary X-ray optics in surface topography

International Nuclear Information System (INIS)

Zhao, Guangcui; Sun, Tianxi; Liu, Zhiguo; Yuan, Hao; Li, Yude; Liu, Hehe; Zhao, Weigang; Zhang, Ruixia; Min, Qin; Peng, Song

2013-01-01

A confocal micro-X-ray fluorescence (MXRF) technology based on polycapillary X-ray optics was proposed for determining surface topography. This confocal topography method involves elemental sensitivity and can be used to classify the objects according to their elemental composition while obtaining their surface topography. To improve the spatial resolution of this confocal topography technology, the center of the confocal micro-volume was overlapped with the output focal spot of the polycapillary X-ray, focusing the lens in the excitation channel. The input focal spot of the X-ray lens parallel to the detection channel was used to determine the surface position of the sample. The corresponding surface adaptive algorithm was designed to obtain the surface topography. The surface topography of a ceramic chip was obtained. This confocal MXRF surface topography method could find application in the materials sciences

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...

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement

International Nuclear Information System (INIS)

Taylor, D.P.; Slattery, J.; Leopold, A.C.

1996-01-01

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units. (author)

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.

Selective Killing of Breast Cancer Cells by Doxorubicin-Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET.

Science.gov (United States)

Chattoraj, Shyamtanu; Amin, Asif; Jana, Batakrishna; Mohapatra, Saswat; Ghosh, Surajit; Bhattacharyya, Kankan

2016-01-18

Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin-loaded AuNCs cause the highly selective and efficient killing (90 %) of breast cancer cells (MCF7) (IC50 =155 nm). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin-loaded AuNCs is only 40 % (IC50 =4500 nm). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490-515 nm) and lifetime (≈2 ns), of the AuNCs inside the cells correspond to Au10-13 . The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Science.gov (United States)

Martisek, Dalibor; Prochazkova, Jana

2017-12-01

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

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

Directory of Open Access Journals (Sweden)

Martisek Dalibor

2017-12-01

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

The N-salicylidene aniline mesogen: Microscopic and macroscopic properties

International Nuclear Information System (INIS)

Nesrullazade, A.

2004-01-01

The vast majority of compounds exhibiting Iiquid crystalline phases may be regarded as having a rigid molecular central group with one or two flexible terminal alkyl or alkyloxy chains. The N-saIicyIidene anilines are very interesting and important materials both from fundamental and application points of view. These materials are on the one hand the ligands used to obtain metal containing complexes and on the other hand they are materials having the thermotropic mesomorphism. In this work we present investigations of microscopic and macroscopic properties of the 4-(Octyloxy)-N-(4-hexylphenyl)-2-hydrobenzaIimine (8SA) compound which was synthesized by our group. The 8SA compound shows the smectic C and nematic mesophases. These mesophases are enantiotropic and display specific confocal and schlieren textures, respectively. Thermotropic and thermodynamical properties of the straight and reverse phase transitions between smectic C and nematic mesophases and between nematic mesophase and isotropic liquid have been investigated

Collection of trace evidence of explosive residues from the skin in a death due to a disguised letter bomb. The synergy between confocal laser scanning microscope and inductively coupled plasma atomic emission spectrometer analyses.

Science.gov (United States)

Turillazzi, Emanuela; Monaci, Fabrizio; Neri, Margherita; Pomara, Cristoforo; Riezzo, Irene; Baroni, Davide; Fineschi, Vittorio

2010-04-15

In most deaths caused by explosive, the victim's body becomes a depot for fragments of explosive materials, so contributing to the collection of trace evidence which may provide clues about the specific type of device used with explosion. Improvised explosive devices are used which contain "homemade" explosives rather than high explosives because of the relative ease with which such components can be procured. Many methods such as chromatography-mass spectrometry, scanning electron microscopy, stereomicroscopy, capillary electrophoresis are available for use in the identification of explosive residues on objects and bomb fragments. Identification and reconstruction of the distribution of explosive residues on the decedent's body may give additional hints in assessing the position of the victim in relation to the device. Traditionally these residues are retrieved by swabbing the body and clothing during the early phase, at autopsy. Gas chromatography-mass spectrometry and other analytical methods may be used to analyze the material swabbed from the victim body. The histological examination of explosive residues on skin samples collected during the autopsy may reveal significant details. The information about type, quantity and particularly about anatomical distribution of explosive residues obtained utilizing confocal laser scanning microscope (CLSM) together with inductively coupled plasma atomic emission spectrometer (ICP-AES), may provide very significant evidence in the clarification and reconstruction of the explosive-related events. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

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

Science.gov (United States)

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

2015-07-01

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

eSIP: A Novel Solution-Based Sectioned Image Property Approach for Microscope Calibration.

Directory of Open Access Journals (Sweden)

Malte Butzlaff

Full Text Available Fluorescence confocal microscopy represents one of the central tools in modern sciences. Correspondingly, a growing amount of research relies on the development of novel microscopic methods. During the last decade numerous microscopic approaches were developed for the investigation of various scientific questions. Thereby, the former qualitative imaging methods became replaced by advanced quantitative methods to gain more and more information from a given sample. However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups. Multispectral beads with sub-resolution size are often used to describe the point spread function and thus the optical properties of the microscope. More recently, a fluorescent layer was utilized to describe the axial profile for each pixel, which allows a spatially resolved characterization. However, fabrication of a thin fluorescent layer with matching refractive index is technically not solved yet. Therefore, we propose a novel type of calibration concept for sectioned image property (SIP measurements which is based on fluorescent solution and makes the calibration concept available for a broader number of users. Compared to the previous approach, additional information can be obtained by application of this extended SIP chart approach, including penetration depth, detected number of photons, and illumination profile shape. Furthermore, due to the fit of the complete profile, our method is less susceptible to noise. Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy.

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.

3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

International Nuclear Information System (INIS)

Meakin, J.P.; Speight, J.D.; Sheridan, R.S.; Bradshaw, A.; Harris, I.R.; Williams, A.J.; Walton, A.

2016-01-01

Highlights: • Room temperature atmospheric oxidation behaviour of sintered NdFeB. • 3D laser confocal microscopy measurement of oxide phase growth. • Significant height increase of oxide phase only observed at triple points. • Raman spectroscopy identified oxide phase to be Nd 2 O 3 . • Diffusion coefficient determined to be 4 × 10 −13 cm 2 /s. - Abstract: Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.— computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd 2 O 3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10 −13 cm 2 /sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated

3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

Energy Technology Data Exchange (ETDEWEB)

Meakin, J.P., E-mail: jxm764@bham.ac.uk; Speight, J.D.; Sheridan, R.S.; Bradshaw, A.; Harris, I.R.; Williams, A.J.; Walton, A.

2016-08-15

Highlights: • Room temperature atmospheric oxidation behaviour of sintered NdFeB. • 3D laser confocal microscopy measurement of oxide phase growth. • Significant height increase of oxide phase only observed at triple points. • Raman spectroscopy identified oxide phase to be Nd{sub 2}O{sub 3}. • Diffusion coefficient determined to be 4 × 10{sup −13} cm{sup 2}/s. - Abstract: Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.— computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd{sub 2}O{sub 3} and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10{sup −13} cm{sup 2}/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth

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

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.

Characterization of the main error sources of chromatic confocal probes for dimensional measurement

International Nuclear Information System (INIS)

Nouira, H; El-Hayek, N; Yuan, X; Anwer, N

2014-01-01

Chromatic confocal probes are increasingly used in high-precision dimensional metrology applications such as roughness, form, thickness and surface profile measurements; however, their measurement behaviour is not well understood and must be characterized at a nanometre level. This paper provides a calibration bench for the characterization of two chromatic confocal probes of 20 and 350 µm travel ranges. The metrology loop that includes the chromatic confocal probe is stable and enables measurement repeatability at the nanometer level. With the proposed system, the major error sources, such as the relative axial and radial motions of the probe with respect to the sample, the material, colour and roughness of the measured sample, the relative deviation/tilt of the probe and the scanning speed are identified. Experimental test results show that the chromatic confocal probes are sensitive to these errors and that their measurement behaviour is highly dependent on them. (paper)

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.

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....

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).

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.

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

Directory of Open Access Journals (Sweden)

Ana Mladenovič

2008-06-01

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

MACROSCOPICAL AND MICROSCOPICAL STUDIES ON THE ...

African Journals Online (AJOL)

Caesalpinia crista leaves are bipinnate of about six pairs with alternate leaflets while the stem us fibrous, cylindrical hollow and prickly. Microscopical examination revealed the presence of strained cuticle, straight-walled epidermal cells, paracytic stomata, unicellular covering trichomes, fibres, prisms as well as cluster of ...

Synovial membrane involvement in osteoarthritic temporomandibular joints - A light microscopic study

NARCIS (Netherlands)

Dijkgraaf, LC; Liem, RSB; deBont, LGM

Objective. To study the light microscopic characteristics of the synovial membrane of osteoarthritic temporomandibular joints to evaluate synovial membrane involvement in the osteoarthritic process. Study design. Synovial membrane biopsies were obtained during unilateral arthroscopy in 40 patients.

Comparative Confocal and Histopathological Study of Corneal Changes in Multiple Myeloma.

Science.gov (United States)

Micali, Antonio; Roszkowska, Anna M; Postorino, Elisa I; Rania, Laura; Aragona, Emanuela; Wylegala, Edward; Nowinska, Anna; Ieni, Antonio; Calimeri, Sebastiano; Pisani, Antonina; Aragona, Pasquale; Puzzolo, Domenico

2017-01-01

Corneal opacities rarely occur in multiple myeloma (MM). Our study correlates the findings of in vivo confocal microscopy (IVCM), a useful diagnostic tool, with histopathological features of corneal opacities appearing in a patient with MM. Case report. A 53-year-old man developed corneal opacities in both eyes, more pronounced in the left eye. After IVCM examination, he underwent penetrating keratoplasty in the left eye, and the button was processed for light and electron microscopy and immunohistochemistry. The diagnosis of MM was made, as confirmed by the elevation of IgGk light chains. IVCM demonstrated hyperreflective areas at the epithelial level, hyperreflective keratocytes of dendritic and lamellar morphology in whole stroma, and hyperreflective endothelial cells. Histopathological examination disclosed many vacuoles in the epithelial cell cytoplasm and a homogenous granular material in the Bowman layer. In stroma, keratocytes of different shape and size, with vesicles laden with an abnormal material, were evident. In Descemet membrane, the posterior nonbanded zone had a honeycomb appearance because of the presence of many roundish spaces among wide-spaced collagen fibers. Endothelial cells demonstrated vesicles filled with a material of uneven electron density. Immunohistochemical analysis showed strong positivity for IgGk light chains in keratocytes and among stromal lamellae. This is the first study describing a correspondence between IVCM features and histopathological alterations observed in corneal opacities in MM. The results of this study improve the current understanding of the pictures obtained by IVCM studies.

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...

Single Cell Confocal Raman Spectroscopy of Human Osteoarthritic Chondrocytes: A Preliminary Study

Directory of Open Access Journals (Sweden)

Rajesh Kumar

2015-04-01

Full Text Available A great deal of effort has been focused on exploring the underlying molecular mechanism of osteoarthritis (OA especially at the cellular level. We report a confocal Raman spectroscopic investigation on human osteoarthritic chondrocytes. The objective of this investigation is to identify molecular features and the stage of OA based on the spectral signatures corresponding to bio-molecular changes at the cellular level in chondrocytes. In this study, we isolated chondrocytes from human osteoarthritic cartilage and acquired Raman spectra from single cells. Major spectral differences between the cells obtained from different International Cartilage Repair Society (ICRS grades of osteoarthritic cartilage were identified. During progression of OA, a decrease in protein content and an increase in cell death were observed from the vibrational spectra. Principal component analysis and subsequent cross-validation was able to associate osteoarthritic chondrocytes to ICRS Grade I, II and III with specificity 100.0%, 98.1%, and 90.7% respectively, while, sensitivity was 98.6%, 82.8%, and 97.5% respectively. The overall predictive efficiency was 92.2%. Our pilot study encourages further use of Raman spectroscopy as a noninvasive and label free technique for revealing molecular features associated with osteoarthritic chondrocytes.

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.

