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Sample records for 1-kw confocal gyro-traveling-wave

  1. Demonstration of a 140-GHz 1-kW Confocal Gyro-Traveling-Wave Amplifier

    Joye, Colin D.; Shapiro, Michael A.; Sirigiri, Jagadishwar R.; Temkin, Richard J.

    2009-01-01

    The theory, design, and experimental results of a wideband 140-GHz 1-kW pulsed gyro-traveling-wave amplifier (gyro-TWA) are presented. The gyro-TWA operates in the HE06 mode of an overmoded quasi-optical waveguide using a gyrating electron beam. The electromagnetic theory, interaction theory, design processes, and experimental procedures are described in detail. At 37.7 kV and a 2.7-A beam current, the experiment has produced over 820 W of peak power with a –3-dB bandwidth of 0.8 GHz and a li...

  2. Theory of relativistic gyro-traveling wave devices

    Using a Hamiltonian formalism, nonlinear, fully relativistic, multimode, multifrequency equations are derived which describe gyro-traveling wave devices. Nonuniform waveguides and nonlinearly tapered magnetic fields are incorporated into the analysis. The formalism is used to analyze the effect of velocity spread on Doppler upshifted operation. It is shown that with present technology, gyro-traveling wave devices cannot operate far from cutoff if high efficiency is desired. As an example, the analysis is applied to a 10 GHz, 430 kV, 240 A gyrotwistron operating at the fundamental cyclotron harmonic with a tapered wall radius and magnetic field. A realistic design that achieves over 30% efficiency is produced. The issue of stability of this device with respect to competition from parasitic modes is taken up in a companion paper [Phys. Plasmas 2, 3511 (1995)]. copyright 1995 American Institute of Physics

  3. Performance evaluation of 1 kw PEFC

    Komaki, Hideaki [Ishikawajima-Harima Heavy Industries Co., Ltd. Tokyo (Japan); Tsuchiyama, Syozo [Shipbuilding Research Association, Minato-ky, Tokyo (Japan)

    1996-12-31

    This report covers part of a joint study on a PEFC propulsion system for surface ships, summarized in a presentation to this Seminar, entitled {open_quote}Study on a PEFC Propulsion System for Surface Ships{close_quotes}, and which envisages application to a 1,500 DWT cargo vessel. The aspect treated here concerns the effects brought on PEFC operating performance by conditions particular to shipboard operation. The performance characteristics were examined through tests performed on a 1 kw stack and on a single cell (Manufactured by Fuji Electric Co., Ltd.). The tests covered the items (1) to (4) cited in the headings of the sections that follow. Specifications of the stack and single cell are as given.

  4. Design and development of 1 KW solid state RF amplifier

    Since low power tube based RF amplifiers are complicated, occupy a large space and are bulky, the efforts are on to develop indigenously 1 KW solid state technology based RF Power amplifier. A power level of 1KW is chosen for the initial design because RF power Mosfets upto 250 watt are easily available and by clubbing 3-4 stages the power level of 1 KW can be made. Presently design and testing of 100-watt stage is in progress. The first 2 stages are designed to give 5 Watt RF power using bipolar transistors and are operated in CE, Class A to provide low noise level at the output of the system. The 3rd stage will be MOSFET based MRF 174, which is ideally suited for class A operation and is designed for 100 Watt RF power. The last stage will be MOSFET based ARF446 power MOSFET in TO-247 plastic package. This amplifier will be used in the classical push- pull configuration. This paper describes the design aspects as well as the test results of 100 watt amplifier on 50 Ohm dummy load along with the specifications, design criteria, circuit used, operating parameters of 1 KW Solid State RF power amplifier to be used as driver for 91.2 MHz, 1.5 MW stage for ICRH experiments on SST-1 tokamak .

  5. Investigation of a 140 GHz gryo-backward wave oscillator and a 95 GHz gyro-traveling wave

    There is current interest in developing a high-power source of continuously tunable millimeter wave radiation as a RF driver for high-power gyrotron, CARM, or FEL amplifiers. The gyrotron backward wave oscillator is a voltage-tunable fast-wave device that can satisfy these requirements. This thesis reports on the design and experimental investigation of a 130--145 GHz gyrotron backward wave oscillator Novel operating features of this design include the use of a 80-kV, 6.2-A Pierce-wiggler electron beam source, a broadband motheye window and an overmoded TE1,2 cylindrical interaction waveguide. Although this device has demonstrated voltage-tunable operation over the design range in the TE1,2 mode, the frequency tuning is not continuous, output powers were low and full-beam transmission through the interaction region was not possible. Simulations indicate that the beam has very high velocity spread induced by space charge forces in the compression region. After increasing the diameter of the beam tunnel to achieve full transmission, the TE1,2 was not found. It is felt that the scraping off of the beam edge in the initial experiments allowed a better quality beam to enter the interaction region and inhibited coupling into competing forward wave modes. The need for radar systems with greater resolution has led to renewed interest in the development of efficient high-power amplifiers at 95 GHz. Current sources are limited to 6--8 kW of output power due to ohmic loading on the slow-wave circuit. A gyrotron traveling wave tube amplifier is capable of efficient operation over a wide bandwidth with the added attraction of low ohmic loading on the smooth fast-wave circuit. This thesis reports of the design a 95-GHz gyrotron traveling wave tube amplifier that is capable of high power (125 kW), high gain (38 dB), large bandwidth > 5 GHz and high efficiencies (> 30%)

  6. Confocal laser endomicroscopy

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

    2015-01-01

    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...

  7. Molecular confocal laser endomicroscopy

    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...

  8. Confocal Raman Microscopy

    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.

  9. Laser differential confocal radius measurement.

    Zhao, Weiqian; Sun, Ruoduan; Qiu, Lirong; Sha, Dingguo

    2010-02-01

    A new laser differential confocal radius measurement (DCRM) is proposed for high precision measurement of radius. Based on the property of an axial intensity curve that the absolute zero precisely corresponds to the focus of the objective in a differential confocal system (DCS), DCRM uses the zero point of the DCS axial intensity curve to precisely identify the cat's-eye and confocal positions of the test lens, and measures the accurate distance between the two positions to achieve the high-precision measurement of radius of curvature (ROC). In comparison with the existing measurement methods, DCRM proposed has a high measurement precision, a strong environmental anti-interference capability and a low cost. The theoretical analyses and preliminary experimental results indicate that DCRM has a relative measurement error of better than 5 ppm. PMID:20174065

  10. Twin-Photon Confocal Microscopy

    Simon, D S

    2010-01-01

    A recently introduced two-channel confocal microscope with correlated detection promises up to 50% improvement in transverse spatial resolution [Simon, Sergienko, Optics Express {\\bf 18}, 9765 (2010)]. Here we move further by introducing a triple-confocal correlated microscope, exploiting the correlations present in optical parametric amplifiers. It is based on tight focusing of pump radiation onto a thin sample positioned in front of a nonlinear crystal, followed by coincidence detection of signal and idler photons, each focused onto a pinhole. This approach offers further resolution enhancement in microscopy.

  11. DMD-enabled confocal microendoscopy

    Lane, Pierre M.; Dlugan, Andrew L. P.; MacAulay, Calum E.

    2001-05-01

    Conventional endoscopy is limited to imaging macroscopic views of tissue. The British Columbia Cancer Research Center, in collaboration with Digital Optical Imaging Corp., is developing a fiber-bundle based microendoscopy system to enable in vivo confocal imaging of cells and tissue structure through the biopsy channel of an endoscope, hypodermic needle, or catheter. The feasibility of imaging individual cells and tissue architecture will be presented using both reflectance and tissue auto-fluorescence modes of imaging. The system consists of a coherent fiber bundle, low-magnification high-NA objective lens, Digital Micromirror DeviceTM(DMD), light source, and CCD camera. The novel approach is the precise control and manipulation of light flow into and out of individual optical fibers. This control is achieved by employing a DMD to illuminate and detect light from selected fibers such that only the core of each fiber is illuminated or detected. The objective of the research is to develop a low-cost, clinically viable microendoscopy system for a range of detection, diagnostic, localization and differentiation uses associated with cancer and pre-cancerous conditions. Currently, multi-wavelength reflectance confocal images with 1 micrometers lateral resolution and 1.6 micrometers axial resolution have been achieved using a 0.95 mm bundle with 30,000 fibers.

  12. Quantitative phase-contrast confocal microscope

    Liu, Changgeng; Marchesini, Stefano; Kim, Myung K.

    2014-01-01

    We present a quantitative phase-contrast confocal microscope (QPCCM) by combining a line-scanning confocal system with digital holography (DH). This combination can merge the merits of these two different imaging modalities. High-contrast intensity images with low coherent noise, and the optical sectioning capability are made available due to the confocality. Phase profiles of the samples become accessible thanks to DH. QPCCM is able to quantitatively measure the phase variations of optical s...

  13. QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

    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.

  14. Confocal Endomicroscopy of Colorectal Polyps

    Vivian M. Ussui

    2012-01-01

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

  15. Confocal Annular Josephson Tunnel Junctions

    Monaco, Roberto

    2016-04-01

    The physics of Josephson tunnel junctions drastically depends on their geometrical configurations and here we show that also tiny geometrical details play a determinant role. More specifically, we develop the theory of short and long annular Josephson tunnel junctions delimited by two confocal ellipses. The behavior of a circular annular Josephson tunnel junction is then seen to be simply a special case of the above result. For junctions having a normalized perimeter less than one, the threshold curves in the presence of an in-plane magnetic field of arbitrary orientations are derived and computed even in the case with trapped Josephson vortices. For longer junctions, a numerical analysis is carried out after the derivation of the appropriate motion equation for the Josephson phase. We found that the system is modeled by a modified and perturbed sine-Gordon equation with a space-dependent effective Josephson penetration length inversely proportional to the local junction width. Both the fluxon statics and dynamics are deeply affected by the non-uniform annulus width. Static zero-field multiple-fluxon solutions exist even in the presence of a large bias current. The tangential velocity of a traveling fluxon is not determined by the balance between the driving and drag forces due to the dissipative losses. Furthermore, the fluxon motion is characterized by a strong radial inward acceleration which causes electromagnetic radiation concentrated at the ellipse equatorial points.

  16. Confocal Annular Josephson Tunnel Junctions

    Monaco, Roberto

    2016-09-01

    The physics of Josephson tunnel junctions drastically depends on their geometrical configurations and here we show that also tiny geometrical details play a determinant role. More specifically, we develop the theory of short and long annular Josephson tunnel junctions delimited by two confocal ellipses. The behavior of a circular annular Josephson tunnel junction is then seen to be simply a special case of the above result. For junctions having a normalized perimeter less than one, the threshold curves in the presence of an in-plane magnetic field of arbitrary orientations are derived and computed even in the case with trapped Josephson vortices. For longer junctions, a numerical analysis is carried out after the derivation of the appropriate motion equation for the Josephson phase. We found that the system is modeled by a modified and perturbed sine-Gordon equation with a space-dependent effective Josephson penetration length inversely proportional to the local junction width. Both the fluxon statics and dynamics are deeply affected by the non-uniform annulus width. Static zero-field multiple-fluxon solutions exist even in the presence of a large bias current. The tangential velocity of a traveling fluxon is not determined by the balance between the driving and drag forces due to the dissipative losses. Furthermore, the fluxon motion is characterized by a strong radial inward acceleration which causes electromagnetic radiation concentrated at the ellipse equatorial points.

  17. Diffractive elements performance in chromatic confocal microscopy

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

    2011-01-01

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

  18. Confocal Laser Endomicroscopy in Inflammatory Bowel Disease

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

    2015-01-01

    BACKGROUND AND AIMS: Confocal laser endomicroscopy is an endoscopic method that provides in vivo real-time imaging of the mucosa at a cellular level, elucidating mucosal changes that are undetectable by white light endoscopy. This paper systematically reviews current indications and perspectives...... 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...... 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...

  19. Confocal diffraction phase microscopy of live cells

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

    2008-01-01

    We present a new quantitative phase microscopy technique, confocal diffraction phase microscopy, which provides quantitative phase measurements from localized sites on a sample with high sensitivity. The technique combines common-path interferometry with confocal microscopy in a transmission geometry. The capability of the technique for static imaging is demonstrated by imaging polystyrene microspheres and live HT29 cells, while dynamic imaging is demonstrated by quantifying the nanometer sca...

  20. Needle-based confocal laser endomicroscopy

    Giovannini, Marc

    2015-01-01

    New applications of confocal laser endomicroscopy were developed as pCLE in the bile duct and nCLE for pancreatic cystic tumors, pancreatic masses and lymph nodes. The aim of this paper would be to give you an update in this new technology and to try to define its place in the diagnosis of cystic and solid pancreatic masses. The material used was a 19G EUS-needle in which the stylet was replaced by the Confocal mini-probe. The mini-probe (0.632 mm of diameter) is pre-loaded and screwed by a l...

  1. Neurosurgical confocal endomicroscopy: A review of contrast agents, confocal systems, and future imaging modalities

    Aqib H Zehri

    2014-01-01

    Full Text Available Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM, two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery.

  2. Image inpainting for the differential confocal microscope

    Qiu, Lirong; Wang, Lei; Liu, Dali; Hou, Maosheng; Zhao, Weiqian

    2015-02-01

    In the process of zero-crossing trigger measurement of differential confocal microscope, the sample surface features or tilt will cause the edges can't be triggered. Meanwhile, environment vibration can also cause false triggering. In order to restore the invalid information of sample, and realize high-precision surface topography measurement, Total Variation (TV) inpainting model is applied to restore the scanning images. Emulation analysis and experimental verification of this method are investigated. The image inpainting algorithm based on TV model solves the minimization of the energy equation by calculus of variations, and it can effectively restore the non-textured image with noises. Using this algorithm, the simulation confocal laser intensity curve and height curve of standard step sample are restored. After inpainting the intensity curve below the threshold is repaired, the maximum deviation from ideal situation is 0.0042, the corresponding edge contour of height curve is restored, the maximum deviation is 0.1920, which proves the algorithm is effective. Experiment of grating inpainting indicates that the TV algorithm can restore the lost information caused by failed triggering and eliminate the noise caused by false triggering in zero-crossing trigger measurement of differential confocal microscope. The restored image is consistent with the scanning result of OLYMPUS confocal microscope, which can satisfy the request of follow-up measurement analysis.

  3. Vibrometry using a chromatic confocal sensor

    Berkovic, G.; Zilberman, S.; Shafir, E.; Cohen-Sabban, J.

    2014-05-01

    We demonstrate vibrometry using a chromatic confocal sensor which measures displacements with 0.1 μm resolution at a rate of 10 kHz. This technique was used to study the vibration of a musical tuning fork with a resonance at 523 Hz. Other examples presented include vibration of water waves and multiple point vibrometry of a vibrating steel rod.

  4. Confocal Microscopy Imaging of the Biofilm Matrix

    Schlafer, Sebastian; Meyer, Rikke Louise

    2016-01-01

    . Confocal microscopes are held by many research groups, and a number of methods for qualitative and quantitative imaging of the matrix have emerged in recent years. This review provides an overview and a critical discussion of techniques used to visualize different matrix compounds, to determine the...

  5. Superresolution character of array confocal system

    HUANG Xiang-dong; TAN Jiu-bin

    2006-01-01

    For the incomplete theory of the array confocal system,the more accurate theoretical model is built, and the new type of three-zone amplitude pupil filter is presented to improve the 3D detecting ability of the array confocal system.The 3D imaging equation based on Kirchhoff's diffraction theory in the paper is more accurate,compared with the exciting theory,and it can describe the system imaging process more exactly.Using the evaluation criterion of 3D superresolution,the parameter of the filter pupil is optimized.It can reduce the axial and transversal FWHM of every detecting channel.The results of computer simulation and experiment prove that the filter can improve the axial and transversal resolution at the same time,which can increase the 3D measure capability.

  6. Laser differential confocal lens thickness measurement

    Based on the property that the absolute zero of an axial intensity curve exactly corresponds to the focus of the objective in a differential confocal system (DCS), a new laser differential confocal lens thickness measurement is proposed to achieve the high-precision non-contact measurement of lens thickness. The proposed approach uses the absolute zero of DCS axial response curve to precisely identify the vertexes of the test lens, obtains the central optical thickness of the test lens, and then uses the radius of curvature and refractive index of the test lens and the ray tracing facet iterative calculation to obtain the central geometrical thickness of the test lens. The theoretical analyses and preliminary experiments indicate that the measurement accuracy is better than 0.03%. (paper)

  7. MEMS-Based Dual Axes Confocal Microendoscopy

    Piyawattanametha, Wibool; Wang, Thomas D.

    2010-01-01

    We demonstrate a miniature, near-infrared microscope (λ = 785 nm) that uses a novel dual axes confocal architecture. Scalability is achieved with post-objective scanning, and a MEMS mirror provides real time (>4 Hz) in vivo imaging. This instrument can achieve sub-cellular resolution with deep tissue penetration and large field of view. An endoscope-compatible version can image digestive tract epithelium to guide tissue biopsy and monitor therapy.

  8. Spectrally encoded confocal scanning laser ophthalmoscope

    Tao, Yuankai K.; Izatt, Joseph A.

    2010-02-01

    Fundus imaging has become an essential clinical diagnostic tool in ophthalmology. Current generation scanning laser ophthalmoscopes (SLO) offer advantages over conventional fundus photography and indirect ophthalmoscopy in terms of light efficiency and contrast. As a result of the ability of SLO to provide rapid, continuous imaging of retinal structures and its versatility in accommodating a variety of illumination wavelengths, allowing for imaging of both endogenous and exogenous fluorescent contrast agents, SLO has become a powerful tool for the characterization of retinal pathologies. However, common implementations of SLO, such as the confocal scanning laser ophthalmoscope (CSLO) and line-scanning laser ophthalmoscope (LSLO), require imaging or multidimensional scanning elements which are typically implemented in bulk optics placed close to the subject eye. Here, we apply a spectral encoding technique in one dimension combined with single-axis lateral scanning to create a spectrally encoded confocal scanning laser ophthalmoscope (SECSLO) which is fully confocal. This novel implementation of the SLO allows for high contrast, high resolution in vivo human retinal imaging with image transmission through a single-mode optical fiber. Furthermore, the scanning optics are similar and the detection engine is identical to that of current-generation spectral domain optical coherence tomography (SDOCT) systems, potentially allowing for a simplistic implementation of a joint SECSLO-SDOCT imaging system.

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

    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. PMID:25303106

  10. High harmonic terahertz confocal gyrotron with nonuniform electron beam

    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

  11. High harmonic terahertz confocal gyrotron with nonuniform electron beam

    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.

  12. Analysis of endoplasmic reticulum by confocal microscopy

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

    Prague : Czech Pattern Recognition Society, 2005 - (Hlaváč, V.), s. 22-23 ISBN 80-01-03239-6. [Prague Post Genome Technology Workshop 2005. Prague (CZ), 06.06.2005-07.06.2005] R&D Projects: GA AV ČR(CZ) KJB6011309; GA AV ČR(CZ) IAA100110502 Grant ostatní: CZ-SK(CZ) KONTAKT 139 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal microscopy * endoplasmic reticulum * image analysis Subject RIV: EA - Cell Biology

  13. Quantifying metarefraction with confocal lenslet arrays

    Maceina, Tautvydas; Courtial, Johannes

    2011-01-01

    METATOYs can change the direction of light in ways that appear to, but do not actually, contravene the laws of wave optics. This direction change applies only to part of the transmitted light beam; the remainder gets re-directed differently. For a specific example, namely confocal lenslet arrays, we calculate here the fractions of power of an incident uniform plane wave get re-directed in different ways. This will facilitate assessment of the suitability of METATOYs for applications such as solar concentration.

  14. Line-scanning, stage scanning confocal microscope

    Carucci, John A.; Stevenson, Mary; Gareau, Daniel

    2016-03-01

    We created a line-scanning, stage scanning confocal microscope as part of a new procedure: video assisted micrographic surgery (VAMS). The need for rapid pathological assessment of the tissue on the surface of skin excisions very large since there are 3.5 million new skin cancers diagnosed annually in the United States. The new design presented here is a confocal microscope without any scanning optics. Instead, a line is focused in space and the sample, which is flattened, is physically translated such that the line scans across its face in a direction perpendicular to the line its self. The line is 6mm long and the stage is capable of scanning 50 mm, hence the field of view is quite large. The theoretical diffraction-limited resolution is 0.7um lateral and 3.7um axial. However, in this preliminary report, we present initial results that are a factor of 5-7 poorer in resolution. The results are encouraging because they demonstrate that the linear array detector measures sufficient signal from fluorescently labeled tissue and also demonstrate the large field of view achievable with VAMS.