Performances for confocal X-ray diffraction technology based on polycapillary slightly focusing X-ray optics

Energy Technology Data Exchange (ETDEWEB)

Liu, Hehe; Liu, Zhiguo [The Key Laboratory of Beam Technology and Materials Modification of the Ministry of Education, Beijing Normal University, Beijing 100875 (China); College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); Sun, Tianxi, E-mail: stxbeijing@163.com [The Key Laboratory of Beam Technology and Materials Modification of the Ministry of Education, Beijing Normal University, Beijing 100875 (China); College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); Peng, Song [The Key Laboratory of Beam Technology and Materials Modification of the Ministry of Education, Beijing Normal University, Beijing 100875 (China); College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); Ma, Yongzhong [Center for Disease Control and Prevention of Beijing, Beijing 100013 (China); Sun, Weiyuan; Li, Yude; Lin, Xiaoyan; Zhao, Weigang; Zhao, Guangcui; Luo, Ping; Pan, Qiuli; Ding, Xunliang [The Key Laboratory of Beam Technology and Materials Modification of the Ministry of Education, Beijing Normal University, Beijing 100875 (China); College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China)

2013-09-21

The confocal X-ray diffraction (XRD) technology based on a polycapillary slightly focusing X-ray lens (PSFXRL) in excitation channel and a polycapillary parallel X-ray lens (PPXRL) with a long input focal distance in detection channel was developed. The output focal spot of the PSFXRL and the input focal spot of the PPXRL were adjusted in confocal configuration, and only the X-rays from the volume overlapped by these foci could be accordingly detected. This confocal configuration was helpful in decreasing background. The convergence of the beam focused by the PSFXRL and divergence of the beam which could be collected by the PPXRL with a long input focal distance were both about 9 mrad at 8 keV. This was helpful in improving the resolution of lattice spacing of this confocal XRD technology. The gain in power density of such PSFXRL and PPXRL was about 120 and 7 at 11 keV, respectively, which was helpful in using the low power source to perform XRD analysis efficiently. The performances of this confocal XRD technology were provided, and some common plastics were analyzed. The experimental results demonstrated that the confocal diffraction technology base on polycapillary slightly focusing X-ray optics had wide potential applications.

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.

[In Vivo Study of Chitin in Fungal Hyphae Based on Confocal Raman Microscopy].

Science.gov (United States)

Li, Xiao-li; Luo, Liu-bin; Zhou, Bin-xiong; Hu, Xiao-qian; Sun, Chan-jun; He, Yong

2016-01-01

Chitin is an important structural polysaccharide of fungal cell wall. In this paper, aerial hyphae of Colletotrichum camelliae Massee was first studied by confocal Raman microscopy in vivo. Firstly, the optimal experimental parameters of hyphae for collecting the Raman spectra were determined, and the typical Raman spectra of hyphae, chitin standard and background were acquired. By comparing analysis, characteristic peaks of chitin were found in hyphae. Then, a region of interesting on hyphae was selected for Raman scanning. Through principal component analysis, the Raman signal of hyphae and background in the scanning area can be separated clearly. Combined with loading weight plot, two main characteristic peaks of hyphae were obtained, 1 622 cm(-1) was belong to chitin and 1 368 cm(-1) was assigned to pectic polysaccharide. Finally, two and three dimension chemical images of fungal hyphae were realized based on Raman fingerprint spectra of chitin in a nondestructive way.

Confocal Raman and PL, AFM, and X-ray diffraction studies of CdS:O thin films

International Nuclear Information System (INIS)

Akinori, Suzuki; Kazuki, Wakita; YongGu, Shim; Nazim, Mamedov; Ayaz, Bayramov; Emil, Huseynov

2010-01-01

Full text : CdS has much attention as a window material of thin-film solar cells, for example a CdTe solar cell. In this case, increasing band gap of CdS films leads to rise of conversion efficiency of a solar cell. Recently, it was reported that CdS:O films deposited by rf magnetron sputtering consist of nano-crystals of CdS resulting in increasing the band gap. This work reports confocal Raman and photoluminescence (PL), atomic force microscopy (AFM), and X-ray diffraction studies of CdS:O films deposited by cathode sputtering for formation of nano-crystal of CdS. It was shown that AFM image of CdS:O films annealed at 300, 400 and 500 degrees Celsium. The height of peak and dip on the surface is in the range of 5 and 20 nm in the samples annealed at less than 400 degrees Celsium, while the clear crystalline shape appears in the sample annealed at 500 degrees Celsium. There is also shown X-ray diffraction pattern of CdS:O films. As grown film shows amorphous structure of CdS. On the other hand, the samples annealed at 400 and 500 degrees Celsium display obvious crystalline pattern. The crystal radius of the samples annealed at 300, 400, and 500 degrees Celsium were estimated to be 20, 27, and 37 nm, respectively, according to Scherrers formula. Other results related with the confocal spectroscopy will be also presented.

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.

Transmission positron microscopes

International Nuclear Information System (INIS)

Doyama, Masao; Kogure, Yoshiaki; Inoue, Miyoshi; Kurihara, Toshikazu; Yoshiie, Toshimasa; Oshima, Ryuichiro; Matsuya, Miyuki

2006-01-01

Immediate and near-future plans for transmission positron microscopes being built at KEK, Tsukuba, Japan, are described. The characteristic feature of this project is remolding a commercial electron microscope to a positron microscope. A point source of electrons kept at a negative high voltage is changed to a point source of positrons kept at a high positive voltage. Positional resolution of transmission microscopes should be theoretically the same as electron microscopes. Positron microscopes utilizing trapping of positrons have always positional ambiguity due to the diffusion of positrons

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...

Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope

Science.gov (United States)

Meyer, Stephanie A.; Ozbay, Baris N.; Potcoava, Mariana; Salcedo, Ernesto; Restrepo, Diego; Gibson, Emily A.

2016-06-01

We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ˜81 and ˜44 nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48 nm and 124±43 nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.

Mobile microscope complex GIB-1

International Nuclear Information System (INIS)

Belyakov, A.V.; Gorbachev, A.N.

2002-01-01

To study microstructure in operating pipelines of power units a mobile microscope system is developed and successfully used. The system includes a portable microscope, a monitor, power supply and a portable computer. The monitor is used for surveying images from a video camera mounted on the microscope. The magnification on visual examination constitutes x 100 and x 500. Diameters of pipelines examined should not be less than 130 mm. Surface preparation for microstructural studies includes routine mechanical rough grinding and polishing with subsequent etching [ru

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

Simultaneous measurements of bulk moduli and particle dynamics in a sheared colloidal glass

Science.gov (United States)

Massa, Michael V.; Eisenmann, Christoph; Kim, Chanjoong; Weitz, David A.

2007-03-01

We present a novel study of glassy colloidal systems, using a stress-controlled rheometer in conjunction with a confocal microscope. This experimental setup combines the measurement of bulk moduli, using conventional rheology, with the ability to track the motion of individual particles, through confocal microscopy techniques. We explore the response of the system to applied shear, by simultaneously monitoring the macroscopic relaxation and microscopic particle dynamics, under conditions from the quiescent glass to a shear-melted liquid.

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".

Transmission electron microscopy, fluorescence microscopy, and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L. wood cell wall.

Science.gov (United States)

Ma, Jianfeng; Ji, Zhe; Zhou, Xia; Zhang, Zhiheng; Xu, Feng

2013-02-01

Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

The head-mounted microscope.

Science.gov (United States)

Chen, Ting; Dailey, Seth H; Naze, Sawyer A; Jiang, Jack J

2012-04-01

Microsurgical equipment has greatly advanced since the inception of the microscope into the operating room. These advancements have allowed for superior surgical precision and better post-operative results. This study focuses on the use of the Leica HM500 head-mounted microscope for the operating phonosurgeon. The head-mounted microscope has an optical zoom from 2× to 9× and provides a working distance from 300 mm to 700 mm. The headpiece, with its articulated eyepieces, adjusts easily to head shape and circumference, and offers a focus function, which is either automatic or manually controlled. We performed five microlaryngoscopic operations utilizing the head-mounted microscope with successful results. By creating a more ergonomically favorable operating posture, a surgeon may be able to obtain greater precision and success in phonomicrosurgery. Phonomicrosurgery requires the precise manipulation of long-handled cantilevered instruments through the narrow bore of a laryngoscope. The head-mounted microscope shortens the working distance compared with a stand microscope, thereby increasing arm stability, which may improve surgical precision. Also, the head-mounted design permits flexibility in head position, enabling operator comfort, and delaying musculoskeletal fatigue. A head-mounted microscope decreases the working distance and provides better ergonomics in laryngoscopic microsurgery. These advances provide the potential to promote precision in phonomicrosurgery. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

NARCIS (Netherlands)

Uzunbajakava, N.; Otto, Cornelis

2003-01-01

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous

Transmission electron microscope studies of extraterrestrial materials

Science.gov (United States)

Keller, Lindsay P.

1995-01-01

Transmission Electron Microscopy, X-Ray spectrometry and electron-energy-loss spectroscopy are used to analyse carbon in interplanetary dust particles. Optical micrographs are shown depicting cross sections of the dust particles embedded in sulphur. Selected-area electron diffraction patterns are shown. Transmission Electron Microscope specimens of lunar soil were prepared using two methods: ion-milling and ultramicrotomy. A combination of high resolution TEM imaging and electron diffraction is used to characterize the opaque assemblages. The opaque assemblages analyzed in this study are dominated by ilmenite with lesser rutile and spinel exsolutions, and traces of Fe metal.

Pleomorphic (giant cell) carcinoma of the intestine. An immunohistochemical and electron microscopic study

DEFF Research Database (Denmark)

Bak, Martin; Teglbjaerg, P S

1989-01-01

reaction for neuron-specific enolase (NSE) was found in three tumors and a positive reaction for chromogranin was found in one tumor. On electron microscopic study, intracytoplasmic whorls of intermediate filaments were seen in the perinuclear area. Dense core "neurosecretory" granules were rarely seen......Pleomorphic (giant cell) carcinomas have been described in the lungs, thyroid, pancreas, and gallbladder. Two pleomorphic carcinomas of the small bowel and two of the large bowel are presented. On light microscopic study, the carcinomas were solid, without squamous or glandular differentiation...

Handheld optical coherence tomography-reflectance confocal microscopy probe for detection of basal cell carcinoma and delineation of margins

Science.gov (United States)

Iftimia, Nicusor; Yélamos, Oriol; Chen, Chih-Shan J.; Maguluri, Gopi; Cordova, Miguel A.; Sahu, Aditi; Park, Jesung; Fox, William; Alessi-Fox, Christi; Rajadhyaksha, Milind

2017-07-01

We present a hand-held implementation and preliminary evaluation of a combined optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) probe for detecting and delineating the margins of basal cell carcinomas (BCCs) in human skin in vivo. A standard OCT approach (spectrometer-based) with a central wavelength of 1310 nm and 0.11 numerical aperture (NA) was combined with a standard RCM approach (830-nm wavelength and 0.9 NA) into a common path hand-held probe. Cross-sectional OCT images and enface RCM images are simultaneously displayed, allowing for three-dimensional microscopic assessment of tumor morphology in real time. Depending on the subtype and depth of the BCC tumor and surrounding skin conditions, OCT and RCM imaging are able to complement each other, the strengths of each helping overcome the limitations of the other. Four representative cases are summarized, out of the 15 investigated in a preliminary pilot study, demonstrating how OCT and RCM imaging may be synergistically combined to more accurately detect BCCs and more completely delineate margins. Our preliminary results highlight the potential benefits of combining the two technologies within a single probe to potentially guide diagnosis as well as treatment of BCCs.

Microscopic study of low-lying yrast spectra and deformation ...

Indian Academy of Sciences (India)

73, No. 4. — journal of. October 2009 physics pp. 657–668. Microscopic study of low-lying yrast spectra and deformation systematics in neutron-rich. 98−106Sr isotopes ... with a large and rigid moment of inertia. 98Sr is predicted to have a ... 2 energy as neutron number N changes from 58 to 60. The onset of deformation in ...

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.

Poster - 14: Batch Effect Reduction in in-vitro Raman Microscopic Radiosensitivity Study Using Ovarian Cancer Cells

International Nuclear Information System (INIS)

Moradi, Hamid; Murugkar, Sangeeta; Ahmad, Abrar; Shepherdson, Dean; Nyiri, Balazs; Vuong, Nhung; Niedbala, Gosia; Vanderhyden, Barbara; Eapen, Libni

2016-01-01

Purpose: To improve classification by reducing batch effect in samples from the ovarian carcinoma cell lines A2780s (parental wild type) and A2780cp (cisplatin cross-radio-resistant), before, right after, and 24 hours after irradiation to 10Gy. Methods: Spectra were acquired with a home built confocal Raman microscope in 3 distinct runs of six samples: unirradiated s&cp (control pair), then 0h and 24h after irradiation. The Raman spectra were noise reduced, then background subtracted with SMIRF algorithm. ∼35 cell spectra were collected from each sample in 1024 channels from 700cm-1 to 1618cm-1. The spectra were analyzed by regularized multiclass LDA. For feature reduction the spectra were grouped into 3 overlapping group pairs: s-cp, 0Gy–10Gy0h and 0Gy10–Gy24h. The three features, the three differences of the mean spectra were mapped to the analysis sub-space by the inverse regularized covariance matrix. The batch effect noticeably confounded the dose and time effect. Results: To remove the batch effect, the 2+2=4D subspace extended by the covariance matrix of the means of the 0Gy control groups was subtracted from the spectra of each sample. Repeating the analysis on the spectra with the control group variability removed, the batch effect was dramatically reduced in the dose and time directions enabling sharp linear discrimination. The cell type classification also improved. Conclusions: We identified a efficient batch effect removal technique crucial to the applicability of Raman microscopy to radiosensitivity studies both on cell cultures and potential clinical diagnostic applications.

Poster - 14: Batch Effect Reduction in in-vitro Raman Microscopic Radiosensitivity Study Using Ovarian Cancer Cells

Energy Technology Data Exchange (ETDEWEB)

Moradi, Hamid; Murugkar, Sangeeta; Ahmad, Abrar; Shepherdson, Dean; Nyiri, Balazs; Vuong, Nhung; Niedbala, Gosia; Vanderhyden, Barbara; Eapen, Libni [Carleton University, Carleton University, Carleton University, Carleton University, The Ottawa Hospital Cancer Centre, University of Ottawa, The Ottawa Hospital Cancer Centre, University of Ottawa, The Ottawa Hospital Cancer Centre (Canada)

2016-08-15

Purpose: To improve classification by reducing batch effect in samples from the ovarian carcinoma cell lines A2780s (parental wild type) and A2780cp (cisplatin cross-radio-resistant), before, right after, and 24 hours after irradiation to 10Gy. Methods: Spectra were acquired with a home built confocal Raman microscope in 3 distinct runs of six samples: unirradiated s&cp (control pair), then 0h and 24h after irradiation. The Raman spectra were noise reduced, then background subtracted with SMIRF algorithm. ∼35 cell spectra were collected from each sample in 1024 channels from 700cm-1 to 1618cm-1. The spectra were analyzed by regularized multiclass LDA. For feature reduction the spectra were grouped into 3 overlapping group pairs: s-cp, 0Gy–10Gy0h and 0Gy10–Gy24h. The three features, the three differences of the mean spectra were mapped to the analysis sub-space by the inverse regularized covariance matrix. The batch effect noticeably confounded the dose and time effect. Results: To remove the batch effect, the 2+2=4D subspace extended by the covariance matrix of the means of the 0Gy control groups was subtracted from the spectra of each sample. Repeating the analysis on the spectra with the control group variability removed, the batch effect was dramatically reduced in the dose and time directions enabling sharp linear discrimination. The cell type classification also improved. Conclusions: We identified a efficient batch effect removal technique crucial to the applicability of Raman microscopy to radiosensitivity studies both on cell cultures and potential clinical diagnostic applications.

Oxidation mechanism of nickel particles studied in an environmental transmission electron microscope

DEFF Research Database (Denmark)

Jeangros, Q.; Hansen, Thomas Willum; Wagner, Jakob Birkedal

2014-01-01

The oxidation of nickel particles was studied in situ in an environmental transmission electron microscope in 3.2 mbar of O2 between ambient temperature and 600°C. Several different transmission electron microscopy imaging techniques, electron diffraction and electron energy-loss spectroscopy were...... diffusion of Ni2+ along NiO grain boundaries, self-diffusion of Ni2+ ions and vacancies, growth of NiO grains and nucleation of voids at Ni/NiO interfaces. We also observed the formation of transverse cracks in a growing NiO film in situ in the electron microscope....

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K.

Science.gov (United States)

Lange, M; Guénon, S; Lever, F; Kleiner, R; Koelle, D

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4 He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO 3 . The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K

Science.gov (United States)

Lange, M.; Guénon, S.; Lever, F.; Kleiner, R.; Koelle, D.

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO3. The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

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)

Improved signal model for confocal sensors accounting for object depending artifacts.

Science.gov (United States)

Mauch, Florian; Lyda, Wolfram; Gronle, Marc; Osten, Wolfgang

2012-08-27

The conventional signal model of confocal sensors is well established and has proven to be exceptionally robust especially when measuring rough surfaces. Its physical derivation however is explicitly based on plane surfaces or point like objects, respectively. Here we show experimental results of a confocal point sensor measurement of a surface standard. The results illustrate the rise of severe artifacts when measuring curved surfaces. On this basis, we present a systematic extension of the conventional signal model that is proven to be capable of qualitatively explaining these artifacts.

Note: long-range scanning tunneling microscope for the study of nanostructures on insulating substrates.

Science.gov (United States)

Molina-Mendoza, Aday J; Rodrigo, José G; Island, Joshua; Burzuri, Enrique; Rubio-Bollinger, Gabino; van der Zant, Herre S J; Agraït, Nicolás

2014-02-01

The scanning tunneling microscope (STM) is a powerful tool for studying the electronic properties at the atomic level, however, it is of relatively small scanning range and the fact that it can only operate on conducting samples prevents its application to study heterogeneous samples consisting of conducting and insulating regions. Here we present a long-range scanning tunneling microscope capable of detecting conducting micro and nanostructures on insulating substrates using a technique based on the capacitance between the tip and the sample and performing STM studies.

Note: Long-range scanning tunneling microscope for the study of nanostructures on insulating substrates

Energy Technology Data Exchange (ETDEWEB)

Molina-Mendoza, Aday J., E-mail: aday.molina@uam.es [Departamento de Física de la Materia Condensada, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid (Spain); Rodrigo, José G.; Rubio-Bollinger, Gabino [Departamento de Física de la Materia Condensada, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid (Spain); Condensed Matter Physics Center (IFIMAC) and Instituto Universitario de Ciencia de Materiales “Nicolás Cabrera,” Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid (Spain); Island, Joshua; Burzuri, Enrique; Zant, Herre S. J. van der [Kavli Institute of Nanoscience, Delft University of Technology, P.O. Box 5046, 2600 GA Delft (Netherlands); Agraït, Nicolás [Departamento de Física de la Materia Condensada, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid (Spain); Condensed Matter Physics Center (IFIMAC) and Instituto Universitario de Ciencia de Materiales “Nicolás Cabrera,” Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid (Spain); Instituto Madrileño de Estudios Avanzados en Nanociencia IMDEA-Nanociencia, E-28049 Madrid (Spain)

2014-02-15

The scanning tunneling microscope (STM) is a powerful tool for studying the electronic properties at the atomic level, however, it is of relatively small scanning range and the fact that it can only operate on conducting samples prevents its application to study heterogeneous samples consisting of conducting and insulating regions. Here we present a long-range scanning tunneling microscope capable of detecting conducting micro and nanostructures on insulating substrates using a technique based on the capacitance between the tip and the sample and performing STM studies.

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.

TCSPC upgrade of a confocal FCS microscope

Czech Academy of Sciences Publication Activity Database

Benda, Aleš; Hof, Martin; Wahl, M.; Patting, M.; Erdmann, R.; Kapusta, P.

2005-01-01

Roč. 76, č. 3 (2005), 033106-1-033106-4 ISSN 0034-6748 R&D Projects: GA ČR(CZ) GA203/05/2308; GA AV ČR(CZ) 1ET400400413 Institutional research plan: CEZ:AV0Z40400503 Keywords : fluorescence correlation spectroscopy * photon * TCSPC Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.235, year: 2005

Analysis of the effects of cerium on calcium ion in the protoplasts of ...

African Journals Online (AJOL)

The laser-scanning confocal microscopy has become a routine technique and indispensable tool for cell biological studies. In this study, the probe Fluo-3 AM was used to research the instantaneous changes of calcium ion (Ca2+) in the protoplasts of Arabidopsis thaliana. The laser-scanning mode of confocal microscope is ...

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

infiltrating carcinoma, and were comparable to standard histologic sections of the same tissue. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images. Additional studies are needed to 1. establish correlation of the confocal and traditional histologic images for the various diseases of the breast; 2. validate diagnostic use of CSLM and; 3. further define features of borderline lesions such as well-differentiated ductal CIS vs. atypical hyperplasia.

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

Science.gov (United States)

2009-01-01

were comparable to standard histologic sections of the same tissue. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images. Additional studies are needed to 1.) establish correlation of the confocal and traditional histologic images for the various diseases of the breast; 2.) validate diagnostic use of CSLM and; 3.) further define features of borderline lesions such as well-differentiated ductal CIS vs. atypical hyperplasia. PMID:19650910

Nitrogen implantation with a scanning electron microscope.

Science.gov (United States)

Becker, S; Raatz, N; Jankuhn, St; John, R; Meijer, J

2018-01-08

Established techniques for ion implantation rely on technically advanced and costly machines like particle accelerators that only few research groups possess. We report here about a new and surprisingly simple ion implantation method that is based upon a widespread laboratory instrument: The scanning electron microscope. We show that it can be utilized to ionize atoms and molecules from the restgas by collisions with electrons of the beam and subsequently accelerate and implant them into an insulating sample by the effect of a potential building up at the sample surface. Our method is demonstrated by the implantation of nitrogen ions into diamond and their subsequent conversion to nitrogen vacancy centres which can be easily measured by fluorescence confocal microscopy. To provide evidence that the observed centres are truly generated in the way we describe, we supplied a 98% isotopically enriched 15 N gas to the chamber, whose natural abundance is very low. By employing the method of optically detected magnetic resonance, we were thus able to verify that the investigated centres are actually created from the 15 N isotopes. We also show that this method is compatible with lithography techniques using e-beam resist, as demonstrated by the implantation of lines using PMMA.

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.

Measurement of chemical and geometrical surface changes in a wear track by a confocal height sensor and confocal Raman spectroscopy

NARCIS (Netherlands)

Winogrodzka, A.; Valefi, Mahdiar; de Rooij, Matthias B.; Schipper, Dirk J.

2014-01-01

Geometrical and chemical changes in the wear track can cause a drift in friction level. In this paper, chemical and geometrical surface changes in wear tracks are analyzed. For this, a setup with a confocal height sensor was developed to measure the local height changes on the wear track, combined

Confocal Raman spectrocopy for the analysis of nail polish evidence.

Science.gov (United States)

López-López, Maria; Vaz, Joana; García-Ruiz, Carmen

2015-06-01

Nail polishes are cosmetic paints that may be susceptible of forensic analysis offering useful information to assist in a crime reconstruction. Although the nail polish appearance could allow a quick visual identification of the sample, this analysis is subjected to the perception and subjective interpretation of the forensic examiner. The chemical analysis of the nail polishes offers great deal of information not subjected to analyst interpretation. Confocal Raman spectroscopy is a well-suited technique for the analysis of paints due to its non-invasive and non-destructive nature and its ability to supply information about the organic and inorganic components of the sample. In this work, 77 regular and gel nail polishes were analyzed with confocal Raman spectroscopy using two laser wavelengths (532 and 780 nm). The sample behavior under the two laser wavelengths and the differences in the spectra taken at different points of the sample were studied for each nail polish. Additionally, the spectra obtained for all the nail polishes were visually compared. The results concluded that the longer laser wavelength prevents sample burning and fluorescence effects; the similarity among the spectra collected within the sample is not directly related with the presence of glitter particles; and 64% of the samples analyzed showed a characteristic spectrum. Additionally, the use of confocal Raman spectroscopy for the forensic analysis of nail polishes evidence in the form of flakes or smudges on different surfaces were studied. The results showed that both types of evidence can be analyzed by the technique. Also, two non-invasive sampling methods for the collection of the evidence from the nails of the suspect or the victim were proposed: (i) to use acetone-soaked cotton swabs to remove the nail varnishes and (ii) to scrape the nail polish from the nail with a blade. Both approaches, each exhibiting advantages and drawbacks in terms of transport and handling were appropriate

Robotic autopositioning of the operating microscope.