  15. Confocal Raman imaging for cancer cell classification

    Mathieu, Evelien; Van Dorpe, Pol; Stakenborg, Tim; Liu, Chengxun; Lagae, Liesbet

    2014-05-01

    We propose confocal Raman imaging as a label-free single cell characterization method that can be used as an alternative for conventional cell identification techniques that typically require labels, long incubation times and complex sample preparation. In this study it is investigated whether cancer and blood cells can be distinguished based on their Raman spectra. 2D Raman scans are recorded of 114 single cells, i.e. 60 breast (MCF-7), 5 cervix (HeLa) and 39 prostate (LNCaP) cancer cells and 10 monocytes (from healthy donors). For each cell an average spectrum is calculated and principal component analysis is performed on all average cell spectra. The main features of these principal components indicate that the information for cell identification based on Raman spectra mainly comes from the fatty acid composition in the cell. Based on the second and third principal component, blood cells could be distinguished from cancer cells; and prostate cancer cells could be distinguished from breast and cervix cancer cells. However, it was not possible to distinguish breast and cervix cancer cells. The results obtained in this study, demonstrate the potential of confocal Raman imaging for cell type classification and identification purposes.

  16. Confocal Raman microspectroscopy of the skin.

    Förster, Matthias; Bolzinger, Marie-Alexandrine; Montagnac, Gilles; Briançon, Stéphanie

    2011-01-01

    Confocal Raman spectroscopy is a technique with considerable potential for the non-invasive study of biological tissues and skin samples in vitro or in vivo. It can be used to study skin physiology and possible pathological conditions and to obtain data about molecular composition and the structure of skin, for example, water content, moisturization and changes in the skin barrier function can all be observed. In-depth measurements also allow biopharmaceutical studies, such as analyzing the rate of penetration of a drug and the biochemical changes that may be induced by an applied formulation. Confocal Raman microspectroscopy is now at such a stage of refinement that it opens up new vistas. The big leap forward in its ease of use enables this technology to be used as an analytical method by more and more non-specialist laboratories. This review gives an overview of the state of the art of this technology by presenting an update on the principles of Raman spectroscopy and then by looking at examples of new developments in in vivo and in vitro applications. PMID:21914580

  17. Biological applications of confocal fluorescence polarization microscopy

    Bigelow, Chad E.

    Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine

  18. Distance measurements by differential confocal optical ranging.

    Corle, T R; Fanton, J T; Kino, G S

    1987-06-15

    A new technique is described for measuring the distance between a lens and reflecting surface extremely accurately. It is based on the sharply peaked depth response of type II confocal systems. By dithering either the sample or the optical system, a differential measurement is generated, placing a zero-crossing at the peak of the depth response and improving the ranging accuracy. The technique is independent of surface roughness or tilt and hence is useful for robotics or machining applications. Sensitivities to surface vibrations of 0.01 nm and thin film measurements to 0.04 microm demonstrated. Signal-to-noise calculations are presented, and the procedure for measuring the thickness of transparent films is outlined. PMID:20489885

  19. Reflectance confocal microscopy in infectious diseases.

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

    2015-10-01

    In vivo reflectance confocal microscope (RCM) is a high-resolution non-invasive imaging technique that was initially focused on the diagnosis of skin cancers. A rising number of other indications have been later described for the diagnosis and management of inflammatory and infectious dermatological disorders. RCM can identify cutaneous parasites that are not visible to naked eye such as Sarcoptes scabiei and Demodex folliculorum and it allows to better identify the different body parts of bigger parasites such as ticks. Fungal filaments can also be identified as elongated bright structures in the cutaneous upper layers. RCM cannot observe virus directly. However, the cytopathic effect associated with some virus can be recognized. In addition of being helpful for the diagnosis and follow-up after treatment, thanks to its non-invasiveness, RCM allows pathophysiological studies. PMID:26129682

  20. Automated cellular pathology in noninvasive confocal microscopy

    Ting, Monica; Krueger, James; Gareau, Daniel

    2014-03-01

    A computer algorithm was developed to automatically identify and count melanocytes and keratinocytes in 3D reflectance confocal microscopy (RCM) images of the skin. Computerized pathology increases our understanding and enables prevention of superficial spreading melanoma (SSM). Machine learning involved looking at the images to measure the size of cells through a 2-D Fourier transform and developing an appropriate mask with the erf() function to model the cells. Implementation involved processing the images to identify cells whose image segments provided the least difference when subtracted from the mask. With further simplification of the algorithm, the program may be directly implemented on the RCM images to indicate the presence of keratinocytes in seconds and to quantify the keratinocytes size in the en face plane as a function of depth. Using this system, the algorithm can identify any irregularities in maturation and differentiation of keratinocytes, thereby signaling the possible presence of cancer.

  1. The Jefferson Lab 1 KW IR FEL

    D. Douglas for the Jefferson Lab IR Demo FEL Team

    2000-08-01

    The Jefferson Lab (JLab) IR Demo Free Electron Laser (FEL) has completed commissioning and is initiating user service. The FEL - a high repetition rate, low extraction efficiency wiggler-driven optical cavity resonator - produces over 1 kW of tuneable light on intervals in a 3-6 lim wavelength range. It is driven by a 35-48 MeV, 5 mA superconducting RF (SRF) based energy-recovering continuous wave (CW) electron linac. The driver accelerator meets requirements imposed by low energy, high current, and a demand for stringent beam control at the wiggler and during energy recovery. These constraints are driven by the need for six-dimensional phase space management, the existence of deleterious collective phenomena (space charge, wake-fields, beam break-up, and coherent synchrotron radiation), and interactions between the FEL and the accelerator RF. The authors detail the system design, relate commissioning highlights, and discuss present performance.

  2. Confocal Raman Microspectroscopy of Oral Streptococci

    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

  3. Confocal scanner for vertical particle tracks in the nuclear photoemulsion

    A confocal scanner for selective observation of the vertical particle tracks in the nuclear photoemulsion is described. The particle track being searched for is imaging at an angle of 45 deg with respect to the optical axis of the system. The confocal scanner is provided with a new optical element, an 'image orthogonalizator', by means of which the extended image of the inclined vertical particle track is rotated over an angle of 90 deg. The stereoscopic version of the confocal scanner is presented as well. The described systems will be used in the experiments for investigation of the neutrino oscillations in the accelerators experiments

  4. Confocal supercritical angle fluorescence microscopy for cell membrane imaging

    Sivankutty, Siddharth; Mayet, Céline; Dupuis, Guillaume; Fort, Emmanuel; Lévêque-Fort, Sandrine

    2013-01-01

    We demonstrate sub-wavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fuorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modi?cation on the detection channel. Confocal-SAF microscopy would be a powerful tool to achieve high resolution surface imaging, especially for membrane imaging in biological samples

  5. Advantages of chromatic-confocal spectral interferometry in comparison to chromatic confocal microscopy

    Chromatic confocal microscopy (CCM) and spectral interferometry (SI) are established and robust sensor principles. CCM is a focus-based measurement principle, whose lateral and axial resolutions depend on the sensor's numerical aperture (NA), while the measurement range is given by the spectral bandwidth and the chromatic dispersion in the axial direction. Although CCM is a robust principle, its accuracy can be reduced by self-imaging effects or asymmetric illumination of the sensor pupil. Interferometric principles based on the evaluation of the optical path difference, e.g., SI, have proven to be robust against self-imaging. The disadvantage of SI is its measurement range, which is limited by the depth of focus. Hence, the usable NA and the lateral resolution are restricted. Chromatic-confocal spectral interferometry (CCSI) is a combination of SI and CCM, which overcomes these restrictions. The increase of robustness of CCSI compared to CCM due to the interferometric gain has been demonstrated before. In this contribution the advantages of CCSI in comparison to CCM concerning self-imaging artifacts will be demonstrated. Therefore, a new phase-evaluation algorithm with higher resolution concerning classical SI-based evaluation algorithms is presented. For the comparison of different sensor systems, a chirp comparison standard is used. (paper)

  6. In vivo confocal microscopy in chloroquine-induced keratopathy

    Iacopo Paladini

    2013-01-01

    Full Text Available In vivo confocal microscopy is becoming a mandatory examination to study corneal abnormalities such as drug deposits in systemic disease. A female diagnosed with fibromyalgia on systemic chloroquine for 9 months presented for an ophthalmic examination. Confocal microscopy was performed using the Confoscan 4 (Nidek Co. Ltd., Gamagori, Japan and multiple highly reflective deposits in the epithelial basal cells were found, that were consistent with choloquine. Deposits were also present in the wing cell layer. In the anterior stroma these deposits were rare. Atypically shaped and branched nerves were also present in the anterior stroma. Corneal deposits of chloroquine can be evaluated by confocal microscopy. Confocal microscopy provides information on corneal metabolism and physiology. Chloroquine keratopathy can affect the anterior stroma in addition to the epithelium.

  7. THE PARALLEL CONFOCAL DETECTING SYSTEM USING OPTICAL FIBER PLATE

    2005-01-01

    Objective Focusing on the problem such as slow scanning speed, complex system design and low light efficiency, a new parallel confocal 3D profile detecting method based on optical fiber technology, which realizes whole-field confocal detecting, is proposed. Methods The optical fiber plate generates an 2D point light source array, which splits one light beam into N2 subbeams and act the role of pinholes as point source and point detecting to filter the stray light and reflect light. By introducing the construction and working principle of the multi-beam 3D detecting system, the feasibility is investigated. Results Experiment result indicates that the optical fiber technology is applicable in rotation. The measuring parameters that influence the detecting can easily be adapted to satisfy different requirments of measurement. Compared with the conventional confocal method, the parallel confocal detecting system using optical fiber plate is simple in the mechanism, the measuring field is larger and the speed is faster.

  8. A New Multichannel Spectral Imaging Laser Scanning Confocal Microscope

    Yunhai Zhang

    2013-01-01

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

  9. Combined confocal Raman and quantitative phase microscopy system for biomedical diagnosis

    Kang, Jeon Woong; Lue, Niyom; Kong, Chae-Ryon; Barman, Ishan; Dingari, Narahara C.; Goldfless, Stephen J.; Niles, Jacquin C.; Dasari, Ramachandra R.; Michael S. Feld

    2011-01-01

    We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exoge...

  10. Digital adaptive optics line-scanning confocal imaging system

    Liu, Changgeng; Kim, Myung K.

    2015-11-01

    A digital adaptive optics line-scanning confocal imaging (DAOLCI) system is proposed by applying digital holographic adaptive optics to a digital form of line-scanning confocal imaging system. In DAOLCI, each line scan is recorded by a digital hologram, which allows access to the complex optical field from one slice of the sample through digital holography. This complex optical field contains both the information of one slice of the sample and the optical aberration of the system, thus allowing us to compensate for the effect of the optical aberration, which can be sensed by a complex guide star hologram. After numerical aberration compensation, the corrected optical fields of a sequence of line scans are stitched into the final corrected confocal image. In DAOLCI, a numerical slit is applied to realize the confocality at the sensor end. The width of this slit can be adjusted to control the image contrast and speckle noise for scattering samples. DAOLCI dispenses with the hardware pieces, such as Shack-Hartmann wavefront sensor and deformable mirror, and the closed-loop feedbacks adopted in the conventional adaptive optics confocal imaging system, thus reducing the optomechanical complexity and cost. Numerical simulations and proof-of-principle experiments are presented that demonstrate the feasibility of this idea.

  11. Confocal Raman microscopy of protein adsorbed in chromatographic particles.

    Xiao, Yuewu; Stone, Thomas; Bell, David; Gillespie, Christopher; Portoles, Marta

    2012-09-01

    Confocal Raman microscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure conformation and distribution of protein adsorbed in wetted chromatographic particles. Monoclonal antibody was loaded into the Fractogel EMD SO(3) (M) cation exchanger at 2 mS/cm or 10 mS/cm. Amide I and III frequencies in the Raman spectrum of the adsorbed protein suggest that there are no detectable changes of the original β-sheet conformation in the chromatographic particles. Protein depth profile measurements indicate that, when the conductivity is increased from 2 mS/cm to 10 mS/cm, there is a change in mass transport mechanism for protein adsorption, from the shrinking-core model to the homogeneous-diffusion model. In this study, the use of confocal Raman microscopy to measure protein distribution in chromatographic particles fundamentally agrees with previous confocal laser scanning microscopic investigations, but confocal Raman spectroscopy enjoys additional advantages: use of unlabeled protein to eliminate fluorescent labeling, ability for characterization of protein secondary structure, and ability for spectral normalization to provide a nondestructive experimental approach to correct light attenuation effects caused by refractive index (RI) mismatching in semiopaque chromatographic particles. PMID:22803776

  12. Colloidal structural evolution of asphaltene studied by confocal microscopy

    Hung, Jannett; Castillo, Jimmy A.; Reyes, A.

    2004-10-01

    In this work, a detail analysis of the flocculation kinetic of asphaltenes colloidal particles has been carried out usng confocal microscopy. The colloidal structural evolution of the asphaltene flocculated has had varies postulated; however, the aggregation process of asphaltene is still not fully understood. In a recent paper, using Confocal microscope (homemade), we reported high-resolution micrographic images of asphaltenes flocculated and the correlation between crude oil stability and flocculation process. This technique permitted visualizes directly the physical nature of asphaltene flocculated. In this work, a detail analysis of the flocculation kinetic of asphaltene colloidal particles has been carried out using confocal microscopy. The physical nature of asphaltene flocculated from different crude oils is showed through of high-resolution image micrographies and its colloidal structural evolution.

  13. A Simple Model for Nonlinear Confocal Ultrasonic Beams

    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.

  14. EUS-Guided Needle-Based Confocal Laser Endomicroscopy

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

    2015-01-01

    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...

  15. Spectral confocal reflection microscopy using a white light source

    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.

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

    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.

  17. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR CALIBRATION, QUANTITATION AND SPECTROSCOPY

    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...

  18. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY AND FOUNDATIONS FOR QUANTITATION

    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...

  19. Study of liquid jet instability by confocal microscopy

    Yang, Lisong; Adamson, Leanne J.; Bain, Colin D.

    2012-07-01

    The instability of a liquid microjet was used to measure the dynamic surface tension of liquids at the surface ages of ≤1 ms using confocal microscopy. The reflected light from a laser beam at normal incidence to the jet surface is linear in the displacement of the surface near the confocal position, leading to a radial resolution of 4 nm and a dynamic range of 4 μm in the surface position, thus permitting the measurement of amplitude of oscillation at the very early stage of jet instability. For larger oscillations outside the linear region of the confocal response, the swell and neck position of the jet can be located separately and the amplitude of oscillation determined with an accuracy of 0.2 μm. The growth rate of periodically perturbed water and ethanol/water mixture jets with a 100-μm diameter nozzle and mean velocity of 5.7 m s-1 has been measured. The dynamic surface tension was determined from the growth rate of the instability with a linear, axisymmetric, constant property model. Synchronisation of the confocal imaging system with the perturbation applied to the jet permitted a detailed study of the temporal evolution of the neck into a ligament and eventually into a satellite drop.

  20. Identifying brain neoplasms using dye-enhanced multimodal confocal imaging

    Wirth, Dennis; Snuderl, Matija; Sheth, Sameer; Kwon, Churl-Su; Frosch, Matthew P.; Curry, William; Yaroslavsky, Anna N.

    2012-02-01

    Brain tumors cause significant morbidity and mortality even when benign. Completeness of resection of brain tumors improves quality of life and survival; however, that is often difficult to accomplish. The goal of this study was to evaluate the feasibility of using multimodal confocal imaging for intraoperative detection of brain neoplasms. We have imaged different types of benign and malignant, primary and metastatic brain tumors. We correlated optical images with histopathology and evaluated the possibility of interpreting confocal images in a manner similar to pathology. Surgical specimens were briefly stained in 0.05 mg/ml aqueous solution of methylene blue (MB) and imaged using a multimodal confocal microscope. Reflectance and fluorescence signals of MB were excited at 642 nm. Fluorescence emission of MB was registered between 670 and 710 nm. After imaging, tissues were processed for hematoxylin and eosin (H&E) histopathology. The results of comparison demonstrate good correlation between fluorescence images and histopathology. Reflectance images provide information about morphology and vascularity of the specimens, complementary to that provided by fluorescence images. Multimodal confocal imaging has the potential to aid in the intraoperative detection of microscopic deposits of brain neoplasms. The application of this technique may improve completeness of resection and increase patient survival.

  1. Solar Confocal interferometers for Sub-Picometer-Resolution Spectral Filters

    Gary, G. Allen; Pietraszewski, Chris; West, Edward A.; Dines. Terence C.

    2007-01-01

    The confocal Fabry-Perot interferometer allows sub-picometer spectral resolution of Fraunhofer line profiles. Such high spectral resolution is needed to keep pace with the higher spatial resolution of the new set of large-aperture solar telescopes. The line-of-sight spatial resolution derived for line profile inversions would then track the improvements of the transverse spatial scale provided by the larger apertures. In particular, profile inversion allows improved velocity and magnetic field gradients to be determined independent of multiple line analysis using different energy levels and ions. The confocal interferometer's unique properties allow a simultaneous increase in both etendue and spectral power. The higher throughput for the interferometer provides significant decrease in the aperture, which is important in spaceflight considerations. We have constructed and tested two confocal interferometers. A slow-response thermal-controlled interferometer provides a stable system for laboratory investigation, while a piezoelectric interferometer provides a rapid response for solar observations. In this paper we provide design parameters, show construction details, and report on the laboratory test for these interferometers. The field of view versus aperture for confocal interferometers is compared with other types of spectral imaging filters. We propose a multiple etalon system for observing with these units using existing planar interferometers as pre-filters. The radiometry for these tests established that high spectral resolution profiles can be obtained with imaging confocal interferometers. These sub-picometer spectral data of the photosphere in both the visible and near-infrared can provide important height variation information. However, at the diffraction-limited spatial resolution of the telescope, the spectral data is photon starved due to the decreased spectral passband.

  2. Theoretical investigation on Raman induced Kerr effect spectroscopy in nonlinear confocal microscopy

    2008-01-01

    The imaging theory of Raman induced Kerr effect spectroscopy (RIKES) in nonlinear confocal microscopy is presented in this paper. Three-dimensional point spread function (3D-PSF) of RIKES nonlinear confocal microscopy in isotropic media is derived with Fourier imaging theory and RIKES theory. The impact of nonlinear property of RIKES on the spatial resolution and imaging properties of confocal microscopy have been analyzed in detail. It is proved that RIKES nonlinear confocal microscopy can simultaneously provide more information than two-photon confocal microscopy concerning molecular vibration mode, vibration orientation and optically induced molecular reorientation, etc. It is shown that RIKES nonlinear confocal microscopy significantly enhances the spatial resolution and imaging quality of confocal microscopy and achieves much higher resolution than that of two-photon confocal microscopy.

  3. Theoretical investigation on Raman induced Kerr effect spectroscopy in nonlinear confocal microscopy

    Gun LiNa; TANG ZhiLie; XING Da

    2008-01-01

    The imaging theory of Raman induced Kerr effect spectroscopy (RIKES) in nonlinear confocal microscopy is presented in this paper. Three-dimensional point spread function (3D-PSF) of RIKES nonlinear confocal microscopy in isotropic media is derived with Fourier imaging theory and RIKES theory. The impact of nonlinear property of RIKES on the spatial resolution and imaging properties of confocal microscopy have been analyzed in detail. It is proved that RIKES nonlinear confocal microscopy can simultaneously provide more information than twophoton confocal microscopy concerning molecular vibration mode, vibration orientation and optically induced molecular reorientation, etc. It is shown that RIKES nonlinear confocal microscopy significantly enhances the spatial resolution and imaging quality of confocal microscopy and achieves much higher resolution than that of two-photon confocal microscopy.

  4. A laser reflection confocal large-radius measurement

    We propose a new laser reflection confocal large-radius measurement (RCLRM) method. By utilizing the precise correspondence relationship between the peak point of the confocal curve and the convergence point of the multi-reflected measuring beam, we identify the position of the test lens. With a distance interferometer, we obtain the position variation of the test lens with different reflection times. Therefore, a fast and precise large-radius measurement is achieved with a shorter measuring system. Additionally, the RCLRM significantly enhances the measurement accuracy by using conic fitting. The theoretical analyses and experiments indicate that the relative expanded uncertainty is better than 0.008% (k  =  2). (paper)

  5. Integrated photoacoustic, confocal, and two-photon microscope

    Rao, Bin; Soto, Florentina; Kerschensteiner, Daniel; Wang, Lihong V.