Science.gov (United States)

Oppenlander, Mark E; Chowdhry, Shakeel A; Merkl, Brandon; Hattendorf, Guido M; Nakaji, Peter; Spetzler, Robert F

2014-06-01

Use of the operating microscope has become pervasive since its introduction to the neurosurgical world. Neuronavigation fused with the operating microscope has allowed accurate correlation of the focal point of the microscope and its location on the downloaded imaging study. However, the robotic ability of the Pentero microscope has not been utilized to orient the angle of the microscope or to change its focal length to hone in on a predefined target. To report a novel technology that allows automatic positioning of the operating microscope onto a set target and utilization of a planned trajectory, either determined with the StealthStation S7 by using preoperative imaging or intraoperatively with the microscope. By utilizing the current motorized capabilities of the Zeiss OPMI Pentero microscope, a robotic autopositioning feature was developed in collaboration with Surgical Technologies, Medtronic, Inc. (StealthStation S7). The system is currently being tested at the Barrow Neurological Institute. Three options were developed for automatically positioning the microscope: AutoLock Current Point, Align Parallel to Plan, and Point to Plan Target. These options allow the microscope to pivot around the lesion, hover in a set plane parallel to the determined trajectory, or rotate and point to a set target point, respectively. Integration of automatic microscope positioning into the operative workflow has potential to increase operative efficacy and safety. This technology is best suited for precise trajectories and entry points into deep-seated lesions.

Comparison of stromal corneal nerves between normal and keratoconus patients using confocal microscopy.

Science.gov (United States)

Ramírez Fernández, M; Hernández Quintela, E; Naranjo Tackman, R

2014-08-01

To evaluate the differences in stromal corneal nerves between normal patients and keratoconus patients. A total of 140 eyes of 70 normal patients (group A) and 122 eyes of 87 keratoconus patients (group B) were examined with the confocal microscope, with a central scan of the total corneal thickness being taken. The morphology and thickness of the corneal stromal nerves were evaluated by using the Navis v. 3.5.0. software. Nerve thickness was obtained from the mean between the widest and the narrowest portions of each stromal nerve. Corneal stromal nerves were observed as irregular linear hyper-reflective structures with wide and narrow portions in all cases. Mean corneal stromal nerves thickness in group A was 5.7±1.7 (range from 3.3 to 10.4 μ), mean corneal stromal nerves thickness in group B was 7.2±1.9 (range from 3.5 to 12.0 μ). There was a statistical significant difference (P<.05) in stromal corneal nerves thickness between group A and group B. Stromal corneal nerves morphology was similar in both groups, but stromal nerves were thicker in keratoconus patients. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Confocal laser scanning microscopy to estimate nanoparticles' human skin penetration in vitro.

Science.gov (United States)

Zou, Ying; Celli, Anna; Zhu, Hanjiang; Elmahdy, Akram; Cao, Yachao; Hui, Xiaoying; Maibach, Howard

2017-01-01

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. 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. 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. 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.

Scanning confocal slit photon counter measurements of post-PRK haze in two-year study

Science.gov (United States)

Taboada, John; Gaines, David; Perez, Mary A.; Waller, Steve G.; Ivan, Douglas J.; Baldwin, J. Bruce; LoRusso, Frank; Tutt, Ronald C.; Thompson, B.; Perez, Jose; Tredici, Thomas; Johnson, Dan A.

2001-06-01

In our study, a group of 80 United States Air Force, non- flying personnel will undergo photorefractive corneal surgery for moderate levels of myopia (< 6 diopters) and 20 will serve as controls. As of this report, approximately 56 have had the treatment. Of these, only about 59% of the treated eyes showed even a trace (.5) level of clinically assessed haze at any time. We report on the use of a recently developed instrument designed for the objective measurement of these low levels of haze in treated corneas. The sensitivity of the instrument is derived from the use of a scanning confocal slit photon counter. The use of a physical standard for calibration secures accuracy and reproducibility over an extensive period of time. Our haze measurements in this study revealed a very low level increase from baseline values for these patients. The typical increase over baseline was of the same magnitude as the variability in the observations, although the inherent variability in the measurements was approximately 0.25 times the value of the patient's haze variability.

A landmark-based method for the geometrical 3D calibration of scanning microscopes

Energy Technology Data Exchange (ETDEWEB)

Ritter, M.

2007-04-27

This thesis presents a new strategy and a spatial method for the geometric calibration of 3D measurement devices at the micro-range, based on spatial reference structures with nanometersized landmarks (nanomarkers). The new method was successfully applied for the 3D calibration of scanning probe microscopes (SPM) and confocal laser scanning microscopes (CLSM). Moreover, the spatial method was also used for the photogrammetric self-calibration of scanning electron microscopes (SEM). In order to implement the calibration strategy to all scanning microscopes used, the landmark-based principle of reference points often applied at land survey or at close-range applications has been transferred to the nano- and micro-range in the form of nanomarker. In order to function as a support to the nanomarkers, slope-shaped step pyramids have been developed and fabricated by focused ion beam (FIB) induced metal deposition. These FIB produced 3D microstructures have been sized to embrace most of the measurement volume of the scanning microscopes. Additionally, their special design allows the homogenous distribution of the nanomarkers. The nanomarkers were applied onto the support and the plateaus of the slope-step pyramids by FIB etching (milling) as landmarks with as little as several hundreds of nanometers in diameter. The nanomarkers are either of point-, or ring-shaped design. They are optimized so that they can be spatially measured by SPM and CLSM, and, imaged and photogrammetrically analyzed on the basis of SEM data. The centre of the each nanomarker serves as reference point in the measurement data or images. By applying image processing routines, the image (2D) or object (3D) coordinates of each nanomarker has been determined with subpixel accuracy. The correlative analysis of the SPM, CLSM and photogrammetric SEM measurement data after 3D calibration resulted in mean residues in the measured coordinates of as little as 13 nm. Without the coupling factors the mean

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

The deuteron microscopic optical potential

International Nuclear Information System (INIS)

Lu Congshan; Zhang Jingshang; Shen Qingbiao

1991-01-01

The two particle Green's function is introduced. When the direct interaction between two nucleons is neglected, the first and second order mass operators of two particles are the sum of those for each particle. The nucleon microscopic optical potential is calculated by applying nuclear matter approximation and effective Skyrme interaction. Then the deuteron microscopic optical potential (DMOP) is calculated by using fold formula. For improvement of the theory, the two particle polarization diagram contribution to the imaginary part of the deuteron microscopic optical potential is studied

Immunoelectron Microscopic Study of Podoplanin Localization in Mouse Salivary Gland Myoepithelium

Science.gov (United States)

Hata, Minoru; Amano, Ikuko; Tsuruga, Eichi; Kojima, Hiroshi; Sawa, Yoshihiko

2010-01-01

We have recently reported that salivary gland cells express the lymphatic endothelial cell marker podoplanin. The present study was aimed to immunohistochemically investigate the expression of the myoepithelial cell marker α-smooth muscle actin (SMA) on podoplanin-positive cells in mouse parotid and sublingual glands, and to elucidate podoplanin localization in salivary gland myoepithelial cells by immunoelectron microscopic study. The distribution of myoepithelial cells expressing podoplanin and α-SMA was examined by immunofluorescent staining, and the localization of reaction products of anti-podoplanin antibody was investigated by pre-embedded immunoelectron microscopic method. In immunohistochemistry, the surfaces of both the mucous acini terminal portion and ducts were covered by a number of extensive myoepithelial cellular processes expressing podoplanin, and the immunostaining level with anti-podoplanin antibody to myoepithelial cells completely coincided with the immunostaining level with anti-α-SMA antibody. These findings suggest that podoplanin is a salivary gland myoepithelial cell antigen, and that the detection level directly reflects the myoepithelial cell distribution. In immunoelectron microscopic study, a number of reaction products with anti-podoplanin antibody were found at the Golgi apparatus binding to the endoplasmic reticulum in the cytoplasm of myoepithelial cells between sublingual gland acinar cells, and were also found at the myoepithelial cell membrane. These findings suggest that salivary gland myoepithelial cells constantly produce podoplanin and glycosylate at the Golgi apparatus, and transport them to the cell membrane. Podoplanin may be involved in maintaining the homeostasis of myoepithelial cells through its characteristic as a mucin-type transmembrane glycoprotein. PMID:20514295

Comparison of microscopic and endoscopic view of the internal acoustic meatus: A cadaveric study.

Science.gov (United States)

Montibeller, Guilherme Ramina; Hendrix, Philipp; Fries, Fabian N; Becker, Kurt W; Oertel, Joachim

2018-04-01

The endoscope is thought to provide an improved exposure of the internal acoustic meatus after retrosigmoid craniotomy for microsurgical resection of intrameatal tumors. The aim of this study is to quantify the differences in internal acoustic meatus (IAM) exposure comparing microscopic and endoscopic visualization. A retrosigmoid approach was performed on 5 cadaver heads. A millimeter gauge was introduced into the internal acoustic meatus, and examinations with a surgical microscope and 0°, 30° and 70° rigid endoscopes were performed. The extent of IAM depth visualized with the microscope and the different angled endoscopes were analyzed. The microscopic view allowed an average IAM depth visualization of 2.8 mm. The endoscope allowed an improved exposure of IAM in all cases. The 0°, 30° and 70° endoscopes permitted an exposure that was respectively 96% (5.5 mm), 139% (6.7 mm) and 200% (8.4 mm) more lateral than the microscopic view. Angled optics, however, provided an image distortion, specifically the 70° endoscope. The endoscope provides a superior visualization of the IAM compared to the microscope when using a retrosigmoid approach. The 30° endoscope represented an ideal compromise of superior visualization with marginal image distortion. Additional implementation of the endoscope into microsurgery of intrameatal tumors likely facilitates complete tumor removal and might spare facial and vestibulocochlear function. Clin. Anat. 31:398-403, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

An optical study of single pentacene molecules in n-tetradecane

NARCIS (Netherlands)

Durand, Yannig; Bloeß, Andreas; Oijen, Antoine M. van; Köhler, Jürgen; Groenen, Edgar J.J.; Schmidt, Jan

2000-01-01

We report the spectroscopic observation of single pentacene molecules in the matrices n-tetradecane and n-hexadecane, using a confocal microscope operating at liquid-helium temperatures. A maximum detected photon emission rate of only 30 counts per second (cps) is found for pentacene in n-hexadecane

A fluorescence scanning electron microscope

International Nuclear Information System (INIS)

Kanemaru, Takaaki; Hirata, Kazuho; Takasu, Shin-ichi; Isobe, Shin-ichiro; Mizuki, Keiji; Mataka, Shuntaro; Nakamura, Kei-ichiro

2009-01-01

Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.

Spectroscopic, microscopic, and internal stress analysis in cadmium telluride grown by close-space sublimation

International Nuclear Information System (INIS)

Manciu, Felicia S.; Salazar, Jessica G.; Diaz, Aryzbe; Quinones, Stella A.

2015-01-01

High quality materials with excellent ordered structure are needed for developing photovoltaic and infrared devices. With this end in mind, the results of our research prove the importance of a detailed, comprehensive spectroscopic and microscopic analysis in assessing cadmium telluride (CdTe) characteristics. The goal of this work is to examine not only material crystallinity and morphology, but also induced stress in the deposit material. A uniform, selective growth of polycrystalline CdTe by close-space sublimation on patterned Si(111) and Si(211) substrates is demonstrated by scanning electron microscopy images. Besides good crystallinity of the samples, as revealed by both Raman scattering and Fourier transform infrared absorption investigations, the far-infrared transmission data also show the presence of surface optical phonon modes, which is direct evidence of confinement in such a material. The qualitative identification of the induced stress was achieved by performing confocal Raman mapping microscopy on sample surfaces and by monitoring the existence of the rock-salt and zinc-blende structural phases of CdTe, which were associated with strained and unstrained morphologies, respectively. Although the induced stress in the material is still largely due to the high lattice mismatch between CdTe and the Si substrate, the current results provide a direct visualization of its partial release through the relaxation effect at crystallite boundaries and of preferential growth directions of less strain. Our study, thus offers significant value for improvement of material properties, by targeting the needed adjustments in the growth processes. - Highlights: • Assessing the characteristics of CdTe deposited on patterned Si substrates • Proving the utility of confocal Raman microscopy in monitoring the induced stress • Confirming the partial stress release through the grain boundary relaxation effect • Demonstrating the phonon confinement effect in low

Using corneal confocal microscopy to track changes in the corneal layers of dry eye patients after autologous serum treatment.