    2014-01-01

    Abstract. The invention of green fluorescent protein and other molecular fluorescent probes has promoted applications of confocal and two-photon fluorescence microscopy in biology and medicine. However, exogenous fluorescence contrast agents may affect cellular structure and function, and fluorescence microscopy cannot image nonfluorescent chromophores. We overcome this limitation by integrating optical-resolution photoacoustic microscopy into a modern Olympus IX81 confocal, two-photon, fluorescence microscope setup to provide complementary, label-free, optical absorption contrast. Automatically coregistered images can be generated from the same sample. Imaging applications in ophthalmology, developmental biology, and plant science are demonstrated. For the first time, in a familiar microscopic fluorescence imaging setting, this trimodality microscope provides a platform for future biological and medical discoveries. PMID:24589986

  6. Polarization conversion in confocal microscopy with radially polarized illumination.

    Tang, Wai Teng; Yew, Elijah Y S; Sheppard, Colin J R

    2009-07-15

    The effects of using radially polarized illumination in a confocal microscope are discussed, and the introduction of a polarization mode converter into the detection optics of the microscope is proposed. We find that with such a configuration, bright-field imaging can be performed without losing the resolution advantage of radially polarized illumination. The detection efficiency can be increased by three times without having to increase the pinhole radius and sacrificing the confocality of the system. Furthermore, the merits of such a setup are also discussed in relation to surface plasmon microscopy and single-molecule orientation studies, where the doughnut point spread function can be engineered into a single-lobed point spread function. PMID:19823530

  7. Analysis of 3D confocal images of capillaries

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

    Saint-Etienne : International society for stereology, 2007, s. 12-15. [International congress for stereology /12./. Saint-Etienne (FR), 03.09.2007-07.09.2007] R&D Projects: GA AV ČR(CZ) IAA100110502 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10190503 Keywords : capillaries * confocal microscopy * image analysis Subject RIV: EA - Cell Biology

  8. Corneal confocal microscopy in chronic inflammatory demyelinating polyneuropathy

    Stettner, Mark; Hinrichs, Lena; Guthoff, Rainer; Bairov, Silja; Petropoulos, Ioannis N.; Warnke, Clemens; Hartung, Hans‐Peter; Malik, Rayaz A.; Kieseier, Bernd C.

    2015-01-01

    Abstract Objective There is an unmet need for better diagnostic tools to further delineate clinical subsets of heterogeneous chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and multifocal motor neuropathy (MMN) to facilitate treatment decisions. Corneal confocal microscopy (CCM) is a noninvasive and reproducible nerve imaging technique. This study evaluates the potential of CCM as a diagnostic surrogate in CIDP and MMN. Methods In a cross‐sectional prospective approach, 182 p...

  9. IV Neuropathy: An In Vivo Confocal Microscopic Study

    Almodovar, Jorge L.; Schifitto, Giovanni; McDermott, Michael P; Ferguson, Michele; Herrmann, David N.

    2012-01-01

    Several approaches exist for quantitative assessment of human immunodeficiency virus (HIV) associated distal sensory polyneuropathy (DSP). While useful, each has some limitations. This study evaluated non-invasive, in vivo reflectance confocal microscopy (RCM) of Meissner Corpuscles (MCs) as a measure of HIV-DSP. Forty-eight adults (29 HIV-infected, 19 controls) underwent RCM of MC density (MCs/mm2) at the arch, fingertip and thenar eminence (TE), ankle skin biopsy to measure epidermal nerve ...

  10. Optical Characterization and Confocal Imaging of Mechanochromic Polymers [video

    Naval Postgraduate Schoo; Van Horn, Matthew

    2013-01-01

    Mechanochromic molecules open new pathways for the study of micro-damage in polymers. Their application stress and temperature diagnostics, however, requires measurement techniques capable of detecting the activated force- or temperature-sensitive chemical species with spatial resolution. Confocal imaging techniques offer excellent spatial resolution but, due to the increased energy-input, may affect the activation state of the mechanochromic polymer systems. Here we present a systematic stud...

  11. Towards drug quantification in human skin with confocal Raman microscopy.

    Franzen, Lutz; Selzer, Dominik; Fluhr, Joachim W; Schaefer, Ulrich F.; Windbergs, Maike

    2013-01-01

    Understanding the penetration behaviour of drugs into human skin is a prerequisite for the rational development and evaluation of effective dermal drug delivery. The general procedure for the acquisition of quantitative drug penetration profiles in human skin is performed by sequential segmentation and extraction. Unfortunately, this technique is destructive, laborious and lacks spatial resolution. Confocal Raman microscopy bares the potential of a chemically selective, label free and nondest...

  12. Combining confocal and BSE SEM imaging for bone block surfaces

    Boyde, A.; Lovicar, L.; Zamecnik, J.

    2005-01-01

    The present report presents a method for the correlation of qualitative and quantitative BSE SEM imaging with confocal scanning light microscopy (CSLM) imaging modes applied to bone samples embedded in PMMA. The SEM has a proper digital scan generator: we leave the BSE image unchanged, and match the CSLM image to it, because the CSLM scan mechanism is not digital, though the signal is digitised. Our overlapping program uses a linear transformation matrix which projects one system to the other...

  13. Application of Reflectance Confocal Microscopy in Dermatology Practice

    Ayşe Esra Koku Aksu; Mehmet Salih Gürel

    2015-01-01

    In vivo reflectance confocal microscopy (RCM) is a non-invasive method, imaging cellular structures in living skin at a level close to the histological resolution. It is easier to diagnose melanocytic and non-melanocytic skin tumors especially in difficult cases when RCM features have been identified. Determination of the cellular features, presence of cellular and structural atypia with RCM allows the discrimination of benign and malignant lesions. Preoperative differential diagnosis of mali...

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

    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.

  15. Confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry

    Jun, Brian; Giarra, Matthew; Yang, Haisheng; Main, Russell; Vlachos, Pavlos

    2016-01-01

    We present a new particle image correlation technique for resolving nanoparticle flow velocity using confocal laser scanning microscopy (CLSM). The two primary issues that complicate nanoparticle scanning laser image correlation (SLIC) based velocimetry are (1) the use of diffusion dominated nanoparticles as flow tracers, which introduce a random decorrelating error into the velocity estimate, and (2) the effects of the scanning laser image acquisition, which introduces a bias error. To date,...

  16. Confocal Imaging of Early Heart Development in Xenopus laevis

    Kolker, Sandra J.; Tajchman, Urszula; Weeks, Daniel L.

    2000-01-01

    Xenopus laevis provides a number of advantages for studies on cardiovascular development. The embryos are fairly large, easy to obtain, and can develop at ambient temperature in simple buffer solutions. Although classic descriptions of heart development exist, the ability to use whole mount immunohistochemical methods and confocal microscopy may enhance the ability to understand both normal and experimentally perturbed cardiovascular development. We have started to examine the early stages of...

  17. Application of confocal X-ray fluorescence micro-spectroscopy to the investigation of paint layers

    A confocal micro X-ray fluorescence (MXRF) spectrometer based on polycapillary X-ray optics was used for the identification of paint layers. The performance of the confocal MXRF was studied. Multilayered paint fragments of a car were analyzed nondestructively to demonstrate that this confocal MXRF instrument could be used in the discrimination of the various layers in multilayer paint systems. - Hihglights: • The performance of the confocal micro X-ray fluorescence was studied. • Confocal micro X-ray fluorescence was used for identifying paint layers. • The multilayered paint fragments of a car were analyzed nondestructively

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

    TANG; Zhilie; XING; Da; LIU; Songhao

    2004-01-01

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

  19. In-vivo multi-spectral confocal microscopy

    Rouse, Andrew R.; Udovich, Joshua A.; Gmitro, Arthur F.

    2005-03-01

    A multi-spectral confocal microendoscope (MCME) for in-vivo imaging has been developed. The MCME employs a flexible fiber-optic catheter coupled to a slit-scan confocal microscope with an imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The focus mechanism allows for imaging to a maximum tissue depth of 200 microns. 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 3 micron lateral resolution and 30 micron axial resolution. The system incorporates two laser sources and is therefore capable of simultaneous acquisition of spectra from multiple dyes using dual excitation. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 8nm to 16nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersion characteristics 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. In-vitro, and ex-vivo multi-spectral results are presented.

  20. Characterization of a hazardous eyeliner (kohl) by confocal Raman microscopy

    A new method of analyzing kohl, a cosmetic eyeliner, using confocal Raman microscopy is reported. This technique offers an important alternative to conventional spectroscopic techniques that provide elemental/atomic composition. Raman spectra of three kohl samples have been measured between 150 and 3000 cm-1 at room temperature. The main component of two kohl samples was found to be lead(II) sulfide (PbS). Kohl is used as a traditional cosmetic and remedy in the Middle East, Far East, and Northern Africa. Since kohl products contain very high concentrations of lead, they constitute a risk for public health, particularly for children

  1. Spatial resolution of confocal XRF technique using capillary optics

    Dehlinger, Maël; Fauquet, Carole; Lavandier, Sebastien; Aumporn, Orawan; Jandard, Franck; Arkadiev, Vladimir; Bjeoumikhov, Aniouar; Tonneau, Didier

    2013-01-01

    XRF (X-ray fluorescence) is a powerful technique for elemental analysis with a high sensitivity. The resolution is presently limited by the size of the primary excitation X-ray beam. A test-bed for confocal-type XRF has been developed to estimate the ultimate lateral resolution which could be reached in chemical mapping using this technique. A polycapillary lens is used to tightly focus the primary X-ray beam of a low power rhodium X-ray source, while the fluorescence signal is collected by a...

  2. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    Kim, B-M.; Stoller, P.; Reiser, K.; Eichler, J.; Yan, M.; Rubenchik, A.; Da Silva, L.

    2000-03-06

    A confocal microscopy imaging system was devised to selectively detect Second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.

  3. Confocal microscopy through a multimode fiber using optical correlation

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe

    2015-01-01

    We report on a method to obtain confocal imaging through multimode fibers using optical correlation. First, we measure the fiber's transmission matrix in a calibration step. This allows us to create focused spots at one end of the fiber by shaping the wavefront sent into it from the opposite end. These spots are scanned over a sample, and the light coming back from the sample via the fiber is optically correlated with the input pattern. We show that this achieves spatial selectivity in the detection. The technique is demonstrated on microbeads, a dried epithelial cell, and a cover glass.

  4. Atherosclerotic plaque detection by confocal Brillouin and Raman microscopies

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

    2015-02-01

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

  5. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    A confocal microscopy imaging system was devised to selectively detect Second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing

  6. Confocal epifluorescence detection for microspheres delivered on disposable microfluidic chip

    Honghua Hu; Xiyun Hou; Guoguang Yang

    2006-01-01

    @@ The laser induced fluorescence (LIF) detection system for 5-μm microspheres delivered on microfluidic chip is presented employing confocal optical scheme. The parameters of the optical system are specifically optimized for single microsphere detection. With the excitation laser spot size of 4.6 μm and optical sectioning power of 27 μm, the lowest concentration detection limit is 0.45 nmol/L, corresponding to only 122 molecules in probe volume. The microsphere detection is carried on successfully with the maximum signal-to-noise ratio (SNR) of 55.7, which provides good detection sensitivity.

  7. Volume visualization of biological tissue specimens using confocal microscopy

    Čapek, Martin; Janáček, Jiří; Kubínová, Lucie; Smrčka, P.; Hána, K.

    2006-01-01

    Roč. 36, č. 2 (2006), s. 240-244. ISSN 0301-5491. [Biomedical Engineering Conference of Young Biomedical Engineers and Researchers /2./. Kladno, 19.07.2006-21.07.2006] R&D Projects: GA MŠk(CZ) LC06063; GA AV ČR(CZ) IAA100110502; GA AV ČR(CZ) IAA500200510; GA ČR(CZ) GA304/05/0153 Institutional research plan: CEZ:AV0Z50110509 Keywords : 3D reconstruction * confocal microscopy Subject RIV: JC - Computer Hardware ; Software

  8. The confocal plane grating spectrometer at BESSY II

    Highlights: ► At the electron storage ring BESSY II a confocal plane grating RIXS endstation with a spot size of 4 μm × 1 μm is presently being installed. ► A resolving power above 10,000 is expected for low energy excitations below 500 eV. ► The sample will be excited with a photon flux up to 1015 photons/(s 300 mA 0.1%bandwidth). ► Sample environments for solid, gaseous and liquid samples will be provided. ► A fast detecting system is being set up for future pump-probe experiments. -- Abstract: At BESSY II a confocal plane grating spectrometer for resonant inelastic X-ray scattering (RIXS) is currently under commissioning. The new endstation operates with a source size of 4 × 1 μm2 provided by its dedicated beamline. The RIXS-spectrometer covers an energy range from 50 eV to 1000 eV, providing a resolving power E/ΔE of 5000–15,000. The beamline allows full polarization control and gives a photon flux of up to 7 × 1014 photons/s/0.1 A/0.1%bandwidth by offering a resolving power E/ΔE of 4000–12,000

  9. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    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.

  10. CCDiode: an optimal detector for laser confocal microscopes

    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.

  11. Combining in vivo reflectance with fluorescence confocal microscopy provides additive information on skin morphology

    Skvara, Hans; Plut, Ulrike; Schmid, Johannes A; Jonak, Constanze

    2012-01-01

    Background: Within the last decade, confocal microscopy has become a valuable non-invasive diagnostic tool in imaging human skin in vivo. Of the two different methods that exist, reflectance confocal microscopy (RCM) displays the backscattering signal of naturally occurring skin components, whereas fluorescence confocal microscopy (FCM) provides contrast by using an exogenously applied fluorescent dye. Methodology: A newly developed multilaser device, in which both techniques are implemented,...

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

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

    2014-01-01

    We report the first pilot qualitative confocal microscopic analysis of a laser in situ keratomileusis (Lasik) treatment combined with sequential high-fluence accelerated corneal collagen cross-linking, denominated Lasik XTra, by means of HRT II laser scanning in vivo confocal microscopy after a 6-month follow-up. After obtaining approval from the Siena University Hospital Institutional Review Board, a 33-year-old female patient underwent a Lasik XTra procedure in her left eye. Confocal analys...

  13. Investigation of a tabletop confocal micro X-ray fluorescence setup

    A new tabletop confocal micro x-ray fluorescence setup with an MCBM 50-0.6B x-ray tube is assembled. The confocal micro x-ray fluorescence setup includes two lenses, a polycapillary full lens in the excitation channel and a polycapillary half lens in the detection channel. A Ni-Cr wire in diameter 25 μm is used to investigate the FWHM of three-dimensional confocal volume, A basso-relievo capital letter of a 1-jiao RMB coin of 2005 version is studied with this confocal micro x-ray fluorescence setup. (authors)

  14. Low-power, Confocal Imaging of Protein Localization in Living Cells (7214-150) Project

    National Aeronautics and Space Administration — The proposed technology genetically labels intracellular structures and visualizes protein interactions in living cells using a compact, confocal microscope with...

  15. Mechanical scanner-less multi-beam confocal microscope with wavefront modulation

    Takiguchi, Yu; Seo, Min-Woong; Kagawa, Keiichiro; Takamoto, Hisayoshi; Inoue, Takashi; Kawahito, Shoji; Terakawa, Susumu

    2016-04-01

    We propose a novel full-electronically controlled laser confocal microscope in which a liquid-crystal-on-silicon spatial light modulator and a custom CMOS imaging sensor are synchronized for performing multi-beam confocal imaging. Adaptive wavefront modulation for functional multi-beam excitation can be achieved by displaying appropriate computer generated holograms on the spatial light modulator, in consideration of the numerical aperture of the focusing objective. We also adopted a custom CMOS imaging sensor to realize multi-beam confocal microscopy without any physical pinhole. The confocality of this microscope was verified by improvements in transverse and axial resolutions of fluorescent micro-beads.

  16. 3D imaging of neutron tracks using confocal microscopy

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

    Neutron detection and neutron flux assessment are important aspects in monitoring nuclear energy production. Neutron flux measurements can also provide information on potential biological damage from exposure. In addition to the applications for neutron measurement in nuclear energy, neutron detection has been proposed as a method of enhancing neutrino detectors and cosmic ray flux has also been assessed using ground-level neutron detectors. Solid State Nuclear Track Detectors (or SSNTDs) have been used extensively to examine cosmic rays, long-lived radioactive elements, radon concentrations in buildings and the age of geological samples. Passive SSNTDs consisting of a CR-39 plastic are commonly used to measure radon because they respond to incident charged particles such as alpha particles from radon gas in air. They have a large dynamic range and a linear flux response. We have previously applied confocal microscopy to obtain 3D images of alpha particle tracks in SSNTDs from radon track monitoring (1). As a charged particle traverses through the polymer it creates an ionisation trail along its path. The trail or track is normally enhanced by chemical etching to better expose radiation damage, as the damaged area is more sensitive to the etchant than the bulk material. Particle tracks in CR-39 are usually assessed using 2D optical microscopy. In this study 6 detectors were examined using an Olympus OLS4100 LEXT 3D laser scanning confocal microscope (Olympus Corporation, Japan). The detectors had been etched for 2 hours 50 minutes at 85 °C in 6.25M NaOH. Post etch the plastics had been treated with a 10 minute immersion in a 2% acetic acid stop bath, followed by rinsing in deionised water. The detectors examined had been irradiated with a 2mSv neutron dose from an Am(Be) neutron source (producing roughly 20 tracks per mm2). We were able to successfully acquire 3D images of neutron tracks in the detectors studied. The range of track diameter observed was between 4

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

    Sedgewick, Jerry

    2014-01-01

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

  18. Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

    Lork, Marie; Delvaeye, Mieke; Gonçalves, Amanda; Van Hamme, Evelien; Beyaert, Rudi

    2016-01-01

    Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy. PMID:27613040

  19. Reflectance confocal microscopy for cutaneous infections and infestations.

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

    2016-05-01

    Reflectance confocal microscopy (RCM) is a high-resolution emerging imaging technique that allows non-invasive diagnosis of several cutaneous disorders. A systematic review of the literature on the use of RCM for the study of infections and infestations has been performed to evaluate the current use of this technique and its possible future applications in this field. RCM is particularly suitable for the identification of Sarcoptes scabies, Demodex folliculorum, Ixodes, Dermatophytes and Candida species in the clinical practice and for the follow-up after treatment. The cytopathic effect of herpes simplex virus, varicella zoster virus and molluscipoxvirus is also detectable by this imaging technique even in a pre-vesicular stage. In addition, thanks to its non-invasiveness, RCM allows pathophysiological studies. PMID:26387660

  20. Surface microstructure profilometry based on laser confocal feedback

    Wang, Weiping; Zhang, Shulian; Li, Yan

    2015-10-01

    We demonstrate a surface microstructure profile measurement method, which utilizes the positioning ability of confocal technology and the high sensitivity of frequency-shift feedback of a microchip laser. The surface profile is measured by combination of the amplitude and phase information of the feedback light reflected by the sample. The amplitude information is used for coarse measurement and to determine the integral number of half lasing wavelengths contained in the sample profile variation. The phase information is used for fine measurement and to determine the fractional number. The measurement realizes both a large axial measuring range of tens of microns and a high axial resolution of ˜2 nm. Meanwhile, a heterodyne phase measurement approach is introduced to compensate for environmental disturbance and to realize high axial resolution measurement under common room conditions. The surface profile of a grating is measured and proves the feasibility of the method.

  1. Three-dimensional chemical mapping with a confocal XRF setup.

    Lühl, Lars; Mantouvalou, Ioanna; Schaumann, Ina; Vogt, Carla; Kanngießer, Birgit

    2013-04-01

    A new approach for the nondestructive reconstruction of stratified systems with constant elemental composition but with varying chemical compounds has been developed. The procedure is based on depth scans with a confocal X-ray fluorescence setup at certain energies near absorption edges. These so-called marker energies, where XAFS signals of the involved chemical compounds differ significantly, can also be used to uncover the chemical composition and its topology. A prominent field of application is homogeneous material that is degraded due to chemical reactions like oxidation or reduction. A procedure for the semiquantitative reconstruction of stratified material by means of depth scans at marker energies is elaborated and validated and a three-dimensional mapping is presented. PMID:23445459

  2. Analysis of fiber-like structures using confocal microscopy

    Janáček, Jiří; Radochová, Barbora; Demjénová, E.; Mao, X. W.; Archambeau, P.; Schwarzerová, K.; Tomori, Z.; Karen, Petr; Kubínová, Lucie

    Vol. 2. Kraków : Polish Society for Stereology, 2005 - (Chrapoński, J.; Cwajna, J.; Wojnar, L.), s. 79-85 ISBN 83-917834-4-8. [European Congress on Stereology and Image Analysis /9./ and International Conference on Stereology and Image Analysis in Materials Science STERMAT /7./. Zakopane (PL), 10.05.2005-13.05.2005] R&D Projects: GA AV ČR(CZ) KJB6011309; GA AV ČR(CZ) IAA100110502 Grant ostatní: CZ-SK(CZ) KONTAKT 139; NASA SRHP(US) 02-OBPR 02-18 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal microscopy * endoplasmic reticulum * mathematical morphology Subject RIV: EA - Cell Biology

  3. Confocal imaging of ionised calcium in living plant cells.

    Williams, D A; Cody, S H; Gehring, C A; Parish, R W; Harris, P J

    1990-04-01

    Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualize Ca2(+)-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively. PMID:2113832

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

    Jaikrit Bhutani

    2015-01-01

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

  5. Confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry

    Jun, Brian; Yang, Haisheng; Main, Russell; Vlachos, Pavlos

    2016-01-01

    We present a new particle image correlation technique for resolving nanoparticle flow velocity using confocal laser scanning microscopy (CLSM). The two primary issues that complicate nanoparticle scanning laser image correlation (SLIC) based velocimetry are (1) the use of diffusion dominated nanoparticles as flow tracers, which introduce a random decorrelating error into the velocity estimate, and (2) the effects of the scanning laser image acquisition, which introduces a bias error. To date, no study has quantified these errors or demonstrated a means to deal with them in SLIC velocimetry. In this work, we build upon the robust phase correlation (RPC) and existing methods of SLIC to quantify and mitigate these errors. First, we implement an ensemble RPC instead of using an ensemble standard cross correlation, and develop an SLIC optimal filter that maximizes the correlation strength in order to reliably and accurately detect the correlation peak representing the most probable average displacement of the nano...

  6. Total internal reflection fluorescence microscopy imaging-guided confocal single-molecule fluorescence spectroscopy

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

    2012-01-01

    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...

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

    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

  8. Development of a Confocal Optical System Design for Molecular Imaging Applications of Biochip

    Guoliang Huang

    2007-08-01

    Full Text Available A novel confocal optical system design and a dual laser confocal scanner have been developed to meet the requirements of highly sensitive detection of biomolecules on microarray chips, which is characterized by a long working distance (wd>3.0 mm, high numerical aperture (NA=0.72, and only 3 materials and 7 lenses used. This confocal optical system has a high scanning resolution, an excellent contrast and signal-to-noise ratio, and an efficiency of collected fluorescence of more than 2-fold better than that of other commercial confocal biochip scanners. The scanner is as equally good for the molecular imaging detection of enclosed biochips as for the detection of biological samples on a slide surface covered with a cover-slip glass. Some applications of gene and protein imagings using the dual laser confocal scanner are described.

  9. Diffusion of photoacid generators by laser scanning confocal microscopy

    Zhang, Ping L.; Webber, Stephen E.; Mendenhall, J.; Byers, Jeffrey D.; Chao, Keith K.

    1998-06-01

    Diffusion of the photogenerated acid during the period of time between exposure and development can cause contrast loss and ultimately loss of the latent image. This is especially relevant for chemically amplified photoresists that require a post-exposure baking step, which in turn facilitates acid diffusion due to the high temperature normally employed. It is thus important to develop techniques with good spatial resolution to monitor the photogeneration of acid. More precisely, we need techniques that provide two distinct types of information: spatial resolution on various length scales within the surface layer and also sufficient depth resolution so that one can observe the transition from very surface layer to bulk structure in the polymer blend coated on silicon substrate. Herein laser scanning confocal microscopy is used to evaluate the resist for the first time. We report the use of the confocal microscopy to map the pag/dye distribution in PHS matrices, with both reflectance images and fluorescence images. A laser beam is focused onto a small 3D volume element, termed a voxel. It is typically 200 nm X 200 nm laterally and 800 nm axially. The illuminated voxel is viewed such that only signals emanating from this voxel are detected, i.e., signal from outside the probed voxel is not detected. By adjusting the vertical position of the laser focal point, the voxel can be moved to the designated lateral plane to produce an image. Contrast caused by topology difference between the exposed and unexposed area can be eliminated. Bis-p-butylphenyl iodonium triflat (7% of polyhydroxystyrene) is used as photoacid generators. 5% - 18% (by weight, PHS Mn equals 13 k) resist in PGMEA solution is spin cast onto the treated quartz disk with thickness of 1.4 micrometers , 5 micrometers space/10 micrometers pitch chrome mask is used to generate the pattern with mercury DUV illumination. Fluoresceinamine, the pH-sensitive dye, is also used to enhance the contrast of

  10. Confocal laser scanning microscopy in study of bone calcification

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

    2012-12-01

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

  11. Confocal laser endomicroscopy in gastrointestinal and pancreatobiliary diseases.

    Nakai, Yousuke; Isayama, Hiroyuki; Shinoura, Susumu; Iwashita, Takuji; Samarasena, Jason B; Chang, Kenneth J; Koike, Kazuhiko

    2014-01-01

    Confocal laser endomicroscopy (CLE) is an emerging diagnostic procedure that enables in vivo pathological evaluation during ongoing endoscopy. There are two types of CLE: endoscope-based CLE (eCLE), which is integrated in the tip of the endoscope, and probe-based CLE (pCLE), which goes through the accessory channel of the endoscope. Clinical data of CLE have been reported mainly in gastrointestinal (GI) diseases including Barrett's esophagus, gastric neoplasms, and colon polyps, but, recently, a smaller pCLE, which goes through a catheter or a fine-needle aspiration needle, was developed and clinical data in the diagnosis of biliary stricture or pancreatic cysts have been increasingly reported. The future application of this novel technique expands beyond the pathological diagnosis to functional or molecular imaging. Despite these promising data, the generalizability of the procedure should be confirmed especially in Japan and other Asian countries, where the current diagnostic yield for GI luminal diseases is high. Given the high cost of CLE devices, cost-benefit analysis should also be considered. PMID:24033351

  12. Spatial resolution of confocal XRF technique using capillary optics.

    Dehlinger, Maël; Fauquet, Carole; Lavandier, Sebastien; Aumporn, Orawan; Jandard, Franck; Arkadiev, Vladimir; Bjeoumikhov, Aniouar; Tonneau, Didier

    2013-01-01

    XRF (X-ray fluorescence) is a powerful technique for elemental analysis with a high sensitivity. The resolution is presently limited by the size of the primary excitation X-ray beam. A test-bed for confocal-type XRF has been developed to estimate the ultimate lateral resolution which could be reached in chemical mapping using this technique. A polycapillary lens is used to tightly focus the primary X-ray beam of a low power rhodium X-ray source, while the fluorescence signal is collected by a SDD detector through a cylindrical monocapillary. This system was used to characterize the geometry of the fluorescent zone. Capillary radii ranging from 50 μm down to 5 μm were used to investigate the fluorescence signal maximum level This study allows to estimate the ultimate resolution which could be reached in-lab or on a synchrotron beamline. A new tool combining local XRF and scanning probe microscopy is finally proposed. PMID:23758858

  13. Application of Reflectance Confocal Microscopy in Dermatology Practice

    Ayşe Esra Koku Aksu

    2015-03-01

    Full Text Available In vivo reflectance confocal microscopy (RCM is a non-invasive method, imaging cellular structures in living skin at a level close to the histological resolution. It is easier to diagnose melanocytic and non-melanocytic skin tumors especially in difficult cases when RCM features have been identified. Determination of the cellular features, presence of cellular and structural atypia with RCM allows the discrimination of benign and malignant lesions. Preoperative differential diagnosis of malignant lesions, determining preoperative lesion borders in complicated cases, identification of local recurrence after excision of malignant lesions, monitoring the treatment efficacy in patients using topical treatment and who can not be operated, are the main areas of RCM in tumoral lesions. Besides, RCM is helpful in the establishing the diagnosis of inflammatory disease like psoriasis, contact dermatitis, lichen planus and in evaluation of therapeutic efficacy, detecting of infestation like tinea, skabiyes, demodicosis and determining the level of bullae in bullous disease. Due to being noninvasive, RCM is preferred in cosmetology, in clinical research and practice for the evaluation of the effectiveness of cosmetic products and cosmetic procedures.

  14. Comparison of in vivo confocal endomicroscopy with other diagnostic modalities to detect intracellular helicobacters.

    Sharman, M; Bacci, B; Simpson, K; Mansfield, C

    2016-07-01

    Intracellular colonisation may serve as a protected niche where Helicobacter spp. organisms evade effective treatment. In dogs, non-Helicobacter pylori-helicobacters are frequently intracellular. Confocal endomicroscopy allows in vivo gastrointestinal imaging and has aided real-time identification of Helicobacter pylori and other intracellular and mucosally associated bacteria. The objectives of this study were: (1) to determine the utility of confocal endomicroscopy to identify non-Helicobacter pylori-helicobacters compared with other diagnostic modalities, and (2) to assess its ability to identify intracellular organisms. Fourteen clinically healthy dogs underwent standard gastroduodenoscopy followed by confocal endomicroscopy using topical acriflavine. Confocal images were obtained from at least five gastric sites. Endoscopic biopsies were obtained for histopathology, PCR and fluorescence in situ hybridisation (FISH). Methodologies were compared for their ability to determine the presence and spatial distribution of gastric helicobacters in dogs. Confocal endomicroscopy provided high quality images allowing in vivo identification of non-Helicobacter pylori-helicobacters in 13 dogs. Histopathology identified helicobacters in 11 dogs. Organisms were identified within the superficial gastric mucus and within gastric pits, and distribution throughout the stomach was diffuse and multi-focal. Confocal endomicroscopy findings correlated with PCR and FISH post-procedure analysis. Only FISH identified intracellular organisms, which were present in 13/14 dogs. Confocal endomicroscopy provided in vivo histology images and was capable of identifying non-Helicobacter pylori-helicobacters during gastroscopy, but was unable to identify intracellular organisms using the current fluorophore protocol. PMID:27240920

  15. Innovative confocal laser method for exact dioptric power measurement of intraocular lens implants Invited Paper

    Ilko K. Ilev; Robert W. Faaland; Do-Hyun Kim; Robert H. James; Don Calogero

    2008-01-01

    We present a novel confocal laser method (CLM) for precise testing of the dioptric power of both positive and negative intraocular lens (IOL) implants. The CLM principle is based on a simple fiber-optic confocal laser design including a single-mode fiber coupler that serves simultaneously as a point light source used for formation of a collimated Gaussian laser beam, and as a highly sensitive confocal point receiver. The CLM approach provides an accurate, repeatable, objective, and fast method for IOL dioptric power measurement over the range from 0 D to greater than =t=30 D under both dry and in-situ simulated conditions.

  16. Confocal laser scanning microscopy in study of bone calcification

    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.

  17. Optimum Combined Lenses for Confocal Biochip Scanning System

    黄国亮; 程京; 周玉祥; 冯继宏; 刘诚迅; 金国藩; 邬敏贤; 严瑛白; 张腾飞; 李林

    2002-01-01

    Laboratory-on-a-chip technology has attracted wide interest in recent years, where the sample preparation, bio-chemical reaction, separation, detection and analysis are performed in a small biochip of the size of a fingernail. To obtain a high detection sensitivity of 1 fluors/μm2 (one fluorescence molecule per square micrometer) in biochip scanning systems, the scanning objective lens is required to have a high numerical aperture (>0.5), very small focal spot (3 mm). This study presents the design of optimum combined lenses including scanning objective and fluorescence focal lenses. The scanning objective had a high numerical aperture (NA) of 0.72, a very small focal spot of 1.67 μm, a long back focal length of 3.2 mm, and a high resolving power of 760 lines/mm. The fluorescence focal lenses had an NA of 0.3, a fluorescence focal spot of 16 μm, a long back focal length of 16.7 mm and a resolving power of 590 lines/mm. The phase aberrations of the combined lenses, including the aspherical aberration and the chromatic aberration corresponding to wavelengths of 532, 570, 635, and 670 nm, were well-corrected. The encircled energy diagram of the lenses was within the diffraction limit. The study also included the focal spot diagram, the optical path difference diagram, the transverse ray fan plot, and the modulation transfer function. A confocal biochip scanning system with designed combined lenses was developed and some experiments were conducted on a multi-channel biochip.

  18. Confocal laser scanning microscopy in study of bone calcification

    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.

  19. Anti-translational research: from the bedside back to the bench for reflectance confocal microscopy

    Gareau, Daniel

    2014-03-01

    The reflectance confocal microscope has made translational progress in dermatology. 0.5 micrometer lateral resolution, 0.75mm field-of-view and excellent temporal resolution at ~15 frames/second serve the VivaScope well in the clinic, but it may be overlooked in basic research. This work reviews high spatiotemporal confocal microscopy and presents images acquired of various samples: zebra fish embryo where melanocytes with excellent contrast overly the spinal column, chicken embryo, where myocardium is seen moving at 15 frames/ second, calcium spikes in dendrites (fluorescence mode) just beyond the temporal resolution, and human skin where blood cells race through the artereovenous microvasculature. For an introduction to confocal microscopy, see: http://dangareau.net.s69818.gridserver.com/science/confocal-microscopy

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

    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. ...

  1. Confocal Optical Imaging Systems and Their Applications in Microscopy and Range Sensing

    Xiao, Guoqing

    1990-11-01

    Confocal optical imaging systems have been the subject of much recent studies. They have found their applications in biomedical imaging and integrated circuit metrology. Confocal systems differ from the standard optical imaging systems in their use of point illumination and point detection, gaining an improved transverse resolution and superior depth resolution. The depth discrimination capability allows confocal imaging systems to optically cross section translucent objects or to image three-dimensional structures. The improvement in transverse resolution permits them to image structures with more detail and better contrast. This thesis has focused on the design and implementation of the confocal optical imaging systems and their applications. A nonparaxial confocal optical imaging theory is developed based on the scalar Rayleigh-Sommerfeld diffraction theory and Sine Condition without the normally-used thin-lens approximation. Two confocal optical range sensors and a Real-time Confocal Scanning Optical Microscope (RSOM) are demonstrated. It is shown that our RSOM has tremendous advantages over other confocal microscopes both in scanning speed and in the ease of use and alignment. The dependence of the imaging characteristics on the pinhole size and the lens is fully discussed. Experimental measurements are compared with the theoretical calculations. Good agreement is obtained. Also demonstrated in this thesis are numerous applications of the RSOM in integrated circuit metrology and biomedical imaging. Deep trenches as narrow as 1 μm and deep as 6 mu m are observed with the RSOM. The RSOM is not only able to measure the trench depth but, is also able to inspect individual defects inside the trench. Linewidth measurement is also investigated. The RSOM is shown to have an excellent optical cross-sectioning capability. Sectioned images of bones, teeth, and the unprepared cornea of a rabbit eye have been observed. Well-defined sectioned images have been obtained

  2. Confocal Bioluminescence Imaging for Living Tissues with a Caged Substrate of Luciferin.

    Hattori, Mitsuru; Kawamura, Genki; Kojima, Ryosuke; Kamiya, Mako; Urano, Yasuteru; Ozawa, Takeaki

    2016-06-21

    Fluorescence imaging can elucidate morphological organization and coordinal networks, but its background luminescence degrades the image contrast. Our confocal bioluminescence imaging system uses a luciferase caged substrate, with light passing through multipinhole arrays, causing bioluminescence at a focal plane. After a charge-coupled device camera captures luminescence, the imaging system acquires confocal images of multilayered cells with depth information, supporting quantitative analysis of spatial cellular localization in living tissues. PMID:27216493

  3. Confocal laser microscopy of dystrophin localization in guinea pig skeletal muscle fibers

    1992-01-01

    A confocal laser microscope was used to analyze the localization pattern of dystrophin along the sarcolemma in guinea pig skeletal muscle fibers. Hind leg muscles of the normal animals were freshly dissected and frozen for cryostat sections, which were then stained with a monoclonal antidystrophin antibody. In confocal laser microscopy, immunofluorescence staining in relatively thick sections could be sharply imaged in thin optical sections. When longitudinal and transverse sections of muscle...

  4. Diagnostic accuracy of microbial keratitis with in vivo scanning laser confocal microscopy

    Hau, Scott; Dart, John; Vesaluoma, Minna; Parmar, Dipak; Claerhout, Ilse; Bibi, Kanom; Larkin, Frank

    2010-01-01

    Abstract Aims To determine the accuracy of diagnosing microbial keratitis by masked medical and non-medical observers using the Heidelberg Retina Tomograph II / Rostock Cornea Module in vivo confocal microscope. Methods Confocal images were selected for 62 eyes with culture or biopsy proven infections. The cases comprised of 26 Acanthamoeba, 12 fungus, 3 Microsporidia, 2 Nocardia, and 19 bacterial infections (controls). The reference standard for comparison was a pos...

  5. Spectrally encoded confocal microscopy of esophageal tissues at 100 kHz line rate

    Schlachter, Simon C.; Kang, DongKyun; Gora, Michalina J.; Vacas-jacques, Paulino; Wu, Tao; Carruth, Robert W.; Eric J Wilsterman; Bouma, Brett E.; Woods, Kevin; Tearney, Guillermo J.

    2013-01-01

    Spectrally encoded confocal microscopy (SECM) is a reflectance confocal microscopy technology that uses a diffraction grating to illuminate different locations on the sample with distinct wavelengths. SECM can obtain line images without any beam scanning devices, which opens up the possibility of high-speed imaging with relatively simple probe optics. This feature makes SECM a promising technology for rapid endoscopic imaging of internal organs, such as the esophagus, at microscopic resolutio...

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

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

    2013-01-01

    The gastric mucosa ischemic tissular damage plays an important role in critical care patients' outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized,...

  7. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    Ramla Gary; Giovani Carbone; Gia Petriashvili; Maria Penelope De Santo; Riccardo Barberi

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra ...

  8. Clinical applications of in vivo fluorescence confocal laser scanning microscopy

    Oh, Chilhwan; Park, Sangyong; Kim, Junhyung; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Chun, Byungseon; Gweon, Daegab

    2008-02-01

    Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In

  9. Latest developments and opportunities for 3D analysis of biological samples by confocal mu-XRF

    Perez, Roberto D., E-mail: danperez@famaf.unc.edu.a [FaMAF, Universidad Nacional de Cordoba, Ciudad Universitaria, 5000 Cordoba (Argentina); CONICET, Rivadavia 1917, 1033 Buenos Aires (Argentina); Sanchez, Hector J. [FaMAF, Universidad Nacional de Cordoba, Ciudad Universitaria, 5000 Cordoba (Argentina); CONICET, Rivadavia 1917, 1033 Buenos Aires (Argentina); Perez, Carlos A. [Laboratorio Nacional de Luz Sincrotron-LNLS, POB 6192, 13084-971 Campinas, SP (Brazil); Rubio, Marcelo [FaMAF, Universidad Nacional de Cordoba, Ciudad Universitaria, 5000 Cordoba (Argentina); CONICET, Rivadavia 1917, 1033 Buenos Aires (Argentina); CEPROCOR, Ministerio de Ciencia y Tecnologia de Cordoba, 5164 Santa Maria de Punilla, Cordoba (Argentina)

    2010-02-15

    X-ray fluorescence analysis performed with a primary radiation focused in the micrometer range is known as micro-X-ray fluorescence (mu-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 mu-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 mu-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 mu-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 mu-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 mu-XRF was introduced by Malzer and Kanngiebetaer [2005. A model for the

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

    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

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

    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

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

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

    2013-01-01

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

  13. Feasibility of confocal endomicroscopy in the diagnosis of pediatric gastrointestinal disorders

    Krishnappa Venkatesh; Marta Cohen; Clair Evans; Peter Delaney; Steven Thomas; Christopher Taylor; Ashraf Abou-Taleb; Ralf Kiesslich; Mike Thomson

    2009-01-01

    AIM: To evaluate the feasibility and utility of confocal laser endomicroscopy (CLE) in the description of normal gastrointestinal (GI) mucosa and in the diagnosis of GI disorders in children, in comparison to histology. METHODS: Forty-four patients (19 female) median age 10.9 years (range 0.7-16.6 years) with suspected or known GI pathology underwent esophago-gastroduodenoscopy (OGD) ( n = 36) and/or ileocolonoscopy (IC) ( n = 31) with CLE using sodium fluorescein and acriflavine as contrast agents. Histological sections were compared with same site confocal images by two experienced pediatric and GI histopathologists and endoscopists, respectively. RESULTS: Duodenum and ileum were intubated in all but one patient undergoing OGD and IC. The median procedure time was 16.4 min (range 7-25 min) for OGD and 27.9 min (range 15-45 min) for IC. A total of 4798 confocal images were compared with 153 biopsies from the upper GI tract from 36 procedures, and 4661 confocal images were compared with 188 biopsies from the ileocolon from 31 procedures. Confocal images were comparable to conventional histology both in normal and in pathological conditions such as esophagitis, Helicobacter pylori gastritis, celiac disease, inflammatory bowel disease, colonic heterotopia, and graft versus host disease. CONCLUSION: CLE offers the prospect of targeting biopsies to abnormal mucosa, thereby increasing diagnostic yield, reducing the number of biopsies, decreasing the burden on the histopathological services, and reducing costs.

  14. Confocal scanning laser ophthalmoscopic imaging resolution of secondary retinal effects induced by laser radiation

    Zwick, Harry; Lund, David J.; Stuck, Bruce E.; Zuclich, Joseph A.; Elliot, Rowe; Schuschereba, Steven T.; Gagliano, Donald A.; Belkin, M.; Glickman, Randolph D.