Science.gov (United States)

Mahelkova, Gabriela; Jirsova, Katerina; Seidler Stangova, Petra; Palos, Michalis; Vesela, Viera; Fales, Ivan; Jiraskova, Nada; Dotrelova, Dagmar

2017-05-01

In vivo corneal confocal microscopy allows the examination of each layer of the cornea in detail and the identification of pathological changes at the cellular level. The purpose of this study was to identify the possible effects of a three-month treatment with autologous serum eye-drops in different corneal layers of patients with severe dry eye disease using corneal confocal microscopy. Twenty-six patients with dry eye disease were included in the study. Corneal fluorescein staining was performed. The corneas of the right eyes were examined using in vivo corneal confocal microscopy before and after a three-month treatment with autologous serum drops. The densities of superficial and basal epithelial cells, Langerhans cells, the keratocytes and activated keratocytes, the density of endothelial cells and the status of the sub-basal nerve plexus fibres were evaluated. A significant decrease in corneal fluorescein staining was found after the three-month autologous serum treatment (p = 0.0006). The basal epithelial cell density decreased significantly (p = 0.001), while the density of superficial epithelial cells did not change significantly (p = 0.473) nor did the number of Langerhans cells or activated keratocytes (p = 0.223; p = 0.307, respectively). There were no differences in the other corneal cell layers or in the status of the nerve fibres. The results demonstrate the ability of corneal confocal microscopy to evaluate an improvement in the basal epithelial cell layer of the cornea after autologous serum treatment in patients with dry eye disease. More studies with longer follow-up periods are needed to elucidate the suitability of corneal confocal microscopy to follow the effect of autologous serum treatment on nerve fibres or other corneal layers in dry eye disease patients. © 2016 Optometry Australia.

Europium Uptake and Partitioning in Oat (Avena sativa) Roots as studied By Laser-Induced Fluorescence Spectroscopy and Confocal Microscopy Profiling Technique

International Nuclear Information System (INIS)

Fellows, Robert J.; Wang, Zheming; Ainsworth, Calvin C.

2003-01-01

The uptake of Eu3+ by elongating oat plant roots was studied by fluorescence spectroscopy, fluorescence lifetime measurement, as well as laser excitation time-resolved confocal fluorescence profiling technique. The results of this work indicated that the initial uptake of Eu(III) by oat root was most evident within the apical meristem of the root just proximal to the root cap. Distribution of assimilated Eu(III) within the roots differentiation and elongation zone was non-uniform. Higher concentrations were observed within the vascular cylinder, specifically in the phloem and developing xylem parenchyma. Elevated levels of the metal were also observed in the root hairs of the mature root. The concentration of assimilated Eu3+ dropped sharply from the apical meristem to the differentiation and elongation zone and then gradually decreased as the distance from the root cap increased. Fluorescence spectroscopic characteristics of the assimilated Eu3+ suggested that the Eu3+ exists a s inner-sphere mononuclear complexes inside the root. This work has also demonstrated the effectiveness of a time-resolved Eu3+ fluorescence spectroscopy and confocal fluorescence profiling techniques for the in vivo, real-time study of metal[Eu3+] accumulation by a functioning intact plant root. This approach can prove valuable for basic and applied studies in plant nutrition and environmental uptake of actinide radionuclides

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.

Transmission electron microscope studies of crystalline LiNbO3

International Nuclear Information System (INIS)

Pareja, R.; Gonzalez, R.; Chen, Y.

1984-01-01

Transmission electron microscope investigations in both as-grown and hydrogen-reduced LiNbO 3 reveal that niobium oxide precipitates can be produced by in situ irradiations in the electron microscope. The precipitation process is produced by a combined effect of ionizing electrons and the thermal heating of the specimens during irradiation. It is proposed that the composition of the precipitates is primarily Nb 2 O 5

Microscopic hyperspectral imaging studies of normal and diabetic retina of rats

Institute of Scientific and Technical Information of China (English)

2008-01-01

A microscopic hyperspectral imager was developed based on the microscopic technology and the spectral imaging technology. Some microscopic hyperspectral images of retina sections of the normal, the diabetic, and the treated rats were collected by the new imager. Single-band images and pseudo-color images of each group were obtained and the typical transmittance spectrums were ex-tracted. The results showed that the transmittance of outer nuclear layer cells of the diabetic group was generally higher than that of the normal. A small absorption peak appeared near the 180th band in the spectrum of the diabetic group and this peak weakened or disappeared in the spectrum of the treated group. Our findings indicate that the microscopic hyperspectral images include wealthy information of retina sections which is helpful for the ophthalmologist to reveal the pathogenesis of diabetic reti-nopathy and explore the therapeutic effect of drugs.

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope.

Science.gov (United States)

Trägårdh, J; Macrae, K; Travis, C; Amor, R; Norris, G; Wilson, S H; Oppo, G-L; McConnell, G

2015-07-01

We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic-scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light-collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife-edge method has several advantages over alternative knife-edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

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

Science.gov (United States)

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

2014-10-01

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

Craniovertebral junction 360°: A combined microscopic and endoscopic anatomical study

Directory of Open Access Journals (Sweden)

Sukhdeep Singh Jhawar

2016-01-01

Conclusion: With advances in endoscopic and microscopic techniques, access to lesions and bony anomalies around CVJ is becoming easier and straightforward. A combination of microscopic and endoscopic techniques is more useful to understand this anatomy and may aid in the development of future combined approaches.

3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

Science.gov (United States)

Meakin, J. P.; Speight, J. D.; Sheridan, R. S.; Bradshaw, A.; Harris, I. R.; Williams, A. J.; Walton, A.

2016-08-01

Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.- computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd2O3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10-13 cm2/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated temperatures in the literature. This indicates that the growth of the room temperature oxidation products are likely defect enhanced processes at the NdFeB triple junctions.

Microscopic study of neutron-rich dysprosium isotopes

International Nuclear Information System (INIS)

Vargas, Carlos E.; Velazquez, Victor; Lerma, Sergio

2013-01-01

Microscopic studies in heavy nuclei are very scarce due to large valence spaces involved. This computational problem can be avoided by means of the use of symmetry-based models. Ground-state, γ and β bands, and their B(E2) transition strengths in 160-168 Dy isotopes, are studied in the framework of the pseudo-SU(3) model which includes the preserving symmetry Q . Q term and the symmetry-breaking Nilsson and pairing terms, systematically parametrized. Additionally, three rotor-like terms are considered, whose free parameters, fixed for all members of the chain, are used to fine tune the moment of inertia of rotational bands and the band head of γ and β bands. The model succesfully describes in a systematic way rotational features in these nuclei and allows to extrapolate toward the midshell nucleus 170 Dy. The results presented show that it is possible to study a full chain of isotopes or isotones in the region with the present model. (orig.)

Microscopic study of neutron-rich dysprosium isotopes

Energy Technology Data Exchange (ETDEWEB)

Vargas, Carlos E. [Universidad Veracruzana, Facultad de Fisica e Inteligencia Artificial, Xalapa (Mexico); Universidad Nacional Autonoma de Mexico, Facultad de Ciencias, Apartado Postal 70-542, Mexico D.F. (Mexico); Velazquez, Victor [Universidad Nacional Autonoma de Mexico, Facultad de Ciencias, Apartado Postal 70-542, Mexico D.F. (Mexico); Lerma, Sergio [Universidad Veracruzana, Facultad de Fisica e Inteligencia Artificial, Xalapa (Mexico)

2013-01-15

Microscopic studies in heavy nuclei are very scarce due to large valence spaces involved. This computational problem can be avoided by means of the use of symmetry-based models. Ground-state, {gamma} and {beta} bands, and their B(E2) transition strengths in {sup 160-168}Dy isotopes, are studied in the framework of the pseudo-SU(3) model which includes the preserving symmetry Q . Q term and the symmetry-breaking Nilsson and pairing terms, systematically parametrized. Additionally, three rotor-like terms are considered, whose free parameters, fixed for all members of the chain, are used to fine tune the moment of inertia of rotational bands and the band head of {gamma} and {beta} bands. The model succesfully describes in a systematic way rotational features in these nuclei and allows to extrapolate toward the midshell nucleus {sup 170}Dy. The results presented show that it is possible to study a full chain of isotopes or isotones in the region with the present model. (orig.)

Atomic force microscopy and confocal laser scanning microscopy on the cytoskeleton of permeabilised and embedded cells

International Nuclear Information System (INIS)

Meller, Karl; Theiss, Carsten

2006-01-01

We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 o C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton

Selection of bioindicators to detect lead pollution in Ebro delta microbial mats, using high-resolution microscopic techniques

Energy Technology Data Exchange (ETDEWEB)

Maldonado, J.; Sole, A.; Puyen, Z.M. [Departament de Genetica i Microbiologia, Facultat de Biociencies, Universitat Autonoma de Barcelona, Edifici C, Campus de la UAB, Cerdanyola del Valles, Bellaterra (Spain); Esteve, I., E-mail: isabel.esteve@uab.cat [Departament de Genetica i Microbiologia, Facultat de Biociencies, Universitat Autonoma de Barcelona, Edifici C, Campus de la UAB, Cerdanyola del Valles, Bellaterra (Spain)

2011-07-15

Lead (Pb) is a metal that is non-essential to any metabolic process and, moreover, highly deleterious to life. In microbial mats - benthic stratified ecosystems - located in coastal areas, phototrophic microorganisms (algae and oxygenic phototrophic bacteria) are the primary producers and they are exposed to pollution by metals. In this paper we describe the search for bioindicators among phototrophic populations of Ebro delta microbial mats, using high-resolution microscopic techniques that we have optimized in previous studies. Confocal laser scanning microscopy coupled to a spectrofluorometric detector (CLSM-{lambda}scan) to determine in vivo sensitivity of different cyanobacteria to lead, and scanning electron microscopy (SEM) and transmission electron microscopy (TEM), both coupled to energy dispersive X-ray microanalysis (EDX), to determine the extra- and intracellular sequestration of this metal in cells, were the techniques used for this purpose. Oscillatoria sp. PCC 7515, Chroococcus sp. PCC 9106 and Spirulina sp. PCC 6313 tested in this paper could be considered bioindicators for lead pollution, because all of these microorganisms are indigenous, have high tolerance to high concentrations of lead and are able to accumulate this metal externally in extracellular polymeric substances (EPS) and intracellularly in polyphosphate (PP) inclusions. Experiments made with microcosms demonstrated that Phormidium-like and Lyngbya-like organisms selected themselves at the highest concentrations of lead assayed. In the present study it is shown that all cyanobacteria studied (both in culture and in microcosms) present PP inclusions in their cytoplasm and that these increase in number in lead polluted cultures and microcosms. We believe that the application of these microscopic techniques open up broad prospects for future studies of metal ecotoxicity.

Selection of bioindicators to detect lead pollution in Ebro delta microbial mats, using high-resolution microscopic techniques

International Nuclear Information System (INIS)

Maldonado, J.; Sole, A.; Puyen, Z.M.; Esteve, I.

2011-01-01

Lead (Pb) is a metal that is non-essential to any metabolic process and, moreover, highly deleterious to life. In microbial mats - benthic stratified ecosystems - located in coastal areas, phototrophic microorganisms (algae and oxygenic phototrophic bacteria) are the primary producers and they are exposed to pollution by metals. In this paper we describe the search for bioindicators among phototrophic populations of Ebro delta microbial mats, using high-resolution microscopic techniques that we have optimized in previous studies. Confocal laser scanning microscopy coupled to a spectrofluorometric detector (CLSM-λscan) to determine in vivo sensitivity of different cyanobacteria to lead, and scanning electron microscopy (SEM) and transmission electron microscopy (TEM), both coupled to energy dispersive X-ray microanalysis (EDX), to determine the extra- and intracellular sequestration of this metal in cells, were the techniques used for this purpose. Oscillatoria sp. PCC 7515, Chroococcus sp. PCC 9106 and Spirulina sp. PCC 6313 tested in this paper could be considered bioindicators for lead pollution, because all of these microorganisms are indigenous, have high tolerance to high concentrations of lead and are able to accumulate this metal externally in extracellular polymeric substances (EPS) and intracellularly in polyphosphate (PP) inclusions. Experiments made with microcosms demonstrated that Phormidium-like and Lyngbya-like organisms selected themselves at the highest concentrations of lead assayed. In the present study it is shown that all cyanobacteria studied (both in culture and in microcosms) present PP inclusions in their cytoplasm and that these increase in number in lead polluted cultures and microcosms. We believe that the application of these microscopic techniques open up broad prospects for future studies of metal ecotoxicity.

Selection of bioindicators to detect lead pollution in Ebro delta microbial mats, using high-resolution microscopic techniques.