    1996-02-01

    We have evaluated secondary laser induced retinal effects in non-human primates with a Rodenstock confocal scanning laser ophthalmoscope. A small eye animal model, the Garter snake, was employed to evaluate confocal numerical aperture effects in imaging laser retinal damage in small eyes vs. large eyes. Results demonstrate that the confocal image resolution in the Rhesus monkey eye is sufficient to differentiate deep retinal scar formation from retinal nerve fiber layer (NFL) damage and to estimate the depth of the NFL damage. The best comparison with histological depth was obtained for the snake retina, yielding a ratio close to 1:1 compared to 2:1 for the Rhesus. Resolution in the Garter snake allows imaging the photoreceptor matrix and therefore, evaluation of the interrelationship between the primary damage site (posterior retina), the photoreceptor matrix, and secondary sites in the anterior retina such as the NFL and the epiretinal vascular system. Alterations in both the retinal NFL and epiretinal blood flow rate were observed within several minutes post Argon laser exposure. Unique aspects of the snake eye such as high tissue transparency and inherently high contrast cellular structures, contribute to the confocal image quality. Such factors may be nearly comparable in primate eyes suggesting that depth of resolution can be improved by smaller confocal apertures and more sensitive signal processing techniques.

  15. Theoretical analysis of a rotating-disk partially confocal scanning microscope.

    Conchello, J A; Lichtman, J W

    1994-02-01

    Confocal scanning microscopy is widely used for three-dimensional (3-D) visualization of fixed specimens but has found only a limited 3-D reconstruction application for living specimens because the high intensity of the excitation often damages the specimen or causes the fluorescent dye to bleach. Computational optical-sectioning microscopy also suffers from drawbacks because nonconfocal 3-D imaging is fundamentally constrained by an artifactual elongation in the optical axis imposed by the so-called missing cone. We investigate the imaging properties of a new rotating-disk partially confocal scanning microscope (PCSM) that greatly reduces collection time by using multiple apertures for both excitation and detection, effectively working as many confocal microscopes in parallel. We show that this PCSM behaves as a hybrid microscope; near the in-focus plane it behaves near the theoretical optimum for confocal microscopy, and away from this plane its behavior approaches that of a nonconfocal microscope. We also show that the rotating-disk PCSM does not suffer from a missing cone. In fact, the optical transfer function of the theoretically optimal confocal microscope and the rotating-disk PCSM have practically the same bandpass in the spatial-frequency domain. PMID:20862053

  16. Depth elemental imaging of forensic samples by confocal micro-XRF method.

    Nakano, Kazuhiko; Nishi, Chihiro; Otsuki, Kazunori; Nishiwaki, Yoshinori; Tsuji, Kouichi

    2011-05-01

    Micro-XRF is a significant tool for the analysis of small regions. A micro-X-ray beam can be created in the laboratory by various focusing X-ray optics. Previously, nondestructive 3D-XRF analysis had not been easy because of the high penetration of fluorescent X-rays emitted into the sample. A recently developed confocal micro-XRF technique combined with polycapillary X-ray lenses enables depth-selective analysis. In this paper, we applied a new tabletop confocal micro-XRF system to analyze several forensic samples, that is, multilayered automotive paint fragments and leather samples, for use in the criminaliztics. Elemental depth profiles and mapping images of forensic samples were successfully obtained by the confocal micro-XRF technique. Multilayered structures can be distinguished in forensic samples by their elemental depth profiles. However, it was found that some leather sheets exhibited heterogeneous distribution. To confirm the validity, the result of a conventional micro-XRF of the cross section was compared with that of the confocal micro-XRF. The results obtained by the confocal micro-XRF system were in approximate agreement with those obtained by the conventional micro-XRF. Elemental depth imaging was performed on the paint fragments and leather sheets to confirm the homogeneity of the respective layers of the sample. The depth images of the paint fragment showed homogeneous distribution in each layer expect for Fe and Zn. In contrast, several components in the leather sheets were predominantly localized. PMID:21438498

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

    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.

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

    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. (paper)

  19. Reflectance confocal microscopy for scarring and non-scarring alopecia real-time assessment.

    Ardigò, Marco; Agozzino, Marina; Franceschini, Chiara; Donadio, Carlo; Abraham, Leonardo Spagnol; Barbieri, Luca; Sperduti, Isabella; Berardesca, Enzo; González, Salvador

    2016-07-01

    Clinical management of alopecia represents one of the major issues in dermatology. Scalp biopsies are not easily accepted because of the high bleeding and sensitive anatomical area. Trichoscopy is routinely used for diagnosis of alopecia, but in several cases lack to provide sufficient information on the status of the disease. Recently, reflectance confocal microscopy demonstrated its usefulness for the evaluation of several inflammatory skin condition and preliminary reports about alopecia have been proposed in the literature. The aim was to identify the confocal features characterizing scarring and non-scarring alopecia. Reflectance confocal microscopy from 86 patients affected by scarring (28 lichen planopilaris and 9 lupus erythematosus) and non-scarring alopecia (30 androgenic alopecia and 19 alopecia areata), were retrospectively, blinded evaluated. Good concordance between different readers on the confocal criteria has been assessed. Statistical significant features, specific for scarring alopecia and non-scarring alopecia have been identified. In this study, data on reflectance confocal microscopy features useful for the differential diagnosis between scarring and non-scarring alopecia have been identified. Further studies focusing on the use of this non-invasive technique in the therapeutic follow-up and distinction of sub-entities of alopecia are still required. PMID:27225248

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

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

    2015-10-01

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

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

    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.

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

    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.

  3. Imaging theory and resolution improvement of two-photon confocal microscopy

    TANG; Zhilie(唐志列); YANG; Chuping(杨初平); PEI; Hongjin(裴红津); LIANG; Ruisheng(梁瑞生); LIU; Songhao(刘颂豪)

    2002-01-01

    The nonlinear effect of two-photon excitation on the imaging property of two-photonconfocal microscopy has been analyzed by the two-photon fluorescence intensity transfer functionderived in this paper. The two-photon fluorescence intensity transfer function in a confocal micros-copy is given. Furthermore the three-dimensional point spread function (3D-PSF) and thethree-dimensional optical transfer function (3D-OTF) of two-photon confocal microscopy are de-rived based on the nonlinear effect of two-photon excitation. The imaging property of two-photonconfocal microscopy is discussed in detail based on 3D-OTF. Finally the spatial resolution limit oftwo-photon confocal microscopy is discussed according to the uncertainty principle.

  4. Confocal laser endomicroscopy in the " in vivo" histological diagnosis of the gastrointestinal tract

    Giovanni D De Palma

    2009-01-01

    Recent technological advances in miniaturization have allowed for a confocal scanning microscope to be integrated into a conventional flexible endoscope, or into trans-endoscopic probes, a technique now known as confocal endomicroscopy or confocal laser endomicroscopy. This newly-developed technology has enabled endoscopists to collect real-time in vivo histological images or "virtual biopsies" of the gastrointestinal mucosa during endoscopy, and has stimulated significant interest in the application of this technique in clinical gastroenterology. This review aims to evaluate the current data on the technical aspects and the utility of this new technology in clinical gastroenterology and its potential impact in the future, particularly in the screening or surveillance of gastrointestinal neoplasia.

  5. Use of confocal microscopy in the study of ischemia-induced hippocampal neuronal damage

    Radenović Lidija

    2008-01-01

    Full Text Available The present study was undertaken to reveal by means of confocal laser microscopy the cytoarchitecture of hippocampal CA3 neurons in Mongolian gerbils before and after cerebral ischemia of different duration. The common carotid arteries of gerbils were occluded for 5, 10, or 15 min. On the 4th, 14th and 28th day after reperfusion, neuronal damage was examined by laser scanning confocal microscopy in the CA3 region of hippocampus (30 μm slices. Slices were stained with fluorescent Nissl staining and fluorescent membrane tracer DiI. Increased duration of cerebral ischemia resulted in a progressive loss of hippocampal CA3 neurons. Four days after the ischemic insult, neuronal damage in the hippocampal CA3 region was mild but visible. On the 28th day after reperfusion, neuronal damage in the observed brain structure was most severe. These results demonstrate the temporal profile of neuronal damage after an ischemic insult as observed using confocal microscopy.

  6. Nonlocal Effects in the Confocal μ-Raman Characterization of Inhomogeneous Polymer Coatings

    Rodriguez, R.; Vargas, S.; Estevez, M.

    2010-11-01

    The confocal μ-Raman technique was used to characterize the morphology of inhomogeneous anti-graffiti coatings; for these systems, the antiadherent molecules were segregated to the external (exposed) surface forming a layer whose thickness was determined. The confocal data from these inhomogeneous coatings contains nonlocal contributions because the light scattered from sources near the specific specimen under analysis (the focused region) could not be completely rejected by the spatial filter of the confocal device. These nonlocal contributions had important effects in the Raman spectra, modifying the bands height profiles of homogeneous and inhomogeneous materials allowing their identification. Taking into account these nonlocal effects, it was possible to interpret correctly the relative intensities of the Raman bands and characterize properly the inhomogeneous coatings.

  7. Confocal laser endomicroscopy in the in vivo histological diagnosis of the gastrointestinal tract

    Giovanni D De Palma

    2009-12-01

    Full Text Available Recent technological advances in miniaturization have allowed for a confocal scanning microscope to be integrated into a conventional flexible endoscope, or into trans-endoscopic probes, a technique now known as confocal endomicroscopy or confocal laser endomicroscopy. This newly-developed technology has enabled endoscopists to collect real-time in vivo histological images or “virtual biopsies” of the gastrointestinal mucosa during endoscopy, and has stimulated significant interest in the application of this technique in clinical gastroenterology. This review aims to evaluate the current data on the technical aspects and the utility of this new technology in clinical gastroenterology and its potential impact in the future, particularly in the screening or surveillance of gastrointestinal neoplasia.

  8. Supercontinuum ultra wide range confocal microscope for reflectance spectroscopy of living matter and material science surfaces

    Stefano Selci

    2011-09-01

    Full Text Available We report the design and implementation of a new reflectance laser scanning confocal system with spectroscopy imaging capabilities. Confocal spectroscopy is achieved by using a very broad spectral range supercontinuum source capable of high precision reflectance data in the VIS-IR spectral range thanks to an almost achromatic optical layout. With this apparatus we collect each single scanning point as a whole spectrum in a continuous range, associated with the optical section imaging possibilities typical of a confocal set up. While such a microscope has been developed for bio medical analysis of human skin and other similar applications, first test results on solid samples produce spectroscopic results that, compared to analytical models based on the Abelés matrix transfer methods, show a very good agreement, opening new possibilities of a complete spectroscopic fingerprinting of samples with microscopic details.

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

    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+ 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

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

    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.

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

    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. PMID:27515076

  12. Numerical study of a confocal ultrasonic setup for creation of cavitation

    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

  13. Numerical study of a confocal ultrasonic setup for creation of cavitation

    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

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

    Lunter, Dominique; Daniels, Rolf

    2016-03-01

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

  15. Two-photon fluorescence imaging of embryo with much less damage than confocal imaging

    Liu, Bian; Xu, Hui; Jin, Lei; Ma, Hui; Chen, Die Yan

    2002-09-01

    Two-photon Laser Scanning Microscopy (TPLSM) is a novel technique based on the two-photon excitation of fluorophore. In this paper, TPLSM and traditional confocal microscopy are introduced. And the influence of femtosecond near-infrared (NIR) illumination on mouse embryos is investigated for the first time. The result shows that NIR laser has much less damage to embryos than blue laser and proves that TPLSM is superior to conventional confocal microscopy in keeping sample alive. TPLSM enables us to make a continuous observation for a longer time on embryogenesis.

  16. Analysis of plant tissue images obtained by confocal tandem scanning reflected light microscope

    A. Zdunek

    2007-03-01

    Full Text Available The paper presents two methods (automatic and semi-automatic for quantitative evaluation of cell structural parameters of plant tissues. The methods were developed for images obtained by confocal tandem scanning reflected light microscope. Quality of the images is sufficient for semi-automatic analysis. However, the automatic method does not give satisfactory results because it gives mean cell area 30% bigger and 60% fewer cells than in the semi-automatic method. Therefore, we state that for images taken by confocal tandem scanning reflected light microscope semi-automatic analysis is more accurate and proper at this moment.

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

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

    2015-10-01

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

  18. Measurement of Capillary Length from 3D Confocal Images Using Image Analysis and Stereology

    Janáček, Jiří; Saxl, Ivan; Mao, X. W.; Kubínová, Lucie

    Valencia : University of Valencia, 2007. s. 71-71. [Focus on Microscopy FOM 2007. 10.04.2007-13.04.2007, Valencia] Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10190503 Keywords : spo2 * 3D image analysis * capillaries * confocal microscopy Subject RIV: EA - Cell Biology

  19. New classification for probe-based confocal laser endomicroscopy in the colon

    T. Kuiper; F.J.C. van den Broek; S. van Eeden; M.B. Wallace; A.M. Buchner; A. Meining; K. van Hee; P. Fockens; E. Dekker

    2011-01-01

    Background and aims: Probe-based confocal laser endomicroscopy (pCLE; Cellvizio, Mauna Kea Technologies, Paris, France) enables in vivo histology during colonoscopy and may allow endoscopists to make real-time diagnoses. A collaboration of five experts proposed a new pCLE classification for colonic

  20. Hybrid Rayleigh, Raman and two-photon excited fluorescence spectral confocal microscopy of living cells

    Pully, Vishnu Vardhan; Lenferink, Aufried; Otto, Cees

    2010-01-01

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

  1. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR QUANTIFYING CYTOMETRIC APPLICATIONS WITH SPECTROSCOPIC INSTRUMENTS

    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...

  2. A shaped annular beam tri-heterodyne confocal microscope with good anti-environmental interference capability

    Zhao Wei-Qian; Feng Zheng-De; Qiu Li-Rong

    2007-01-01

    A shaped annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a confocal microscope. It simultaneously detects far-, on-, and near-focus signals with given phase differences by dividing the measured light path of the confocal microscope into three sub-paths (signals). Pair-wise real-time heterodyne subtraction of the three signals is used to improve the anti-environmental interference capability, axial resolution, and linearity; and a shaped annular beam super-resolution technique is used to improve lateral resolution. Theoretical analyses and preliminary experiments indicate that an axial resolution of about 1 nm can be achieved with a shaped annular beam tri-heterodyne confocal microscope and its lateral resolution can be better than 0.2μm for λ= 632.8 nm, the numerical aperture of the lens of the microscope is NA = 0.85, and the normalized radius ε= 0.5.

  3. Double-confocal resonator for X-ray generation via intracavity Thomson scattering

    Xie, M. [Lawrence Berkeley Lab., CA (United States)

    1995-12-31

    There has been a growing interest in developing compact X-ray sources through Thomson scattering of a laser beam by a relativistic electron beam. For higher X-ray flux it is desirable to have the scattering to occur inside an optical resonator where the laser power is higher. In this paper I propose a double-confocal resonator design optimized for head-on Thomson scattering inside an FEL oscillator and analyze its performance taking into account the diffraction and FEL gain. A double confocal resonator is equivalent to two confocal resonators in series. Such a resonator has several advantages: it couples electron beam through and X-ray out of the cavity with holes on cavity mirrors, thus allowing the system to be compact; it supports the FEL mode with minimal diffraction loss through the holes; it provides a laser focus in the forward direction for a better mode overlap with the electron beam; and it provides a focus at the same location in the backward direction for higher Thomson scattering efficiency; in addition, the mode size at the focal point and hence the Rayleigh range can be adjusted simply through intracavity apertures; furthermore, it gives a large mode size at the mirrors to reduce power loading. Simulations as well as analytical results will be presented. Also other configurations of intracavity Thomson scattering where the double-confocal resonator could be useful will be discussed.

  4. Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy

    Krause, M.; Riet, J. te; Wolf, K. van der

    2013-01-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness toge

  5. Mapping of geometrical characteristics and 3D visualization of blood capillary network from confocal microscopic images

    Janáček, Jiří; Karen, Petr; Chernyavskiy, Oleksandr; Michálek, Jan; Mao, X. W.; Jirkovská, M.; Kubínová, Lucie

    Urbino: Societa Italiana Scienze Micriscopiche, 2011. s. 89-90 R&D Projects: GA MŠk(CZ) ME09010; GA MŠk(CZ) LC06063; GA ČR(CZ) GA304/09/0733 Institutional research plan: CEZ:AV0Z50110509 Keywords : 3D reconstructions * confocal microscopy * capillaries Subject RIV: BO - Biophysics

  6. Spatial arrangement of genetic loci in human blood cell nuclei studied by confocal cytometry

    Cafourková, Alena; Jirsová, Pavla; Kozubek, Stanislav; Kozubek, Michal; Bártová, Eva; Lukášová, Emilie

    Würzburg : University of Würzburg , 2001, s. P181. [International Chromosome Conference /14./. Würzburg (DE), 04.09.2001-08.09.2001] Institutional research plan: CEZ:AV0Z5004920 Keywords : genetic loci * human blood cell * confocal cytometry Subject RIV: BO - Biophysics

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

    Suihko, C.; Serup, J.

    2008-01-01

    Background: Fibre-optic fluorescence confocal laser scanning microscopy (CLSM) is a novel non-invasive technique for in vivo imaging of skin. The cellular structure of the epidermis can be studied. A fluorophore, e.g. fluorescein sodium, is introduced by an intradermal injection or applied...

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

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

    2015-01-01

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

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

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

    2011-01-01

    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 aim

  10. Correcting the axial shrinkage of skeletal muscle thick sections visualized by confocal microscopy

    Janáček, Jiří; Kreft, M.; Čebašek, V.; Eržen, I.

    2012-01-01

    Roč. 246, č. 2 (2012), s. 107-112. ISSN 0022-2720 R&D Projects: GA MŠk(CZ) MEB090910; GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z50110509 Keywords : capillaries * confocal microscopy * sample deformation * shrinkage * skeletal muscle * 3D Subject RIV: FH - Neurology Impact factor: 1.633, year: 2012

  11. Metrological characterization of optical confocal sensors measurements (20 and 350 travel ranges)

    Confocal sensors are usually used in dimensional metrology applications, like roughness, form, thickness and surface profile measurements. With the progress of technologies, metrological applications require measurements with nanometer-level of accuracy by using ultra-high precision machines, which should present a minimum and stable metrology loop. The loop is equipped with sensors with nanometer-level of resolution and linear residual. The study presented here, is mainly focused on the characterization of Confocal sensors in order to identify their performance practically. Such information is useful to establish a correction model in the digital signal processing (DSP) software. In this context, LNE developed an ultra-high-precision machine, dedicated to the roughness measurement with an uncertainty of a few nanometres (< 30 nm) by using a tactile sensor. In order to match this machine to Confocal sensors, an experiment has been recently developed to characterize the behaviour of two commercial Confocal sensors with the measuring range of 20 μm and 350 μ m. The experiment permits the evaluation of the major error sources: axial and radial motion errors as-well-as the deviation/tilt of the sensors

  12. Calibration Method for Confocal X-Ray Microanalysis with Polychromatic Excitation

    C. Sosa

    2015-01-01

    Full Text Available To apply the fundamental parameters method at the confocal setup the knowledge of the sensitivity of the spectrometer is required which depends on the characteristics of two X-ray lenses: one in the excitation channel and another in the detection channel. For the particular case of polychromatic excitation, the theory shows that the focalization properties of the excitation lens for all incident energies affect the X-ray fluorescence intensity. Therefore the traditional calibration method based on the measurement of standard samples becomes unstable since the number of required fitting parameters is too high. To reduce these parameters a previous characterization of the excitation lens by a simulation program was employed giving rise to a simplified confocal setup calibration. The developed calibration method was applied for a confocal spectrometer implemented in the Brazilian Synchrotron Radiation Source (LNLS with white beam. The experimental parameters of the sensitivity were obtained from depth profile analysis of several pure thin films. The calibrated confocal setup was used to quantify reference standards in order to validate the calibration procedure. Our results for elemental concentrations show relative errors less than 15% for the quantitative analysis of a light matrix reference standard.

  13. Correction of axial chromatic aberrations in confocal Raman microspectroscopic measurements of a single microbial spore.

    Lasch, Peter; Hermelink, Antje; Naumann, Dieter

    2009-06-01

    Herein we describe a strategy for correcting the longitudinal or axial component of chromatic aberration in confocal Raman microspectroscopy. The method is based on measuring a vertical series of confocal Raman sections of samples by a high numerical aperture Raman microscope. Using the known characteristics of the wavelength-dependent focal shift of the optical system, the Raman intensities can be corrected to allow the rearrangement of Raman data from different focal planes. In the present study the computational correction routine was applied to an experimental data set of 4-dimensional (xyz spatial and the spectral dimension) confocal Raman spectra collected from single spores of Bacillus cereus. After correcting the axial component of the chromatic aberration, univariate and multivariate spectral parameters were obtained and used in the following for 3D segmentation and volume rendering on the basis of the structural and compositional information contained in the Raman spectra of the spore. Using univariate Raman intensities from defined functional group frequencies or k-means cluster membership values as a multivariate parameter for volume rendering, we demonstrate a high degree of correlation between confocal Raman microspectroscopy and the spores' morphology. In this paper we will also present cluster mean spectra which will be discussed in light of the presence of proteins and Ca-DPA, a calcium chelate of dipicolinic acid in the spore. PMID:19475143

  14. Advances in combined endoscopic fluorescence confocal microscopy and optical coherence tomography

    Risi, Matthew D.

    Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100 μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (˜1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography

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

    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.

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

    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. PMID:26786962

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

    Zavislan James M

    2009-08-01

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

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

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

  19. Measurement of grain size of polycrystalline materials with confocal energy dispersive micro-X-ray diffraction technology based on polycapillary X-ray optics

    The confocal energy dispersive micro-X-ray diffraction (EDMXRD) based on polycapillary X-ray optics was used to determine the grain size of polycrystalline materials. The grain size of a metallographic specimen of nickel base alloy was measured by using the confocal EDMXRD. The experimental results demonstrated that the confocal EDMXRD had potential applications in measuring large grain size

  20. Development of confocal micro X-ray fluorescence instrument using two X-ray beams

    A new confocal micro X-ray fluorescence instrument was developed. This instrument has two independent micro X-ray tubes with Mo targets. A full polycapillary X-ray lens was attached to each X-ray tube. Another half polycapillary 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 effects of the excitation of two X-ray beams were investigated. The instrument enabled highly sensitive three-dimensional X-ray fluorescence analysis. We confirmed that the X-ray fluorescence intensity from the sample increased by applying the two independent X-ray tubes in confocal configuration. Elemental depth profiling of black wheat was demonstrated with the result that each element in the surface coat of a wheat grain showed unique distribution

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

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

    2015-01-01

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

  2. Confocal, two-photon laser-induced fluorescence technique for the detection of nitric oxide.

    Reeves, M; Musculus, M; Farrell, P

    1998-10-01

    We describe a confocal two-photon laser-induced fluorescence scheme for the detection of gaseous NO. Excitation from a simple YAG-pumped Coumarin 450 dye system near 452.6 nm was used to promote the two-photon NO(A (2)?(+), nu? = 0 ? X (2)?, nu? = 0) transition in the gamma(0, 0) band. Subsequent fluorescence detection in the range 200-300 nm permitted almost total rejection of elastic and geometric scatter of laser radiation for excellent signal/noise ratio characteristics. The goal of the research was to apply NO fluorescence to a relatively realistic limited optical access combustion environment. A confocal optical arrangement was demonstrated for single-point measurements of NO concentration in gas samples and in atmospheric-pressure flames. The technique is suitable for applications that offer only a single direction for optical access and when significant elastic scatter is present. PMID:18301470

  3. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    Gary, Ramla; Carbone, Giovani; Petriashvili, Gia; De Santo, Maria Penelope; Barberi, Riccardo

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. PMID:26907286

  4. Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena

    Quantitative colocalization analysis is an advanced digital imaging tool to examine antigens of interest in immunofluorescence images obtained using confocal microscopes. It employs specialized algorithms to estimate the degree of overlap of fluorescence signals and thus enables acquiring important new information not otherwise obtainable using qualitative approaches alone. As raw confocal images have high levels of background, they should be prepared to become suitable for reliable calculation of colocalization coefficients by correcting it. We provide concise theoretical basis of quantitative colocalization analysis, discuss its limitations, and describe proper use of the technique. The use of quantitative colocalization analysis is demonstrated by studying bile salt export pump and multidrug resistance associated protein 2 in the liver and major basic protein and platelet activating factor receptor antigens in conjunctiva. The review is focused on the applicability and correct interpretation of the results of colocalization coefficients calculations

  5. A confocal rheoscope to study bulk or thin-film material under uniaxial or biaxial shear

    Lin, Neil Y C; Cheng, Xiang; Leahy, Brian; Cohen, Itai

    2016-01-01

    We present a new design of a confocal rheoscope that enables us to precisely impose a uniform uniaxial or biaxial shear. The design consists of two precisely-positioned parallel plates. Our design allows us to adjust the gap between the plates to be as small as 2$\\pm$0.1 $\\mu$m, 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 3D 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.

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

    Truernit Elisabeth

    2008-06-01

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

  7. Sub-diffraction imaging with confocal fluorescence microscopy by stochastic photobleaching

    Wang, Yifan; Kuang, Cuifang; Cai, Huanqing; Li, Shuai; Liu, Wei; Hao, Xiang; Ge, Jianhong; Liu, Xu

    2014-02-01

    We propose a single molecule localization method which takes advantage of stochastic photobleaching to improve the resolution of confocal fluorescence microscopy. By detecting the stochastic intensity loss of fluorophores, each fluorophore in the field can be localized. When all locations are known, a sub-diffraction image can be retrieved through single molecule localization algorithms. A confocal scheme is used to record the bleaching process of the sample. Each fluorophore can be localized from the recorded streaming followed by image subtraction. Compared with other single molecule localization concepts such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM), this method does not require a laser cycling equipment and the pixel size is no longer limited by the size of CCD. This technique works well with common fluorescent dyes and does not require the use of engineered photoactivatable proteins or photoswitchable synthetic dye pairs.

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

    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.

  9. Analysis of cell-tissue grafts under weightless conditions using confocal fluorescence microscopy

    Volova, L. T.; Milyakova, M. N.; Rossinskaya, V. V.; Boltovskaya, V. V.; Kulagina, L. N.; Kurganskaya, L. V.; Timchenko, P. E.; Timchenko, E. V.; Zherdeva Taskina, Larisa A.

    2015-03-01

    The research results of monitoring of viable cells in a cellular-tissue graft using confocal laser fluorescence microscopy at 488 nm and 561 nm with the use of fluorophore propidium iodide (propidium iodide, PI Sigma Aldrich USA) are presented. The processing of the received images was carried out using the software ANDOR. It is experimentally shown that the method of confocal fluorescence microscopy is one of the informational methods for detecting cells populated in a 3-D bio-carrier with a resolution of at least 400 nm. Analysis of the received micrographs suggests that the cells that were in a bio-carrier for 30 days in a synchronous ground-based experiment retained their viability compared to a similar space-based experiment in which the cells were hardly detected in a bio-carrier.

  10. Confocal X-ray fluorescence micro-spectroscopy experiment in tilted geometry

    This paper provides a generalized mathematical model to describe the intensity of primary X-ray fluorescence radiation collected in the tilted confocal geometry mode, where the collimating optics is rotated over an angle relative to a horizontal plane. The influence of newly introduced terms, which take into account the tilted geometry mode, is discussed. The model is verified with a multi-layer test sample scanned in depth. It is proved that for low-Z matrices, the rotation of the detection channel does not induce any significant differences in a reconstruction of the thickness and chemical composition of layers, so that it may safely be ignored. - Highlights: • A mathematical model for confocal XRF spectroscopy in tilted geometry was derived. • Tilted geometry influenced the analytical capabilities of XRF instrument slightly. • Thickness and the chemical composition of multi-layers were determined

  11. Confocal laser endomicroscopy and immunoendoscopy for real-time assessment of vascularization in gastrointestinal malignancies

    Dan Ionu(t)Gheonea; Tatiana Car(ta)n(a); Tudorel Ciurea; Carmen Popescu; Anca B(a)d(a)r(a)u; Adrian S(a)ftoiu

    2011-01-01

    Gastrointestinal cancers represent a major cause of morbidity and mortality,with incomplete response to chemotherapy in the advanced stages and poor prognosis.Angiogenesis plays a crucial part in tumor growth and metastasis,with most gastrointestinal cancers depending strictly on the development of a new and devoted capillary network.Confocal laser endomicroscopy is a new technology which allows in vivo microscopic analysis of the gastrointestinal mucosa and its microvascularization during ongoing endoscopy by using topically or systemically administered contrast agents.Targeting markers of angiogenesis in association with confocal laser endomicroscopic examination(immunoendoscopy),as a future challenge,will add functional analysis to the morphological aspect of the neoplastic process.This review describes previous experience in endomicroscopic examination of the upper and lower digestive tract with emphasis on vascularization,resulting in a broad spectrum of potential clinical applications,and also preclinical research that could be translated to human studies.

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

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

    2014-03-01

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

  13. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy.

    Gary, Ramla; Carbone, Giovani; Petriashvili, Gia; De Santo, Maria Penelope; Barberi, Riccardo

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. PMID:26907286

  14. A Monolithic Confocal Laser Coupler For an Optical Pick-up

    Mizuno, Takeshi; Doi, Masato; YoshinobuHiguchi, YoshinobuHiguchi; Taniguchi, Takehiro; NobukataOkano, NobukataOkano; Nakao, Takashi; Narui, Hironobu; Matsuda, Osamu

    1999-04-01

    We propose a novel optical pick-up using a confocal lasercoupler for an optical disk player. The laser coupler consists of aglass window and a monolithic optical element which includes a laserdiode, 8 photodiodes, and a pyramid-shaped prism mirror positionednear the confocal plane which acts as a knife edge and aphoto-coupler. The focusing-error signal is detected using theconfocal knife edge (CKE) method and the tracking-error signal isdetected using the CKE push-pull method. The jitter of Compact Disc(CD) readout was 6.7 ns at a line velocity of 1.2 m/s, and the DCoffsets of the tracking-error signal were sppressed to less than 1/3for a radial lens displacement of ±400 µm compared to theconventional push-pull method.

  15. Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.

    Kubitscheck, U; Pratsch, L; Passow, H; Peters, R.

    1995-01-01

    The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ATP were released by a short UV light pulse. The time-dependent fluorescence intensity of the calcium indicator was then followed in single ghosts by repetitive confocal imaging. The fluore...

  16. Acidic Microclimate pH Distribution in PLGA Microspheres Monitored by Confocal Laser Scanning Microscopy

    Ding, Amy G.; Schwendeman, Steven P.

    2008-01-01

    The microclimate pH (μpH) distribution inside poly(lactic-co-glycolic acid) (PLGA) microspheres was monitored quantitatively over an acidic range as a function of several formulation variables. A ratiometric method by confocal laser scanning microscopy with Lysosensor yellow/blue® dextran was adapted from those previously reported, and μpH distribution kinetics inside PLGA microspheres was examined during incubation under physiologic conditions for 4 weeks. The effects of polymer molecular we...

  17. Segmentation of Confocal Raman Microspectroscopic Imaging Data Using Edge-Preserving Denoising and Clustering

    Alexandrov, Theodore; Lasch, Peter

    2013-01-01

    Over the past decade, confocal Raman microspectroscopic (CRM) imaging has matured into a useful analytical tool to obtain spatially resolved chemical information on the molecular composition of biological samples and has found its way into histopathology, cytology, and microbiology. A CRM imaging data set is a hyperspectral image in which Raman intensities are represented as a function of three coordinates: a spectral coordinate λ encoding the wavelength and two spatial coordinates x and y. U...

  18. Confocal microscopy study of pertussis toxin and toxoids on CHO-cells

    Tan, Yajun; Fleck, Roland A.; Asokanathan, Catpagavalli; Yuen, Chun-Ting; Xing, Dorothy; Zhang, Shumin; Wang, Junzhi

    2013-01-01

    Pertussis toxin in its detoxified form is a major component of all current acellular pertussis vaccines. Here we report the membrane translocation and internalization activities of pertussis toxin and various pertussis toxoids using Chinese hamster ovary cells and confocal microscopy based on indirect immunofluorescence labeling. Chemically detoxified pertussis toxoids were able to translocate/internalize into cells at the concentration about 1,000 times higher than the native toxin. Pertussi...

  19. Confocal and conventional immunofluorescence and ultrastructural localisation of intracellular strength-giving components of human amniochorion.

    Ockleford, C; Malak, T; A. Hubbard; Bracken, K; Burton, S A; Bright, N.; Blakey, G; Goodliffe, J; Garrod, D; d'Lacey, C

    1993-01-01

    Key cytoskeletal polypeptides of human fetal membranes have been localised at subcellular level using confocal and conventional indirect immunofluorescence microscopy. Correlation with electron microscope data has allowed us to examine how cellular compartments of this multilaminar tissue maintain their mechanical integrity until the time of membrane rupture at parturition. Evidence is presented for myofibroblastic characteristics of cells in both the fibroblast and reticular layers which may...

  20. Visualizing the Tumor Microenvironment of Liver Metastasis by Spinning Disk Confocal Microscopy.

    Babes, Liane; Kubes, Paul

    2016-01-01

    Intravital microscopy has evolved into an invaluable technique to study the complexity of tumors by visualizing individual cells in live organisms. Here, we describe a method for employing intravital spinning disk confocal microscopy to picture high-resolution tumor-stroma interactions in real time. We depict in detail the surgical procedures to image various tumor microenvironments and different cellular components in the liver. PMID:27581024

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

    Truernit Elisabeth; Haseloff Jim

    2008-01-01

    Abstract Background Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy....

  2. In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells

    Vermaas, Wim F. J.; Timlin, Jerilyn A.; Jones, Howland D. T.; Sinclair, Michael B.; Nieman, Linda T.; Hamad, Sawsan W.; Melgaard, David K.; Haaland, David M.

    2008-01-01

    Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (≈0.03-μm3) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exc...

  3. Characterization of tumor microvascular structure and permeability: comparison between magnetic resonance imaging and intravital confocal imaging

    Reitan, Nina Kristine; Thuen, Marte; Goa, Pål Erik; de Lange Davies, Catharina

    2010-01-01

    Solid tumors are characterized by abnormal blood vessel organization, structure, and function. These abnormalities give rise to enhanced vascular permeability and may predict therapeutic responses. The permeability and architecture of the microvasculature in human osteosarcoma tumors growing in dorsal window chambers in athymic mice were measured by confocal laser scanning microscopy (CLSM) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Dextran (40 kDa) and Gadomer were u...

  4. Compensation of illumination inhomogeneities in multi-resolution image acquisition in confocal microscopy

    Tomori, Z.; Čapek, Martin; Michálek, Jan; Kubínová, Lucie

    Moscow: Lomonosov Moscow State University, 2009, s. 108-111. [International Conference on Computer Graphics and Vision /19./. Moscow (RU), 05.10.2009-09.10.2009] R&D Projects: GA ČR(CZ) GA102/08/0691; GA MŠk(CZ) MEB080831 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal microscopy * light attenuation * distribution function Subject RIV: IN - Informatics, Computer Science

  5. A SENSITIVE AND STABLE CONFOCAL FABRY-PEROT INTERFEROMETER FOR SURFACE ULTRASONIC VIBRATION DETECTION

    DING HONG-SHENG; TONG LI-GE; CHEN GENG-HUA

    2001-01-01

    A new confocal Fabry-Pérot interferometer (CFPI) has been constructed. By using both of the conjugate rays,the sensitivity of the system was doubled. Moreover, the negative feedback control loop of a single-chip microcomputer (MCS-51) was applied to stabilize the working point at an optimum position. The system has been used in detecting the piezoelectric ultrasonic vibration on the surface of an aluminium sample.

  6. To see the unseeable: confocal miniprobes for routine microscopic imaging during endoscopy

    Osdoit, A.; Lacombe, F.; Cavé, C.; Loiseau, S.; Peltier, E.

    2007-02-01

    Confocal fluorescence high resolution imaging during standard endoscopic procedures has been presented as a very promising tool to enhance patient care and physician practice by providing supplementary diagnostic information in real-time. The purpose of this paper is to show not only potential, but convincing results of endoscopic microscopy using a catheter-based approach. Mauna Kea Technologies' core technology, Cellvizio, delivers dynamic imaging at 12 frames/second using confocal miniprobes inserted through the operating channel of regular endoscopes. Cellvizio is composed of 3 parts including (a) a Laser Scanning Unit, (b) Confocal Miniprobe TM with the following characteristics: 5-15 μm axial resolution, 2-5 μm lateral resolution, 15-100 μm depth of penetration, field of view of 600x500 μm and (c) a software package with onthe- fly processing capabilities. With several tens of patients examined during routine GI endoscopy procedures, the most relevant clinical parameters could be assessed in a doubled-blinded fashion between the endoscopist and a pathologist and results showing very high accuracy in the differentiation of neoplasia from normal and hyperplastic tissue were obtained. In the field of pulmonology, the micro-autofluorescence properties of tissues could be assessed and structures never before accessed in vivo were observed. Cellvizio® may be useful to study bronchial remodeling in asthma and chronic obstructive pulmonary diseases. Using appropriate topical fluorescent dye, the Confocal Miniprobes may also make it possible to perform optical biopsy of precancerous and superficial bronchial cancers. Cellvizio® is as a new tool towards "targeted biopsies", leading to earlier, more reliable and cost effective diagnostic procedures. Other applications, specifically in molecular imaging are also made possible by the miniaturization of the probe (combination with biopsy needle for solid organs use or lymph node detection) and by the

  7. High-speed line-field confocal holographic microscope for quantitative phase imaging.

    Liu, Changgeng; Knitter, Sebastian; Cong, Zhilong; Sencan, Ikbal; Cao, Hui; Choma, Michael A

    2016-05-01

    We present a high-speed and phase-sensitive reflectance line-scanning confocal holographic microscope (LCHM). We achieved rapid confocal imaging using a fast line-scan CCD camera and quantitative phase imaging using off-axis digital holography (DH) on a 1D, line-by-line basis in our prototype experiment. Using a 20 kHz line scan rate, we achieved a frame rate of 20 Hz for 512x512 pixels en-face confocal images. We realized coherent holographic detection two different ways. We first present a LCHM using off-axis configuration. By using a microscope objective of a NA 0.65, we achieved axial and lateral resolution of ~3.5 micrometers and ~0.8 micrometers, respectively. We demonstrated surface profile measurement of a phase target at nanometer precision and the digital refocusing of a defocused confocal en-face image. Ultrahigh temporal resolution M mode is demonstrated by measuring the vibration of a PZT-actuated mirror driven by a sine wave at 1 kHz. We then report our experimental work on a LCHM using an in-line configuration. In this in-line LCHM, the coherent detection is enabled by moving the reference arm at a constant speed, thereby introducing a Doppler frequency shift that leads to spatial interference fringes along the scanning direction. Lastly, we present a unified formulation that treats off-axis and in-line LCHM in a unified joint spatiotemporal modulation framework and provide a connection between LCHM and the traditional off-axis DH. The presented high-speed LCHM may find applications in optical metrology and biomedical imaging. PMID:27137541

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

    Ulrich, Martina; Lange-Asschenfeldt, Susanne

    2013-06-01

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

  9. Calibration Method for Confocal X-Ray Microanalysis with Polychromatic Excitation

    Sosa, C.; Stoytschew, V.; Leani, J.; H. J. Sánchez; C. A. Pérez; R. D. Perez

    2015-01-01

    To apply the fundamental parameters method at the confocal setup the knowledge of the sensitivity of the spectrometer is required which depends on the characteristics of two X-ray lenses: one in the excitation channel and another in the detection channel. For the particular case of polychromatic excitation, the theory shows that the focalization properties of the excitation lens for all incident energies affect the X-ray fluorescence intensity. Therefore the traditional calibration method bas...

  10. In Vivo Reflectance Confocal Microscopy of Basal Cell Carcinoma: Feasibility of Preoperative Mapping of Cancer Margins

    Pan, Zhan-Yan; Lin, Jing-Ran; Cheng, Ting-Ting; Wu, Jia-Qiang; Wu, Wen-Yu

    2012-01-01

    Reflectance confocal microscopy (RCM) images skin at cellular resolution and has shown utility for the diagnosis of nonmelanoma skin cancer in vivo. It has the potential to define lesion margins before surgical therapy. Objectives To investigate the feasibility of RCM in defining the margins of basal cell carcinoma before surgery. Methods The margins of 10 lesions were evaluated using RCM. Biopsies of the margins were used to confirm the results. A protocol was constructed to define margins. ...

  11. Patterns of morphological variation within myelin internodes of normal peripheral nerve: quantitative analysis by confocal microscopy.

    Reynolds, R. J.; Heath, J. W.

    1995-01-01

    Knowledge of variations in the morphology of normal myelinated peripheral nerve fibres is fundamental to subsequent interpretation of neuropathology. It would be advantageous for structural analysis of normal variations to be based on entire myelin internodes, but acquisition of such data via the classic approach of nerve fibre teasing has been hindered by limitations in optical resolution and specimen preparation. This study addressed these limitations through a new confocal imaging method w...

  12. Multimodal confocal mosaics enable high sensitivity and specificity in screening of in situ squamous cell carcinoma

    Grados Luyando, Maria del Carmen; Bar, Anna; Snavely, Nicholas; Jacques, Steven; Gareau, Daniel S.