Science.gov (United States)

Maldonado, J; Solé, A; Puyen, Z M; Esteve, I

2011-07-01

Lead (Pb) is a metal that is non-essential to any metabolic process and, moreover, highly deleterious to life. In microbial mats - benthic stratified ecosystems - located in coastal areas, phototrophic microorganisms (algae and oxygenic phototrophic bacteria) are the primary producers and they are exposed to pollution by metals. In this paper we describe the search for bioindicators among phototrophic populations of Ebro delta microbial mats, using high-resolution microscopic techniques that we have optimized in previous studies. Confocal laser scanning microscopy coupled to a spectrofluorometric detector (CLSM-λscan) to determine in vivo sensitivity of different cyanobacteria to lead, and scanning electron microscopy (SEM) and transmission electron microscopy (TEM), both coupled to energy dispersive X-ray microanalysis (EDX), to determine the extra- and intracellular sequestration of this metal in cells, were the techniques used for this purpose. Oscillatoria sp. PCC 7515, Chroococcus sp. PCC 9106 and Spirulina sp. PCC 6313 tested in this paper could be considered bioindicators for lead pollution, because all of these microorganisms are indigenous, have high tolerance to high concentrations of lead and are able to accumulate this metal externally in extracellular polymeric substances (EPS) and intracellularly in polyphosphate (PP) inclusions. Experiments made with microcosms demonstrated that Phormidium-like and Lyngbya-like organisms selected themselves at the highest concentrations of lead assayed. In the present study it is shown that all cyanobacteria studied (both in culture and in microcosms) present PP inclusions in their cytoplasm and that these increase in number in lead polluted cultures and microcosms. We believe that the application of these microscopic techniques open up broad prospects for future studies of metal ecotoxicity. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Observation of ice sheet formation on methane and ethane gas hydrates using a scanning confocal microscopy

Energy Technology Data Exchange (ETDEWEB)

Nagao, J.; Shimomura, N.; Ebinuma, T.; Narita, H. [National Inst. of Advanced Industrial Science and Technology, Toyohira, Sapporo (Japan). Methane Hydrate Research Lab.

2008-07-01

Interest in gas hydrates has increased in recent years due to the discovery of large deposits under the ocean floor and in permafrost regions. Natural gas hydrates, including methane, is expected to become a new energy source and a medium for energy storage and transportation. Gas hydrates consist of an open network of water molecules that are hydrogen-bonded in a similar manner to ice. Gas molecules are interstitially engaged under high pressures and low temperatures. Although the dissociation temperature of methane hydrate under atmospheric pressure is about 193 K, studies have shown that methane hydrate can be stored at atmospheric pressure and 267 K for 2 years. Because of this phenomenon, known as self-preservation, transportation and storage of methane hydrate can occur at temperature conditions milder than those for liquefied methane gas at atmospheric pressure. This study examined the surface changes of methane and ethane hydrates during dissociation using an optical microscope and confocal scanning microscope (CSM). This paper reported on the results when the atmospheric gas pressure was decreased. Ice sheets formed on the surfaces of methane and ethane gas hydrates due to depressurizing dissociation of methane and ethane hydrates when the methane and ethane gas pressures were decreased at designated temperatures. The dissociation of methane gas hydrate below below 237 K resulted in the generation of small ice particles on the hydrate surface. A transparent ice sheet formed on the hydrate surface above 242 K. The thickness of the ice sheet on the methane hydrate surface showed the maximum of ca. 30 {mu}m at 253 K. In the case of ethane hydrates, ice particles and ice sheets formed below 262 and 267 respectively. Since the ice particles and ice sheets were formed by water molecules generated during the gas hydrate dissociation, the mechanism of ice sheet formation depends on the dissociation rate of hydrate, ice particle sintering rate, and water molecule

Microscopic study of rock for estimating long-term behavior

International Nuclear Information System (INIS)

Ichikawa, Yasuaki

2004-02-01

Micro-structure of rock plays an essential role for their long-term behavior. For understanding long-term characteristics of granite we here present the followings: 1) observation of microcrack initiation and propagation by Conforcal Laser Scanning Microscope (CLSM) under uniaxial compression (before loading and at each loading stage), 2) characterization of the mechanism of microcrack initiation and propagation observed by stereoscopic microscope under uniaxial/triaxial compression and relaxation tests, and 3) a study of strong discontinuity analysis included in the homogenization theory to predict the long-term behavior of micro/macro-level stress for granite. First, CLSM was used to acquire clearly focused three-dimensional images of granite specimens, and observed the change of microscale structure including the mineral configuration under applying uniaxial compression stress. Then though microcracks have ever thought to be initiated and propagated on intergranular boundaries, we understand through the CLSM observation that new microcracks are generated from the ends of pre-existing cracks which are distributed in quartz and biotite. Second, we showed the results of stress-relaxation test of granite specimens observed by an optical microscope under water-saturated triaxial compression condition. Since microcrack generation and propagation play an essential role to predict the long-term behavior of rock, we managed the experiments with careful attention of 1) keeping constant edge-displacement and constant strain in the whole specimen accurately, and 2) measuring the relaxed stress exactly. Next, in order to simulate the experimental results which indicate that initiation and propagation of microcracks control the stress-relaxation phenomenon, we introduce a homogenization analysis procedure together with the strong discontinuity analysis which has recently established the mechanical implication and mathematical foundation. The numerical results show that we can

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

Confocal Microscopy for Process Monitoring and Wide-Area Height Determination of Vertically-Aligned Carbon Nanotube Forests

Directory of Open Access Journals (Sweden)

Markus Piwko

2015-08-01

Full Text Available Confocal microscopy is introduced as a new and generally applicable method for the characterization of the vertically-aligned carbon nanotubes (VACNT forest height. With this technique process control is significantly intensified. The topography of the substrate and VACNT can be mapped with a height resolution down to 15 nm. The advantages of confocal microscopy, compared to scanning electron microscopy (SEM, are demonstrated by investigating the growth kinetics of VACNT using Al2O3 buffer layers with varying thicknesses. A process optimization using confocal microscopy for fast VACNT forest height evaluation is presented.

Effect of resin coating and occlusal loading on microleakage of Class II computer-aided design/computer-aided manufacturing fabricated ceramic restorations: a confocal microscopic study.

Science.gov (United States)

Kitayama, Shuzo; Nasser, Nasser A; Pilecki, Peter; Wilson, Ron F; Nikaido, Toru; Tagami, Junji; Watson, Timothy F; Foxton, Richard M

2011-05-01

To evaluate the effect of resin coating and occlusal loading on microleakage of class II computer-aided design/computer-aided manufacturing (CAD/CAM) ceramic restorations. Molars were prepared for an mesio-occlusal-distal (MOD) inlay and were divided into two groups: non-coated (controls); and resin-coated, in which the cavity was coated with a combination of a dentin bonding system (Clearfil Protect Bond) and a flowable resin composite (Clearfil Majesty Flow). Ceramic inlays were fabricated using the CAD/CAM technique (CEREC 3) and cemented with resin cement (Clearfil Esthetic Cement). After 24 h of water storage, the restored teeth in each group were divided into two subgroups: unloaded or loaded with an axial force of 80 N at a rate of 2.5 cycles/s for 250,000 cycles while stored in water. After immersion in 0.25% Rhodamine B solution, the teeth were sectioned bucco-lingually at the mesial and distal boxes. Tandem scanning confocal microscopy (TSM) was used for evaluation of microleakage. The locations of the measurements were assigned to the cavity walls and floor. Loading did not have a significant effect on microleakage in either the resin-coated or non-coated group. Resin coating significantly reduced microleakage regardless of loading. The cavity floor exhibited greater microleakage compared to the cavity wall. TSM observation also revealed that microleakage at the enamel surface was minimal regardless of resin coating. In contrast, non-coated dentin showed extensive leakage, whereas resin-coated dentin showed decreased leakage. Resin coating with a combination of a dentin-bonding system and a flowable resin composite may be indicated prior to impression-taking when restoring teeth with CAD/CAM ceramic inlays in order to reduce microleakage at the tooth-resin interface.

A transmission positron microscope and a scanning positron microscope being built at KEK, Japan

International Nuclear Information System (INIS)

Doyama, M.; Inoue, M.; Kogure, Y.; Kurihara, T.; Yagishita, A.; Shidara, T.; Nakahara, K.; Hayashi, Y.; Yoshiie, T.

2001-01-01

This paper reports the plans of positron microscopes being built at KEK (High Energy Accelerator Research Organization), Tsukuba, Japan improving used electron microscopes. The kinetic energies of positron produced by accelerators or by nuclear decays have not a unique value but show a spread over in a wide range. Positron beam will be guided near electron microscopes, a transmission electron microscope (JEM100S) and a scanning electron microscope (JSM25S). Positrons are slowed down by a tungsten foil, accelerated and focused on a nickel sheet. The monochromatic focused beam will be injected into an electron microscope. The focusing of positrons and electrons is achieved by magnetic system of the electron microscopes. Imaging plates are used to record positron images for the transmission electron microscope. (orig.)

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.

Microscopic study of rock for estimating long-term behavior

International Nuclear Information System (INIS)

Ichikawa, Yasuaki

2002-03-01

Micro-structure of rock plays a essential role for their long-term behavior. For elucidating long-term characteristics of granite we here present the followings: 1) Conforcal Laser Scanning Microscope (LSM) observation of joint surfaces of granite and Fourier analysis, 2) characterization of the mechanism of microcrack initiation and propagation observed by stereoscopic microscope under uniaxial/triaxial compression and relaxation tests, 3) observation of microcrack initiation and propagation by LSM under uniaxial compression, and 4) a viscoelastic homogenization theory to predict the long-term behavior of micro/macro-level stress for granite. Rock image processing and analysis become a fundamental procedure to determine rock surface discontinuities. But the complexity of rock surface discontinuities seems beyond the manual image processing method. In Chapter 2 a Conforcal Laser Scanning Microscope that can acquire three-dimensional images is introduced to observe the rock roughness of a discontinuity. Then, scanning three-dimensional images are changed its data form in order to adapt various image analysis programs, and granitic rock roughness of discontinuities are displayed by graphic images. For example, these datas are analyzed by Discrete Fourier Transformation (DFT) program and Inverse Discrete Fourier Transformation (IDFT) program. Microcrack generation and propagation play an essential role to predict the long-term behavior of rock. In Chapter 3 a progressive development of cracking in granite is revealed by using stereoscopic microscope under triaxial compression condition and by using LSM under uniaxial compression condition. With a viscoelastic theory applied in homogenization method, we can calculate macro behavior of medium influenced by its micro structure by analyse the long-term time-dependent behavior of granite under the same condition to the relaxation experiment. (author)

Literature survey on microscopic friction modeling

NARCIS (Netherlands)

Hol, J.

2010-01-01

To better understand contact and friction conditions, experimental and theoretical studies have been performed in order to take microscopic dependencies into account. Friction is developed on microscopic level by adhesion between contacting asperities, the ploughing effect between asperities and the

Investigation of single defects created in crystals by laser emission and hard radiation

International Nuclear Information System (INIS)

Martynovich, E F; Dresvyanskiy, V P; Boychenko, S V; Rakevich, A L; Zilov, S A; Bagayev, S N

2017-01-01

The possibility of identifying radiation-created quantum systems via the characteristics of quantum trajectories of luminescence intensity measured on individual centers by confocal scanning fluorescence microscopy with the time-correlated single photon counting has been studied. Calculations of the quantum trajectories have been carried out by the density matrix method. Experimental studies have been carried out using a confocal microscope. (paper)

Confocal laser scanning microscopy to estimate nanoparticles’ human skin penetration in vitro

Science.gov (United States)

Elmahdy, Akram; Cao, Yachao; Hui, Xiaoying; Maibach, Howard

2017-01-01

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. PMID:29184403

EXAFS study influence of pH on microscopic structure of zinc

International Nuclear Information System (INIS)

Li Xianliang; Chongqing Entry-Exit Inspection and Quarantine Bureau, Chongqing; Pan Gang; Zhu Mengqiang; Chen Hao; Hu Tiandou; Wu Ziyu; Xie Yaning; Du Yonghua

2004-01-01

Microscopic local structures of Zn(II) were studied using extended X-ray absorption fine structure (EXAFS) spectroscopy under different pH conditions. When pH 2+ (aq) was coordinated with six water molecules and the average Zn-O distance was measured to be 2.08 Angstrom, which indicated that hydrated Zn 2+ (aq) ions were in octahedral geometry under acid conditions. Under alkaline conditions, Zn 2+ (aq) was coordinated with four water molecules and the average Zn-O distance was measured to be 1.96 Angstrom, which indicated that hydrated Zn 2+ (aq) ions were in tetrahedral geometry. EXAFS results provided detailed information on the form and microscopic structure of hydrated Zn(II) ions under different pH conditions, which were fundamental for understanding the reactivity of Zn(II) in solutions and at particle-water interfaces. (authors)

Laser confocal measurement system for curvature radius of lenses based on grating ruler

Science.gov (United States)

Tian, Jiwei; Wang, Yun; Zhou, Nan; Zhao, Weirui; Zhao, Weiqian

2015-02-01

In the modern optical measurement field, the radius of curvature (ROC) is one of the fundamental parameters of optical lens. Its measurement accuracy directly affects the other optical parameters, such as focal length, aberration and so on, which significantly affect the overall performance of the optical system. To meet the demand of measurement instruments for radius of curvature (ROC) with high accuracy in the market, we develop a laser confocal radius measurement system with grating ruler. The system uses the peak point of the confocal intensity curve to precisely identify the cat-eye and confocal positions and then measure the distance between these two positions by using the grating ruler, thereby achieving the high-precision measurement for the ROC. The system has advantages of high focusing sensitivity and anti-environment disturbance ability. And the preliminary theoretical analysis and experiments show that the measuring repeatability can be up to 0.8 um, which can provide an effective way for the accurate measurement of ROC.