    2014-02-01

    Screening cancer in excision margins with confocal microscopy may potentially save time and cost over the gold standard histopathology (H and E). However, diagnostic accuracy requires sufficient contrast and resolution to reveal pathological traits in a growing set of tumor types. Reflectance mode images structural details due to microscopic refractive index variation. Nuclear contrast with acridine orange fluorescence provides enhanced diagnostic value, but fails for in situ squamous cell carcinoma (SCC), where the cytoplasm is important to visualize. Combination of three modes [eosin (Eo) fluorescence, reflectance (R) and acridine orange (AO) fluorescence] enable imaging of cytoplasm, collagen and nuclei respectively. Toward rapid intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaics can image wide surgical margins (~1cm) with sub-cellular resolution and mimic the appearance of conventional H and E. Absorption contrast is achieved by alternating the excitation wavelength: 488nm (AO fluorescence) and 532nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H and E, enabling detection of the carcinoma in situ in the epidermal layer The sum mosaic Eo+R is false-colored pink to mimic eosins' appearance in H and E, while the AO mosaic is false-colored purple to mimic hematoxylins' appearance in H and E. In this study, mosaics of 10 Mohs surgical excisions containing SCC in situ and 5 containing only normal tissue were subdivided for digital presentation equivalent to 4X histology. Of the total 16 SCC in situ multimodal mosaics and 16 normal cases presented, two reviewers made 1 and 2 (respectively) type-2 errors (false positives) but otherwise scored perfectly when using the confocal images to screen for the presence of SCC in situ as compared to the gold standard histopathology. Limitations to precisely mimic H and E included occasional elastin staining by AO. These results suggest that

  13. Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy

    Kubínová, Lucie; Janáček, Jiří; Ribarič, S.; Čebašek, V.; Eržen, I.

    2001-01-01

    Roč. 22, č. 3 (2001), s. 217-227. ISSN 0142-4319 R&D Projects: GA AV ČR IAA5011810; GA MŠk ME 256; GA ČR GA304/01/0257 Institutional research plan: CEZ:AV0Z5011922 Keywords : capillaries * confocal microscopy * stereology Subject RIV: EA - Cell Biology Impact factor: 1.460, year: 2001

  14. 3D elemental imaging of the crustacean Ceriodaphnia by means of SR confocal micro-XRF

    De Samber, Björn; Evens, Roel; Boone, Matthieu; De Schamphelaere, Karel; Van Hoorebeke, Luc; Janssen, Colin; Falkenberg, Gerald; Appel, Karen; Vincze, Laszlo

    2011-01-01

    Daphnia is a freshwater crustacean (0.2-5 mm height) used for investigating the toxic effects of toxins (e.g. metals) on an ecosystem. Synchrotron radiation based micro X-ray fluorescence (SR micro-XRF) allows the investigation of the trace level metal distribution within these organisms in an essentially non-destructive manner. Several two-dimensional (2D), computed tomography (CT) and confocal micro-XRF experiments under conventional and cryogenic environments have been performed on Daphnia...

  15. Compensation of illumination inhomogeneities in confocal laser scanning microscope (CLSM) images

    Michálek, Jan; Čapek, Martin; Kubínová, Lucie

    Bologna : Esculapio, 2009 - (Capasso, V.; Aletti, G.; Micheletti, A.), s. 384-389 ISBN 978-88-7488-310-3. [European Congress of Stereology and Image Analysis /10./. Milano (IT), 22.06.2009-26.06.2009] R&D Projects: GA MŠk(CZ) LC06063; GA ČR(CZ) GA102/08/0691 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal laser scanning microscope * illumination inhomogeneity * grayscale mapping Subject RIV: JC - Computer Hardware ; Software

  16. Confocal Raman studies in determining crystalline nature of PECVD grown Si nanowires

    Silicon nanowires of diameter ∼200 nm and length of 2-4 µm are grown in the plasma enhanced chemical vapour deposition technique using nanoclustered Au catalyst assisted vapour-liquid-solid process. The crystallinity in the as-grown and annealed samples is studied using confocal Raman spectroscopic studies. Amorphous phase is formed in the as-grown samples. Structural studies using high resolution transmission electron microscopy confirm the polycrystalline nature in the annealed sample

  17. Confocal Raman studies in determining crystalline nature of PECVD grown Si nanowires

    Ahmed, Nafis; Bhargav, P. Balaji; Ramasamy, P. [SSN Research Centre, Kalavakkam-603110, Tamilnadu (India); Department of Physics, SSN College of Engineering, Kalavakkam-603110, Tamilnadu (India); Sivadasan, A. K.; Tyagi, A. K.; Dhara, S., E-mail: dhara@igcar.gov.in [Surface and Nanoscience Division, Indira Gandhi Centre for Atomic Research, Kalpakkam-603102 (India); Amirthapandian, S.; Panigrahi, B. K. [Materials Physics Division, Indira Gandhi Centre for Atomic Research, Kalpakkam-603102 (India); Bhattacharya, S. [SSN Research Centre, Kalavakkam-603110, Tamilnadu (India)

    2015-06-24

    Silicon nanowires of diameter ∼200 nm and length of 2-4 µm are grown in the plasma enhanced chemical vapour deposition technique using nanoclustered Au catalyst assisted vapour-liquid-solid process. The crystallinity in the as-grown and annealed samples is studied using confocal Raman spectroscopic studies. Amorphous phase is formed in the as-grown samples. Structural studies using high resolution transmission electron microscopy confirm the polycrystalline nature in the annealed sample.

  18. Full-field transmission x-ray imaging with confocal polycapillary x-ray optics

    Sun, Tianxi; MacDonald, C. A.

    2013-01-01

    A transmission x-ray imaging setup based on a confocal combination of a polycapillary focusing x-ray optic followed by a polycapillary collimating x-ray optic was designed and demonstrated to have good resolution, better than the unmagnified pixel size and unlimited by the x-ray tube spot size. This imaging setup has potential application in x-ray imaging for small samples, for example, for histology specimens.

  19. A low error reconstruction method for confocal holography to determine 3-dimensional properties

    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

  20. Research of Confocal Laser Induced Fluorescence Detection System for Micro-fluidic Chip

    FENG Jin-yuan; WANG Xiu-hua; ZHANG Hua-feng

    2007-01-01

    The characteristics such as signal noise ratio(SNR)[1-2] and sensitivity of the fluorescence detection system for micro-fluidic chip influence the performance of the whole system extremely.The confocal laser induced fluorescence detection system is presented.Based on the debugging of optical and circuit modules, the results of detecting the samples are given and analyzed theoretically,and the improved project is put forward.

  1. Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope

    Mukai, Rie; Shirai, Yasuhito; Saito, Naoaki; Yoshida, Ken-ichi; Ashida, Hitoshi

    2009-01-01

    Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning f...

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

    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....

  3. Quantitative assessment of microbicide-induced injury in the ovine vaginal epithelium using confocal microendoscopy

    Vargas Gracie; Patrikeev Igor; Wei Jingna; Bell Brent; Vincent Kathleen; Bourne Nigel; Motamedi Massoud

    2012-01-01

    Abstract Background The development of safe topical microbicides that can preserve the integrity of cervicovaginal tract epithelial barrier is of great interest as this may minimize the potential for increased susceptibility to STI infections. High resolution imaging to assess epithelial integrity in a noninvasive manner could be a valuable tool for preclinical testing of candidate topical agents. Methods A quantitative approach using confocal fluorescence microendoscopy (CFM) for assessment ...

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

    Husen, Peter Rasmussen; Fidorra, Matthias; Hartel, Steffen; Bagatolli, Luis; Ipsen, John Hjort

    2012-01-01

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

  5. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy

    Chao Huang; 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 d...

  6. UNDERSTANDING THE EFFECTS OF SURFACTANT ADDITION ON RHEOLOGY USING LASER SCANNING CONFOCAL MICROSCOPY

    White, T

    2007-05-08

    The effectiveness of three dispersants to modify rheology was examined using rheology measurements and laser scanning confocal microscopy (LSCM) in simulated waste solutions. All of the dispersants lowered the yield stress of the slurries below the baseline samples. The rheology curves were fitted reasonably to a Bingham Plastic model. The three-dimensional LSCM images of simulants showed distinct aggregates were greatly reduced after the addition of dispersants leading to a lowering of the yield stress of the simulated waste slurry solutions.

  7. Classification of histological severity of Helicobacter pylori-associated gastritis by confocal laser endomicroscopy

    2010-01-01

    AIM: To classify the histological severity of Helicobacter pylori (H. pylori) infection-associated gastritis by confocal laser endomicroscopy (CLE). METHODS: Patients with upper gastrointestinal symptoms or individuals who were screened for gastric cancer were enrolled in this study. Histological severity of H. pylori infection-associated gastritis was graded according to the established CLE criteria. Diagnostic value of CLE for histo-logical gastritis was investigated and compared with that of white light ...

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

    KA. Al-Salihi; NABA. Tarmidzi

    2009-01-01

    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 (a...

  9. Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy.

    Galli, Paolo; Strona, Giovanni; Villa, Anna Maria; Benzoni, Francesca; Fabrizio, Stefani; Doglia, Silvia Maria; Kritsky, Delane C

    2006-04-01

    A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes, is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy. PMID:16729702

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

    Zavislan James M; Bonfiglio Thomas A; Boger J Neil; Schiffhauer Linda M; Zuley Margarita; Fox Christi

    2009-01-01

    Abstract Background Needle core biopsy, often in conjunction with ultrasonic or stereotactic guided techniques, is frequently used to diagnose breast carcinoma in women. Confocal scanning laser microscopy (CSLM) is a technology that provides real-time digital images of tissues with cellular resolution. This paper reports the progress in developing techniques to rapidly screen needle core breast biopsy and surgical specimens at the point of care. CSLM requires minimal tissue processing and has...

  11. Fluorescein Punctate Staining Traced to Superficial Corneal Epithelial Cells by Impression Cytology and Confocal Microscopy

    Mokhtarzadeh, Maryam; Casey, Richard; Glasgow, Ben J.

    2011-01-01

    Punctate fluorescein staining is an important sign in ocular surface disease but its basis is controversial. The common view is that the spots reflect small epithelial defects. In this study, clinicocytologic and histopathologic correlation of punctate stains in dry eye disease was performed. The hyperfluorescent spots were traced from slit lamp examination to confocal microscopy of tissue to reveal that fluorescent superficial epithelial cells are basis of punctate staining.

  12. Elemental depth analysis of corroded paint-coated steel by confocal micro-XRF method

    A confocal micro-XRF method combined with two individual polycapillary lenses was applied to steel sheets coated with anti-corrosive paint in order to nondestructively observe 3D elemental distribution of paint steels and corroded paint-coated steels. Nondestructive depth analysis and 3D elemental mapping of the painted steel sheets were demonstrated under the confocal XRF configuration. Three different painted steel sheets were prepared by cation electrodeposition coating for automotive onto flat steel sheets modified with a zinc phosphate conversion coating. These painted sheets were then caused to corrode by means of accelerated exposure to a salt bath (5 mass% NaCl) at 55°C for 240 hours. Depth elemental profiles of Ti, Zn, and Fe obtained by confocal micro-XRF measurements were in excellent agreement with that of the prepared sample. Elemental depth profiles and maps of the corroded painted sheets showed some blisters caused by crevice corrosion, which started from the site of a scratch. The distributions of Ti and Fe were approximately homogeneous in both the paint layer and the steel substrate, while the distributions of Zn, Mn, Ca, and Cl were heterogeneous. (author)

  13. Development of confocal 3D micro-XRF spectrometer with dual Cr-Mo excitation

    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)

  14. Application of a novel confocal imaging technique for early the detection of dental decay

    Rousseau, Christel; Girkin, John M.; Vaidya, Shilpa; Hall, Andrew F.; Whitters, C. J.; Creanor, Steve L.

    2002-06-01

    In order to stop or prevent the progression of dental disease, early detection and quantification of decay are crucially important. Dental decay (caries) detection methods have traditionally involved clinical examination by eye, using probes and dental radiography, but up to 60% of lesions are missed. What the dentist requires is a cheap, reliable method of detection of early disease, ideally with information on the depth and rate of growth or healing. Conventional commercial scanning confocal microscopes are unsuitable for use on dental patients. We report on a fibre optic based confocal microscope designed for in vivo examination of caries lesions. The system utilizes a common fibre both as the source and to detect the reflected confocal signal. The initial system has been optimized using dielectric mirrors and the thickness of the stack has been measured with high precision. Dental samples have been examined and the system has been demonstrated to provide information on the depth and mineral loss of a lesion. Fibre optic microscopy (FOCM) demonstrates a practical route to developing an in vivo caries profiler. In this paper, the FOCM and its applications in caries detection are described and the potential of this scheme as a practical dental probe is discussed.

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

    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

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

    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.

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

    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.

  18. Next generation of optical diagnostics for bladder cancer using probe-based confocal laser endomicroscopy

    Liu, Jen-Jane; Chang, Timothy C.; Pan, Ying; Hsiao, Shelly T.; Mach, Kathleen E.; Jensen, Kristin C.; Liao, Joseph C.

    2012-02-01

    Real-time imaging with confocal laser endomicroscopy (CLE) probes that fit in standard endoscopes has emerged as a clinically feasible technology for optical biopsy of bladder cancer. Confocal images of normal, inflammatory, and neoplastic urothelium obtained with intravesical fluorescein can be differentiated by morphologic characteristics. We compiled a confocal atlas of the urinary tract using these diagnostic criteria to be used in a prospective diagnostic accuracy study. Patients scheduled to undergo transurethral resection of bladder tumor underwent white light cystoscopy (WLC), followed by CLE, and histologic confirmation of resected tissue. Areas that appeared normal by WLC were imaged and biopsied as controls. We imaged and prospectively analyzed 135 areas in 57 patients. We show that CLE improves the diagnostic accuracy of WLC for diagnosing benign tissue, low and high grade cancer. Interobserver studies showed a moderate level of agreement by urologists and nonclinical researchers. Despite morphologic differences between inflammation and cancer, real-time differentiation can still be challenging. Identification of bladder cancer-specific contrast agents could provide molecular specificity to CLE. By using fluorescently-labeled antibodies or peptides that bind to proteins expressed in bladder cancer, we have identified putative molecular contrast agents for targeted imaging with CLE. We describe one candidate agent - anti-CD47 - that was instilled into bladder specimens. The tumor and normal urothelium were imaged with CLE, with increased fluorescent signal demonstrated in areas of tumor compared to normal areas. Thus, cancer-specificity can be achieved using molecular contrast agents ex vivo in conjunction with CLE.

  19. 3-D reconstruction of neurons from multichannel confocal laser scanning image series.

    Wouterlood, Floris G

    2014-01-01

    A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. Scanning of a specimen, with stepwise axial (Z-) movement of the stage in between each scan, produces Z-series of confocal images of a tissue volume, which then can be used 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. PMID:24723320

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

    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

  1. Experimental research on radius of curvature measurement of spherical lenses based on laser differential confocal technique

    Ding, Xiang; Sun, Ruoduan; Li, Fei; Zhao, Weiqian; Liu, Wenli

    2011-11-01

    A new approach based on laser differential confocal technique is potential to achieve high accuracy in radius of curvature (ROC) measurement. It utilizes two digital microscopes with virtual pinholes on the CCD detectors to precisely locate the cat's-eye and the confocal positions, which can enhance the focus-identification resolution. An instrumental system was established and experimental research was carried out to determine how error sources contribute to the uncertainty of ROC measurement, such as optical axis misalignment, dead path of the interferometer, surface figure error of tested lenses and temperature fluctuation, etc. Suggestions were also proposed on how these factors could be avoided or suppressed. The system performance was tested by employing four pairs of template lenses with a serial of ROC values. The relative expanded uncertainty was analyzed and calculated based on theoretical analysis and experimental determination, which was smaller than 2x10-5 (k=2). The results were supported by comparison measurement between the differential confocal radius measurement (DCRM) system and an ultra-high accuracy three-dimensional profilometer, showing good consistency. It demonstrated that the DCRM system was capable of high-accuracy ROC measurement.

  2. A novel piezoelectric microstage with embedded sensor for dual axes confocal endomicroscopy (Conference Presentation)

    Choi, Jongsoo; Qiu, Zhen; Rhee, Choong-Ho; Wang, Thomas D.; Oldham, Kenn R.

    2016-03-01

    A piezoelectric microactuator previously proposed by the authors for laser scanning in dual axes confocal endomicroscopy meets two primary challenges for dual axes confocal imaging: large out-of-plane actuation (~500μm) and a relatively high bandwidth (>100Hz). In order to further reach stage positioning error better than desired imaging resolution of 5 μm and to improve the robustness of actuator performance, a closed-loop controller and thus on-chip sensing, are being incorporated and integrated with system modeling. This work presents these thin-film PZT based microstages where piezoelectric unimorphs are used not only to actuate its central platform but also to estimate its vertical motion. Initial results from on-chip piezoelectric sensing are presented. Although sensing output shows some feed-through from the actuation signal, testing shows detection of AC motion from various vibration modes of the stage. Meanwhile, 3D profiles of the entire actuator structure at different DC voltage levels were obtained and used to form a nonlinear optimization problem to estimate all forces and moments that each component of the device experiences for the prediction of its deflection. A comparison between modeled and experimental deflection of the actuation beams is included. These results will be used to describe the dynamic behavior of the actuation beams, where the sensors are embedded, and to estimate sensing outputs in order to implement a close-loop controller. Prototype stages are currently being assembled into a handheld dual axes confocal imaging system.

  3. Quantifying light scattering with single-mode fiber -optic confocal microscopy

    Haidekker Mark A

    2009-11-01

    Full Text Available Abstract Background Confocal microscopy has become an important option for examining tissues in vivo as a diagnostic tool and a quality control tool for tissue-engineered constructs. Collagen is one of the primary determinants of biomechanical stability. Since collagen is also the primary scattering element in skin and other soft tissues, we hypothesized that laser-optical imaging methods, particularly confocal scattered-light scanning, would allow us to quantify scattering intensity and determine collagen content in biological layers. Methods We built a fully automated confocal scattered-light scanner to examine how light scatters in Intralipid, a common tissue phantom, and three-dimensional collagen gels. Intralipid with 0.5%, 1.0%, 1.5%, and 2.0% concentration was filled between precisely spaced glass coverslips. Collagen gels at collagen concentrations from 0.30 mg/mL to 3.30 mg/mL were prepared, and all samples underwent A-mode scanning with multiple averaged scans. In Intralipid samples, light reflected from the upper fluid-glass interface was measured. In collagen gels, average scattering intensity inside the actual gel was measured. In both cases, intensity was correlated with concentration. Results By measuring light attenuation at interface reflections of various thicknesses using our device, we were able to determine that the scattering coefficient at 660 nm of Intralipid at increasing concentrations in water to be 39 cm-1 for each percent increase of Intralipid. We were also able to measure the amount of scattering of various concentrations of collagen in gels directly using backscattered light. The results show a highly linear relationship with an increase of 8.2 arbitrary units in backscattering intensity for every 1 mg increase of collagen within a 1 mL gel volume. Conclusion The confocal scattered-light scanner allows to accurately quantify scattering in Intralipid and collagen gels. Furthermore, a linear relationship between

  4. Application of confocal laser scanning microscopy in characterization of chemical enhancers in drug-in-adhesive transdermal patches

    Qvist, Michael H.; Hoeck, Ulla; Kreilgaard, Bo; Madsen, Flemming; Hovgaard, Lars; Frokjaer, Sven

    2001-01-01

    The purpose of this study was to evaluate the application of confocal laser scanning microscopy (CLSM) in the examination of the embedment and the release characteristics of chemical permeation enhancers from transdermal drug delivery systems (TDDSs) of the “drug-in-adhesive” type. The enhancer lauric acid and a lauric acid fluorescing probe of the Bodipy type were incorporated into TDDSs consisting of an acrylic, a polyisobutylene, or a silicone polymer adhesive. Three-dimensional confocal i...

  5. Cytosolic pH gradients in cultured neuronal cell lines studied by laser scanning confocal microscopy, real-time confocal microscopy, and spectral imaging microscopy

    Sanchez-Armass, Sergio; Sennoune, Souad; Martinez, Gloria M.; Ortega, Filiberta; Martinez-Zaguilan, Raul

    2002-06-01

    Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.