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)

Development of an ultrasound microscope combined with optical microscope for multiparametric characterization of a single cell.

Science.gov (United States)

Arakawa, Mototaka; Shikama, Joe; Yoshida, Koki; Nagaoka, Ryo; Kobayashi, Kazuto; Saijo, Yoshifumi

2015-09-01

Biomechanics of the cell has been gathering much attention because it affects the pathological status in atherosclerosis and cancer. In the present study, an ultrasound microscope system combined with optical microscope for characterization of a single cell with multiple ultrasound parameters was developed. The central frequency of the transducer was 375 MHz and the scan area was 80 × 80 μm with up to 200 × 200 sampling points. An inverted optical microscope was incorporated in the design of the system, allowing for simultaneous optical observations of cultured cells. Two-dimensional mapping of multiple ultrasound parameters, such as sound speed, attenuation, and acoustic impedance, as well as the thickness, density, and bulk modulus of specimen/cell under investigation, etc., was realized by the system. Sound speed and thickness of a 3T3-L1 fibroblast cell were successfully obtained by the system. The ultrasound microscope system combined with optical microscope further enhances our understanding of cellular biomechanics.

Normal microscopic architecture of acetabular labrum of hip joint: a qualitative original study with clinical aspects.

Science.gov (United States)

Kapetanakis, Stylianos; Gkantsinikoudis, Nikolaos; Dermon, Antonios; Kommata, Vassiliki; Papathanasiou, Jannis; Soukakos, Panagiotis; Dermon, Caterina

2017-01-01

Normal histologic architecture of acetabular labrum, regarding presence of Free Nerve Endings (FNEs) and Nerve End Organs (NEOs) has been four times described. Nevertheless, elderly cadaveric specimens and individuals were recruited, leading to considerably high unreliability probability due to microscopic degenerative alterations. Aim of this paper is to analyze distribution pattern of FNEs and NEOs in acetabular labra of healthy middle-aged individuals, configuring thus more reliably acetabular labrum microscopic profile. Six patients with middle age 52 ± 2.5 years were enrolled in this study. Injury of acetabular labrum and normal hip radiograph were present in all cases. Patients were all subjected to successful hip hemi-arthroplasty and derived acetabular labra were subsequently histologically processed and observed under a compound microscope. FNEs and NEOs were detected in all specimens. All types of NEOs were identified, including Paccini, Golgi-Mazzoni, Ruffini and Krause corpuscles. FNEs and NEOs were both in ventral part and in chondral side of labrum predominantly detected. FNEs and NEOs presence was greater in ventral side of labrum, being thus in partial agreement with previous studies results. Further study is required, in order to elucidate the exact acetabular labrum normal microscopic anatomy. IV.

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

Analysis of the in vivo confocal Raman spectral variability in human skin

Science.gov (United States)

Mogilevych, Borys; dos Santos, Laurita; Rangel, Joao L.; Grancianinov, Karen J. S.; Sousa, Mariane P.; Martin, Airton A.

2015-06-01

Biochemical composition of the skin changes in each layer and, therefore, the skin spectral profile vary with the depth. In this work, in vivo Confocal Raman spectroscopy studies were performed at different skin regions and depth profile (from the surface down to 10 μm) of the stratum corneum, to verify the variability and reproducibility of the intra- and interindividual Raman data. The Raman spectra were collected from seven healthy female study participants using a confocal Raman system from Rivers Diagnostic, with 785 nm excitation line and a CCD detector. Measurements were performed in the volar forearm region, at three different points at different depth, with the step of 2 μm. For each depth point, three spectra were acquired. Data analysis included the descriptive statistics (mean, standard deviation and residual) and Pearson's correlation coefficient calculation. Our results show that inter-individual variability is higher than intraindividual variability, and variability inside the SC is higher than on the skin surface. In all these cases we obtained r values, higher than 0.94, which correspond to high correlation between Raman spectra. It reinforces the possibility of the data reproducibility and direct comparison of in vivo results obtained with different study participants of the same age group and phototype.

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)

Blood capillary length estimation from three-dimensional microscopic data by image analysis and stereology.

Science.gov (United States)

Kubínová, Lucie; Mao, Xiao Wen; Janáček, Jiří

2013-08-01

Studies of the capillary bed characterized by its length or length density are relevant in many biomedical studies. A reliable assessment of capillary length from two-dimensional (2D), thin histological sections is a rather difficult task as it requires physical cutting of such sections in randomized directions. This is often technically demanding, inefficient, or outright impossible. However, if 3D image data of the microscopic structure under investigation are available, methods of length estimation that do not require randomized physical cutting of sections may be applied. Two different rat brain regions were optically sliced by confocal microscopy and resulting 3D images processed by three types of capillary length estimation methods: (1) stereological methods based on a computer generation of isotropic uniform random virtual test probes in 3D, either in the form of spatial grids of virtual "slicer" planes or spherical probes; (2) automatic method employing a digital version of the Crofton relations using the Euler characteristic of planar sections of the binary image; and (3) interactive "tracer" method for length measurement based on a manual delineation in 3D of the axes of capillary segments. The presented methods were compared in terms of their practical applicability, efficiency, and precision.

Atomic force microscopic study of the influence of physical stresses on Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Science.gov (United States)

Adya, Ashok K; Canetta, Elisabetta; Walker, Graeme M

2006-01-01

Morphological changes in the cell surfaces of the budding yeast Saccharomyces cerevisiae (strain NCYC 1681), and the fission yeast Schizosaccharomyces pombe (strain DVPB 1354), in response to thermal and osmotic stresses, were investigated using an atomic force microscope. With this microscope imaging, together with measurements of culture viability and cell size, it was possible to relate topological changes of the cell surface at nanoscale with cellular stress physiology. As expected, when the yeasts were exposed to thermostress or osmostress, their viability together with the mean cell volume decreased in conjunction with the increase in thermal or osmotic shock. Nevertheless, the viability of cells stressed for up to 1 h remained relatively high. For example, viabilities were >50% and >90% for the thermostressed, and >60% and >70% for the osmostressed S. cerevisiae and Schiz. pombe, respectively. Mean cell volume measurements, and bearing and roughness analyses of atomic force microscope images of stressed yeasts indicate that Schiz. pombe may be more resistant to physical stresses than S. cerevisiae. Overall, this study has highlighted the usefulness of atomic force microscope in studies of yeast stress physiology.

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.

Study and application of microscopic depletion model in core simulator of COSINE project

International Nuclear Information System (INIS)

Hu Xiaoyu; Wang Su; Yan Yuhang; Liu Zhanquan; Chen Yixue; Huang Kai

2013-01-01

Microscopic depletion correction is one of the commonly used techniques that could improve the historical effect and attain higher precision of diffusion calculation and alleviate the inaccuracy caused by historical effect. Core simulator of COSINE project (core and system integrated engine for design and analysis) has developed a hybrid macroscopic-microscopic depletion model to track important isotopes during each depletion history and correct the macro cross sections. The basic theory was discussed in this paper. The effect and results of microscopic depletion correction were also analyzed. The preliminary test results demonstrate that the microscopic depletion model is effective and practicable for improving the precision of core calculation. (authors)

Confocal microscopy findings in deep anterior lamellar keratoplasty performed after Descemet's stripping automated endothelial keratoplasty

Directory of Open Access Journals (Sweden)

Pang A

2014-01-01

Full Text Available Audrey Pang,1,2 Karim Mohamed-Noriega,1 Anita S Chan,1,3–5 Jodbhir S Mehta1,3 1Singapore National Eye Centre, 2Department of Ophthalmology, Tan Tock Seng Hospital, 3Singapore Eye Research Institute, 4Department of Histopathology, Pathology, Singapore General Hospital, 5Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Background: This study describes the in vivo confocal microscopy findings in two patients who had deep anterior lamellar keratoplasty (DALK following Descemet's stripping automated endothelial keratoplasty (DSAEK. Methods: The study reviewed the cases of two patients who first underwent DSAEK followed by DALK when their vision failed to improve due to residual stromal scarring. In the first case, a DSAEK was performed for a patient with pseudophakic bullous keratopathy. After surgery, the patient's vision failed to improve satisfactorily due to residual anterior stromal opacity and irregularity. Subsequently, the patient underwent a DALK. The same two consecutive operations were performed for a second patient with keratoconus whose previous penetrating keratoplasty had failed and had secondary graft ectasia. In vivo confocal microscopy was performed 2 months after the DALK surgery in both cases. Results: At 3 months after DALK, the best-corrected visual acuity was 6/30 in case 1 and 6/24 in case 2. In vivo confocal microscopy in both cases revealed the presence of quiescent keratocytes in the stroma layers of the DSAEK and DALK grafts, which was similar in the central and peripheral cornea. There was no activated keratocytes or haze noted in the interface between the grafts. Conclusion: Our short-term results show that performing a DALK after a DSAEK is an effective way of restoring cornea clarity in patients with residual anterior stromal opacity. In vivo confocal microscopy showed that there were no activated keratocytes seen in the interface of the grafts, which suggests

Microscopic approach to nuclear anharmonicities

International Nuclear Information System (INIS)

Matsuo, Masayuki; Shimizu, Yoshifumi; Matsuyanagi, Kenichi

1985-01-01

Present status of microscopic study of nuclear anharmonicity phenomena is reviewed from the viewpoint of the time-dependent Hartree-Bogoliubov approach. Both classical- and quantum-mechanical aspects of this approach are discussed. The Bohr-Mottelson-type collective Hamiltonian for anharmonic gamma vibrations is microscopically derived by means of the self-consistent-collective-coordinate method, and applied to the problem of two-phonon states of 168 Er. (orig.)

Miniaturized integration of a fluorescence microscope

Science.gov (United States)

Ghosh, Kunal K.; Burns, Laurie D.; Cocker, Eric D.; Nimmerjahn, Axel; Ziv, Yaniv; Gamal, Abbas El; Schnitzer, Mark J.

2013-01-01

The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals towards relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including semiconductor light source and sensor. This device enables high-speed cellular-level imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens. PMID:21909102

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

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

Normal microscopic architecture of acetabular labrum of hip joint: a qualitative original study with clinical aspects

Science.gov (United States)

Kapetanakis, Stylianos; Gkantsinikoudis, Nikolaos; Dermon, Antonios; Kommata, Vassiliki; Papathanasiou, Jannis; Soukakos, Panagiotis; Dermon, Caterina

2017-01-01

Summary Background Normal histologic architecture of acetabular labrum, regarding presence of Free Nerve Endings (FNEs) and Nerve End Organs (NEOs) has been four times described. Nevertheless, elderly cadaveric specimens and individuals were recruited, leading to considerably high unreliability probability due to microscopic degenerative alterations. Aim of this paper is to analyze distribution pattern of FNEs and NEOs in acetabular labra of healthy middle-aged individuals, configuring thus more reliably acetabular labrum microscopic profile. Materials and methods Six patients with middle age 52 ± 2.5 years were enrolled in this study. Injury of acetabular labrum and normal hip radiograph were present in all cases. Patients were all subjected to successful hip hemi-arthroplasty and derived acetabular labra were subsequently histologically processed and observed under a compound microscope. Results FNEs and NEOs were detected in all specimens. All types of NEOs were identified, including Paccini, Golgi-Mazzoni, Ruffini and Krause corpuscles. FNEs and NEOs were both in ventral part and in chondral side of labrum predominantly detected. Conclusion FNEs and NEOs presence was greater in ventral side of labrum, being thus in partial agreement with previous studies results. Further study is required, in order to elucidate the exact acetabular labrum normal microscopic anatomy. Level of evidence IV. PMID:29264339

Cryogenic immersion microscope

Science.gov (United States)

Le Gros, Mark; Larabell, Carolyn A.

2010-12-14

A cryogenic immersion microscope whose objective lens is at least partially in contact with a liquid reservoir of a cryogenic liquid, in which reservoir a sample of interest is immersed is disclosed. When the cryogenic liquid has an index of refraction that reduces refraction at interfaces between the lens and the sample, overall resolution and image quality are improved. A combination of an immersion microscope and x-ray microscope, suitable for imaging at cryogenic temperatures is also disclosed.

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.

Electron-microscope study of cloud and fog nuclei

Energy Technology Data Exchange (ETDEWEB)

Ogiwara, S; Okita, T

1952-01-01

Droplets of clouds on a mountain and of fog in an urban area were captured and the form, nature and size of their nuclei were studied by means of an electron-microscope and by a chamber of constant humidity. These nuclei have similar form and nature to the hygroscopic particles in haze and to the artificially produced combustion particles. No sea-salt nuclei were found in our observations, therefore, sea-spray appears to be an insignificant source of condensation nuclei. It was found that both the cloud and the fog nuclei originated in combustion products which were the mixture of hygroscopic and non-hygroscopic substances, and that the greater part of the nuclei did not contain pure sulfuric acid.

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

New microscope produced by Lambda Praha Co. applicable to field studies of microorganisms

Czech Academy of Sciences Publication Activity Database

Žižka, Zdeněk

2008-01-01

Roč. 53, č. 5 (2008), s. 457-461 ISSN 0015-5632 Institutional research plan: CEZ:AV0Z50200510 Keywords : lambda * microscope * field studies Subject RIV: EE - Microbiology, Virology Impact factor: 1.172, year: 2008

Microscopy based studies on the interaction of bio-based silver nanoparticles with Bombyx mori Nuclear Polyhedrosis virus.

Science.gov (United States)

Tamilselvan, Selvaraj; Ashokkumar, Thirunavukkarasu; Govindaraju, Kasivelu

2017-04-01

In the present investigation, silver nanoparticles (AgNPs) interactions with Bombyx mori Nuclear Polyhedrosis virus (BmNPV) were characterized using High-Resolution Scanning Electron Microscopy (HR-SEM), Energy Dispersive X-ray Analysis (EDAX), Transmission Electron Microscopy (TEM), Atomic Force Microcopy (AFM) and Confocal Microscope (CM). HR-SEM study reveals that the biosynthesized AgNPs have interacted with BmNPV and were found on the surface. TEM micrographs of normal and viral polyhedra treated with AgNPs showed that the nanoparticles were accumulated in the membrane and it was noted that some of the AgNPs successfully penetrated the membrane by reaching the capsid of BmNPV. AFM and confocal microscopy studies reveal that the disruption in the shell membrane tends to lose its stability due to exposure of AgNPs to BmNPV. Copyright © 2017 Elsevier B.V. All rights reserved.

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)

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)

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

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...

Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy

Science.gov (United States)

Glazachev, Yu I.; Orlova, D. Y.; Řezníčková, P.; Bártová, E.

2018-05-01

We proposed an effective kinetics scheme of photolysis of green fluorescent protein (GFP) observed in live cells with a commercial confocal fluorescence microscope. We investigated the photolysis of GFP-tagged heterochromatin protein, HP1β-GFP, in live nucleus with the pulse position modulation approach, which has several advantages over the classical pump-and-probe method. At the basis of the proposed scheme lies a process of photoswitching from the native fluorescence state to the intermediate fluorescence state, which has a lower fluorescence yield and recovers back to native state in the dark. This kinetics scheme includes four effective parameters (photoswitching, reverse switching, photodegradation rate constants, and relative brightness of the intermediate state) and covers the time scale from dozens of milliseconds to minutes of the experimental fluorescence kinetics. Additionally, the applicability of the scheme was demonstrated in the cases of continuous irradiation and the classical pump-and-probe approach using numerical calculations and analytical solutions. An interesting finding of experimental data analysis was that the overall photodegradation of GFP proceeds dominantly from the intermediate state, and demonstrated approximately the second-order reaction versus irradiation power. As a practical example, the proposed scheme elucidates the artifacts of fluorescence recovery after the photobleaching method, and allows us to propose some suggestions on how to diminish them.

Proceedings of the workshop on microscopic and phenomenological studies of the interactions between light-heavy ions

International Nuclear Information System (INIS)

Yamaguchi, S.

1993-01-01

The workshop 'Microscopic and Phenomenological Studies of the Interactions between Light-Heavy Ions' was held at Institute for Nuclear Study, University of Tokyo from Dec. 24 to Dec. 26, 1991. The workshop included 1) studies of the nucleus-nucleus interactions of the systems as 16 O- 16 O, 16 O- 15 N, etc., or the studies of the elastic and inelastic scatterings and the transfer reactions in such systems, 2) structure and reactions of neutron-rich nuclei, 3) microscopic derivation of the effective two-nucleon interactions, 4) development of the methods of techniques applied to the heavier systems. (author)

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.

Light Microsopy Module, International Space Station Premier Automated Microscope

Science.gov (United States)

Meyer, William V.; Sicker, Ronald J.; Chiaramonte, Francis P.; Brown, Daniel F.; O'Toole, Martin A.; Foster, William M.; Motil, Brian J.; Abbot-Hearn, Amber Ashley; Atherton, Arthur Johnson; Beltram, Alexander;

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

STM-SQUID probe microscope

International Nuclear Information System (INIS)

Hayashi, Tadayuki; Tachiki, Minoru; Itozaki, Hideo

2007-01-01

We have developed a STM-SQUID probe microscope. A high T C SQUID probe microscope was combined with a scanning tunneling microscope for investigation of samples at room temperature in air. A high permeability probe needle was used as a magnetic flux guide to improve the spatial resolution. The probe with tip radius of less than 100 nm was prepared by microelectropolishing. The probe was also used as a scanning tunneling microscope tip. Topography of the sample surface could be measured by the scanning tunneling microscope with high spatial resolution prior to observation by SQUID microscopy. The SQUID probe microscope image could be observed while keeping the distance from the sample surface to the probe tip constant. We observed a topographic image and a magnetic image of Ni fine pattern and also a magnetically recorded hard disk. Furthermore we have investigated a sample vibration method of the static magnetic field emanating from a sample with the aim of achieving a higher signal-to-noise (S/N) ratio

Photon scanning tunneling microscope in combination with a force microscope

NARCIS (Netherlands)

Moers, M.H.P.; Moers, M.H.P.; Tack, R.G.; van Hulst, N.F.; Bölger, B.; Bölger, B.

1994-01-01

The simultaneous operation of a photon scanning tunneling microscope with an atomic force microscope is presented. The use of standard atomic force silicon nitride cantilevers as near-field optical probes offers the possibility to combine the two methods. Vertical forces and torsion are detected

Characterization of particle deformation during compression measured by confocal laser scanning microscopy.

Science.gov (United States)

Guo, H X; Heinämäki, J; Yliruusi, J

1999-09-20

Direct compression of riboflavin sodium phosphate tablets was studied by confocal laser scanning microscopy (CLSM). The technique is non-invasive and generates three-dimensional (3D) images. Tablets of 1% riboflavin sodium phosphate with two grades of microcrystalline cellulose (MCC) were individually compressed at compression forces of 1.0 and 26.8 kN. The behaviour and deformation of drug particles on the upper and lower surfaces of the tablets were studied under compression forces. Even at the lower compression force, distinct recrystallized areas in the riboflavin sodium phosphate particles were observed in both Avicel PH-101 and Avicel PH-102 tablets. At the higher compression force, the recrystallization of riboflavin sodium phosphate was more extensive on the upper surface of the Avicel PH-102 tablet than the Avicel PH-101 tablet. The plastic deformation properties of both MCC grades reduced the fragmentation of riboflavin sodium phosphate particles. When compressed with MCC, riboflavin sodium phosphate behaved as a plastic material. The riboflavin sodium phosphate particles were more tightly bound on the upper surface of the tablet than on the lower surface, and this could also be clearly distinguished by CLSM. Drug deformation could not be visualized by other techniques. Confocal laser scanning microscopy provides valuable information on the internal mechanisms of direct compression of tablets.

In vitro blood flow in a rectangular PDMS microchannel: experimental observations using a confocal micro-PIV system.

Science.gov (United States)

Lima, Rui; Wada, Shigeo; Tanaka, Shuji; Takeda, Motohiro; Ishikawa, Takuji; Tsubota, Ken-ichi; Imai, Yohsuke; Yamaguchi, Takami

2008-04-01

Progress in microfabricated technologies has attracted the attention of researchers in several areas, including microcirculation. Microfluidic devices are expected to provide powerful tools not only to better understand the biophysical behavior of blood flow in microvessels, but also for disease diagnosis. Such microfluidic devices for biomedical applications must be compatible with state-of-the-art flow measuring techniques, such as confocal microparticle image velocimetry (PIV). This confocal system has the ability to not only quantify flow patterns inside microchannels with high spatial and temporal resolution, but can also be used to obtain velocity measurements for several optically sectioned images along the depth of the microchannel. In this study, we investigated the ability to obtain velocity measurements using physiological saline (PS) and in vitro blood in a rectangular polydimethysiloxane (PDMS) microchannel (300 microm wide, 45 microm deep) using a confocal micro-PIV system. Applying this combination, measurements of trace particles seeded in the flow were performed for both fluids at a constant flow rate (Re = 0.02). Velocity profiles were acquired by successive measurements at different depth positions to obtain three-dimensional (3-D) information on the behavior of both fluid flows. Generally, the velocity profiles were found to be markedly blunt in the central region, mainly due to the low aspect ratio (h/w = 0.15) of the rectangular microchannel. Predictions using a theoretical model for the rectangular microchannel corresponded quite well with the experimental micro-PIV results for the PS fluid. However, for the in vitro blood with 20% hematocrit, small fluctuations were found in the velocity profiles. The present study clearly shows that confocal micro-PIV can be effectively integrated with a PDMS microchannel and used to obtain blood velocity profiles along the full depth of the microchannel because of its unique 3-D optical sectioning ability

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.

In-situ detection of drugs-of-abuse on clothing using confocal Raman microscopy

Energy Technology Data Exchange (ETDEWEB)

Ali, Esam M.A. [Raman Spectroscopy Group, University Analytical Centre, Division of Chemical and Forensic Sciences, University of Bradford, Bradford BD7 1DP (United Kingdom); Edwards, Howell G.M. [Raman Spectroscopy Group, University Analytical Centre, Division of Chemical and Forensic Sciences, University of Bradford, Bradford BD7 1DP (United Kingdom)], E-mail: h.g.m.edwards@bradford.ac.uk; Hargreaves, Michael D.; Scowen, Ian J. [Raman Spectroscopy Group, University Analytical Centre, Division of Chemical and Forensic Sciences, University of Bradford, Bradford BD7 1DP (United Kingdom)

2008-05-12

This study describes the application of confocal Raman microscopy to the detection and identification of drugs-of-abuse in situ on undyed natural synthetic fibres, and coloured textile specimens. Raman spectra were obtained from drug particles trapped between the fibres of the specimens. Pure samples of cocaine hydrochloride and N-methyl-3,4-methylenedioxy-amphetamine HCl (MDMA-HCl) were used in this study. Raman spectra were collected from drug particles of an average size in the range 5-15 {mu}m. Despite the presence of spectral bands arising from the natural and synthetic polymer and dyed textiles, the drugs could be identified by their characteristic Raman bands. If necessary, interfering bands could be successfully removed by spectral subtraction. Furthermore, Raman spectra were recorded from drug particles trapped between the fibres of highly fluorescent specimens. Interference from the fibres, including background fluorescence, was overcome by careful focusing of the confocal beam and the resulting spectra allow ready differentiation from interference from the fibres substrate bands. Spectra of several drugs-of-abuse on dyed and undyed clothing substrates were readily obtained within 3 min with little or no sample preparation and with no alteration of the evidential material.