  6. Confocal Raman microscopy for investigation of the level of differentiation in living neuroblastoma tumor cells

    Scalfi-Happ, Claudia; Jauss, Andrea; Hollricher, Olaf; Fulda, Simone; Hauser, Carmen; Steiner, Rudolf; Rück, Angelika

    2007-07-01

    The investigation of living cells at physiological conditions requires very sensitive, sophisticated, non invasive methods. In this study, Raman spectral imaging is used to identify different biomolecules inside of cells. Raman spectroscopy, a chemically and structurally sensitive measuring technique, is combined with high resolution confocal microscopy. In Raman spectral imaging mode, a complete Raman spectrum is recorded at every confocal image point, giving insight into the chemical composition of each sample compartment. Neuroblastoma is the most common solid extra-cranial tumor in children. One of the unique features of neuroblastoma cells is their ability to differentiate spontaneously, eventually leading to complete remission. Since differentiation agents are currently used in the clinic for neuroblastoma therapy, there is a special need to develop non-invasive and sensitive new methods to monitor neuroblastoma cell differentiation. Neuroblastoma cells at different degrees of differentiation were analysed with the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using a frequency doubled Nd:YAG laser at 532 nm and 10 mW for excitation. Integration time per spectrum was 80-100 ms. A lateral resolution in submicrometer range was achieved by using a 60x water immersion lens with a numerical aperture of 1,0. Raman images of cells were generated from these sets of data by either integrating over specific Raman bands, by basis analysis using reference spectra or by cluster analysis. The automated evaluation of all spectra results in spectral unmixed images providing insight into the chemical composition of the sample. With these procedures, different cell organelles, cytosol, membranes could be distinguished. Since neuroblastoma cells at high degree of differentiation overproduce noradrenaline, an attempt was made to trace the presence of this neurotransmitter as a marker for differentiation. The results of this work may have applications in the

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

    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

  8. Quantitative assessment of microbicide-induced injury in the ovine vaginal epithelium using confocal microendoscopy

    Vargas Gracie

    2012-02-01

    Full Text Available Abstract Background The development of safe topical microbicides that can preserve the integrity of cervicovaginal tract epithelial barrier is of great interest as this may minimize the potential for increased susceptibility to STI infections. High resolution imaging to assess epithelial integrity in a noninvasive manner could be a valuable tool for preclinical testing of candidate topical agents. Methods A quantitative approach using confocal fluorescence microendoscopy (CFM for assessment of microbicide-induced injury to the vaginal epithelium was developed. Sheep were treated intravaginally with one of five agents in solution (PBS; 0.02% benzalkonium chloride (BZK; 0.2% BZK or gel formulation (hydroxyethyl cellulose (HEC; Gynol II nonoxynol-9 gel (N-9. After 24 hours the vaginal tract was removed, labeled with propidium iodide (PI, imaged, then fixed for histology. An automated image scoring algorithm was developed for quantitative assessment of injury and applied to the data set. Image-based findings were validated with histological visual gradings that describe degree of injury and measurement of epithelial thickness. Results Distinct differences in PI staining were detected following BZK and N-9 treatment. Images from controls had uniformly distributed nuclei with defined borders, while those after BZK or N-9 showed heavily stained and disrupted nuclei, which increased in proportion to injury detected on histology. The confocal scoring system revealed statistically significant scores for each agent versus PBS controls with the exception of HEC and were consistent with histology scores of injury. Conclusions Confocal microendoscopy provides a sensitive, objective, and quantitative approach for non-invasive assessment of vaginal epithelial integrity and could serve as a tool for real-time safety evaluation of emerging intravaginal topical agents.

  9. Dark-field scanning confocal microscope for vertical particle tracks in nuclear emulsion

    The principle of the DArk-FIeld Scanning CONfocal (DAFISCON) microscope for selective observation of the vertical particle tracks in nuclear emulsion is described. The construction of the DAFISCON microscope, built on the basis of the 2D measurement microscope, is described. The results of the experimental testing of the DAFISCON microscope, accomplished at high density of the vertical particle tracks, are presented. The 2D plot and the 1D plot of the CCD dark-field image are given. The spatial resolution of our microscope can be increased by using the objective with higher aperture

  10. Cell volume and geometric parameters determination in living cells using confocal microscopy and 3D reconstruction

    sprotocols

    2015-01-01

    Authors: David Hevia, Aida Rodriguez-Garcia, Marta Alonso-Gervós, Isabel Quirós-González, Henar M Cimadevilla, Carmen Gómez-Cordovés, Rosa M Sainz & Juan C Mayo ### Abstract The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO...

  11. Characterization of typical surface effects in additive manufacturing with confocal microscopy

    The surfaces of laser-sintered and laser beam melted parts are characterized by typical surface effects and thus cause difficulties when using the popular tactile profilometry method and conventional two-dimensional surface parameters for analysis. Therefore, this study shows the potential of a new measurement approach by a confocal microscope and three-dimensional surface parameters. Within this study, a special focus is laid on the classification of different surface orientations and on a special issue in laser sintering: orange peel severity. (paper)

  12. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Kecheng Li; Douglas W. Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. Withthe thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration, and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  13. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    KechengLi; DouglasW.Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. With the thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  14. Methods for compensation of the light attenuation with depth of images captured by a confocal microscope

    Čapek, Martin; Janáček, Jiří; Kubínová, Lucie

    2006-01-01

    Roč. 69, č. 8 (2006), s. 624-635. ISSN 1059-910X R&D Projects: GA MŠk(CZ) LC06063; GA AV ČR(CZ) IAA100110502; GA AV ČR(CZ) IAA600110507; GA AV ČR(CZ) IAA500200510; GA AV ČR(CZ) KJB6011309; GA ČR(CZ) GA304/05/0153 Institutional research plan: CEZ:AV0Z50110509 Keywords : image enhancement * confocal microscopy * histogram warping Subject RIV: JC - Computer Hardware ; Software Impact factor: 1.680, year: 2006

  15. Confocal Laser Scanning Microscopy and Two Photon Excitation Microscopy as Tools to Study Testate Amoebae

    Burdíková, Zuzana; Čapek, Martin; Ostašov, Pavel; Mitchell, E.A.D.; Machač, Jiří; Kubínová, Lucie

    2010-01-01

    Roč. 16, Suppl.2 (2010), s. 1142-1143. ISSN 1431-9276. [Microscopy and Microanalysis 2010. Portland, 01.08.2010-05.08.2010] R&D Projects: GA MŠk(CZ) LC06063; GA ČR(CZ) GA102/08/0691; GA ČR(CZ) GA304/09/0733 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z60050516 Keywords : testate amoeba e * confocal microscopy * two-photon microscopy Subject RIV: BH - Optics, Masers, Lasers Impact factor: 2.179, year: 2010

  16. Imaging the Distribution of Sodium Dodecyl Sulfate in Skin by Confocal Raman and Infrared Microspectroscopy

    G. Mao; Flach, C. R.; Mendelsohn, R; Walters, R.M.

    2012-01-01

    ABSTRACT Purpose To image SDS distribution across different skin regions, to compare the permeability difference between porcine and human skin, and to evaluate the interaction between SDS and skin. Methods Full thickness porcine and human skin was treated with acyl chain perdeuterated SDS (SDS-d25) at room temperature and at 34 °C for 3, 24 and 40 h. SDS distribution in skin was monitored by confocal Raman and IR microspectroscopic imaging. Permeation profiles of SDS-d25 in skin were derived...

  17. Compensation and evaluation of errors of 3D reconstructions from confocal microscopic images

    Čapek, Martin; Brůža, Petr; Kocandová, L.; Janáček, Jiří; Kubínová, Lucie; Vagnerová, R.

    Vol. 1. Berlin : Springer, 2008 - (Luysberg, M.; Tillman, K.; Weirich, T.), s. 781-782 ISBN 978-3-540-85154-7. [European Microscopy Congress EMC 2008 /14./. Aachen (DE), 01.09.2008-05.09.2008] R&D Projects: GA MŠk(CZ) LC06063; GA AV ČR(CZ) IAA100110502; GA AV ČR(CZ) IAA500200510; GA ČR(CZ) GA102/08/0691 Institutional research plan: CEZ:AV0Z50110509 Keywords : 3D reconstruction * confocal microscopy * error Subject RIV: JD - Computer Applications, Robotics

  18. Volume Reconstruction Using Confocal Microscopy Images and Correction of Deformations by Elastic Registration

    Čapek, Martin; Brůža, Petr; Kocandová, Lucie; Janáček, Jiří; Kubínová, Lucie; Hozman, J.; Kneppo, P.; Vagnerová, R.

    Bologna : Esculapio, 2009 - (Capasso, V.; Aletti, G.; Micheletti, A.), s. 538-543 ISBN 978-88-7488-310-3. [European Congress of Stereology and Image Analysis /10./. Milano (IT), 22.06.2009-26.06.2009] R&D Projects: GA AV ČR(CZ) IAA100110502; GA ČR(CZ) GA102/08/0691; GA ČR GA304/09/0733; GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z50110509 Keywords : 3D reconstruction * elastic registration * 3D visualization * confocal microscopy Subject RIV: JC - Computer Hardware ; Software

  19. Structural and elemental X-ray microanalysis with synchrotron radiation in confocal geometry

    A spectrometer for 3D structural and multielemental X-ray microanalysis with synchrotron radiation is presented in this work. It is based on the combination of the energy dispersive X-ray fluorescence and diffraction with polycapillary optics. The 3D spatial resolution was achieved by the superposition of the foci of two lenses arranged in confocal geometry. The parameters that affect the performance of the spectrometer were study in detail giving rise to a simplified calibration method for depth profile analysis. Two specific examples were included to illustrate the use of the spectrometer in order to identify their possible application fields

  20. Amiodarone-induced multiorgan toxicity with ocular findings on confocal microscopy

    Ugur Turk

    2015-01-01

    Full Text Available Amiodarone is an antiarrhythmic medication that can adversely effect various organs including lungs, thyroid gland, liver, eyes, skin, and nerves. The risk of adverse effects increases with high doses and prolonged use. We report a 54-year-old female who presented with multiorgan toxicity after 8 months of low dose (200 mg/day amiodarone treatment. The findings of confocal microscopy due to amiodarone-induced keratopathy are described. Amiodarone may cause multiorgan toxicity even at lower doses and for shorter treatment periods.

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

    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)

  2. Using laser confocal scanning microscope to study ischemia-hypoxia injury in rat brain slice

    2000-01-01

    The level of lipid peroxidation and cellular necrosis in rat living brain slices during brain ischemia-hypoxia injury have been observed using a laser confocal scanning microscope (LCSM) with double labeling of fluorescent probes D-399 (2,7-dichlorofluorescin diacetate) and propidium iodide (PI).The hypoxia and/or reoxygenation injury in rat brain slices is markedly decreased by pretreatment with L-NG-nitro-arginine (L-NNA) and N-acetylcysteine (NAC),showing that the nitric oxide (NO) and other free radicals play an important role in brain ischemia-hypoxia injury.

  3. Confocal luminescence study of nitrogen-vacancy distribution within nitrogen-rich single crystal CVD diamond

    Shershulin, V. A.; Samoylenko, S. R.; Kudryavtsev, O. S.; Bolshakov, A. P.; Ashkinazi, E. E.; Yurov, V. Yu; Ralchenko, V. G.; Konov, V. I.; Vlasov, I. I.

    2016-01-01

    Confocal photoluminescence (PL) microscopy was used to study a distribution of negatively charged nitrogen-vacancy (NV-) defects within a surface and in a cross section of a homoepitaxial chemical vapor deposition (CVD) diamond layer intentionally grown with a nitrogen concentration close to the solubility limit. A variation in the PL intensity within the whole sample was found to exceed no more than 30% of the intensity maximum. The diamond layers with densely packed NV- arrays are a promising material platform for the design of highly sensitive magnetic field and temperature sensors, as well as for using this material in quantum optics and informatics technologies based on NV- spins.

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

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

    2010-01-01

    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 polarizat...

  5. Confocal microscopy: A new tool for erosion measurements on large scale plasma facing components in tokamaks

    A diagnostic based on confocal microscopy was developed at CEA Cadarache in order to measure erosion on large plasma facing components during shutdown in situ in Tore Supra. This paper describes the diagnostic and presents results obtained on Beryllium and Carbon Fibre Composite (CFC) materials. Erosion in the range of 800 μm was found on one sector of the Toroidal Pumped Limiter (TPL) which provides, by integration to the full limiter a net carbon erosion of about 900 g over the period 2002–2007

  6. Confocal microscopy: A new tool for erosion measurements on large scale plasma facing components in tokamaks

    Gauthier, E.; Brosset, C.; Roche, H.; Tsitrone, E.; Pégourié, B.; Martinez, A.; Languille, P.; Courtois, X.; Lallier, Y.; Salami, M.

    2013-07-01

    A diagnostic based on confocal microscopy was developed at CEA Cadarache in order to measure erosion on large plasma facing components during shutdown in situ in Tore Supra. This paper describes the diagnostic and presents results obtained on Beryllium and Carbon Fibre Composite (CFC) materials. Erosion in the range of 800 μm was found on one sector of the Toroidal Pumped Limiter (TPL) which provides, by integration to the full limiter a net carbon erosion of about 900 g over the period 2002-2007.

  7. 3D visualisation and analysis of capillaries in human muscles using confocal microscopy

    Eržen, I.; Janáček, Jiří; Kubínová, Lucie

    Vol.2. Graz : Verlag der Technischen Universität, 2009 - (Pabst, M.; Zellnig, G.), s. 191-192 ISBN 978-3-85125-062-6. [MC 2009 - Joint Meeting of Dreiländertagung and Multinational Congress on Microscopy /9./. Graz (AT), 30.08.2009-04.09.2009] R&D Projects: GA MŠk(CZ) MEB090910; GA AV ČR(CZ) IAA100110502 Institutional research plan: CEZ:AV0Z50110509 Keywords : capillaries * muscles * confocal microscopy Subject RIV: EA - Cell Biology

  8. LP-12 Approaches to visualization of 3D structures captured by a confocal microscope

    Čapek, Martin; Janáček, Jiří; Kubínová, Lucie; Hána, K.; Smrčka, P.

    České Budějovice : Czechoslovak microscopy society, 2007 - (Nebesářová, J.; Hozák, P.), s. 453-454 ISBN 978-80-239-9397-4. [Multinational Congress on Microscopy /8./. Prague (CZ), 17.06.2007-21.06.2007] R&D Projects: GA AV ČR(CZ) IAA100110502; GA AV ČR(CZ) IAA500200510; GA ČR(CZ) GA304/05/0153; GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal microscopy * volume visualization Subject RIV: JC - Computer Hardware ; Software

  9. Study of arrangement and orientation of capillaries on 3D confocal images

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

    České Budějovice : Czechoslovak microscopy society, 2007 - (Nebesářová, J.; Hozák, P.). s. 367-368 ISBN 978-80-239-9397-4. [Multinational Congress on Microscopy /8./. 17.06.2007-21.06.2007, Prague] R&D Projects: GA AV ČR(CZ) IAA100110502 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10190503 Keywords : spr2 * confocal microscopy * image analysis * capillaries Subject RIV: EA - Cell Biology

  10. Label-free detection of anticancer drug paclitaxel in living cells by confocal Raman microscopy

    Salehi, H.; Derely, L.; Vegh, A.-G.; Durand, J.-C.; Gergely, C.; Larroque, C.; Fauroux, M.-A.; Cuisinier, F. J. G.

    2013-03-01

    Confocal Raman microscopy, a non-invasive, label-free, and high spatial resolution imaging technique is employed to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel.

  11. Darkfield-Confocal Microscopy detection of nanoscale particle internalization by human lung cells

    Samet James M

    2011-01-01

    Full Text Available Abstract Background Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP. Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm. Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane. Results Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal and scattered transmitted light (Darkfield along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependant internalization process. Conclusions DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron

  12. Iterative diffraction calculations of transverse mode distributions in confocal unstable laser resonators.

    Rensch, D B; Chester, A N

    1973-05-01

    A theoretical investigation has been undertaken to study the transverse modes of two-dimensional positive branch, confocal unstable resonators. Mode amplitude and phase information is obtained from a numerical-iterative type calculation that uses the Fresnel integral for propagating the cavity radiation back and forth between resonator mirrors. Near- and far-field distributions for empty cavity resonators are presented for various resonator Fresnel numbers and magnifications, along with results of resonator mode stability and diffraction losses when cavity perturbations such as mirror misalignment and/or a uniformly saturable gain medium are included. In addition, the diffractive calculations are compared with results obtained from geometric models. PMID:20125458

  13. Coupling to Modes of a Near-Confocal Optical Resonator Using a Digital Light Modulator

    Papageorge, Alexander T; Lev, Benjamin L

    2016-01-01

    Digital Micromirror Devices (DMD) provide a robust platform with which to implement digital holography, in principle providing the means to rapidly generate propagating transverse electromagnetic fields with arbitrary mode profiles at visible and IR wavelengths. We use a DMD to probe a Fabry-P\\'{e}rot cavity in single-mode and near-degenerate confocal configurations. Pumping arbitrary modes of the cavity is possible with excellent specificity by virtue of the spatial overlap between the incident light field and the cavity mode.

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

    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

  15. Volume visualization of large biological tissue specimens captured by a confocal laser scanning microscope

    Čapek, Martin; Janáček, Jiří; Karen, Petr; Kubínová, Lucie; Hána, P.; Smrčka, P.

    Saint-Etienne : International society for stereology, 2007, s. 12-17. [International congress for stereology /12./. Saint-Etienne (FR), 03.09.2007-07.09.2007] R&D Projects: GA MŠk(CZ) LC06063; GA AV ČR(CZ) IAA100110502; GA AV ČR(CZ) IAA600110507; GA ČR(CZ) GA304/05/0153; GA AV ČR(CZ) IAA500200510 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal microscope * volume visualization Subject RIV: JC - Computer Hardware ; Software

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

    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.

  17. Corneal Confocal Microscopy – A Novel, Noninvasive Method to Assess Diabetic Peripheral Neuropathy

    Inceu Georgeta

    2014-12-01

    Full Text Available Background and aims. This article aims to compare corneal confocal microscopy (CCM with acknowledged tests of diabetic peripheral neuropathy (DPN, to assess corneal nerve morphology using CCM in diabetic patients, and to underline possible correlations between clinical and biological parameters, diabetes duration and DPN severity. Material and methods. A total of 90 patients with type 2 diabetes were included in the study for whom we measured anthropometric parameters and we performed laboratory measurements (tests. The patients were assessed for diabetic peripheral neuropathy using Semmes-Weinstein Monofilament Testing (SWMT, Rapid-Current Perception Threshold (R-CPT measurements using the Neurometer®, and CCM. We stratified the patients according to DPN severity, based on four parameters extracted after image analysis. Results. A higher percentage of patients were diagnosed with DPN using CCM (88.8%, compared with SWMT and R-CPT measurement (17.8% and 40% respectively. The incidence of DPN detected with CCM was considerable in patients with normal protective sensation and with normal R-CPT values. Conclusions. Our study showed that corneal confocal microscopy is a useful noninvasive method for diabetic neuropathy assessement in early stages. It was proven to directly quantify small fiber pathology, and to stratify neuropathic severity, and therefore can be used as a new, reliable tool in the diagnosis, clinical evaluation, and follow-up of peripheral diabetic neuropathy.

  18. Confocal imaging and numerical simulations of converging flows in artificial microvessels

    Hitt, Darren L.; Lowe, Mary L.

    1997-03-01

    A number of computational and experimental studies on flows in venular bifurcations have concentrated on the distribution of blood components and the formation of a 'separation surface' between two converging flows with different red blood cell concentrations and viscosities. To better understand these phenomena, we have used microfabrication techniques to construct artificial venular bifurcations with physiologically realistic dimensions. The current apparatus consists of a single bifurcation formed from tubes with semicircular cross sections of radius 50 (mu) . The vessels are fabricated from two glass slides using an abrasive etching process and a low melting point glass as sealant. Using a laser scanning confocal microscope, we have obtained the first three-dimensional images of flow structures in microscopic vessels; in particular we have imaged the separation surface for converging Newtonian fluids. A quantitative comparison is made between finite element simulations and experimental results of the position of the separation surface for different inlet flow ratios. Computed positions of the separation surface at the mid- depth of the outlet branch are in excellent agrement with experiment results obtained by confocal microscopy.

  19. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy

    Han, Jason J.; Kunde, Yuliya A.; Hong-Geller, Elizabeth; Werner, James H.

    2014-01-01

    We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

  20. Design and analysis of multi-color confocal microscopy with a wavelength scanning detector.

    Do, Dukho; Chun, Wanhee; Gweon, Dae-Gab

    2012-05-01

    Spectral (or multi-color) microscopy has the ability to detect the fluorescent light of biological specimens with a broad range of wavelengths. Currently, the acousto-optic tunable filter (AOTF) is widely used in spectral microscopy as a substitute for a multiple-dichroic mirror to divide excitation and emission signals while maintaining sufficient light efficiency. In addition, systems which utilize an AOTF have a very fast switching speed and high resolution for wavelength selection. In this paper, confocal-spectral microscopy is proposed with a particular spectrometer design with a wavelength-scanning galvano-mirror. This enables the detection of broadband (480-700 nm) fluorescence signals by a single point detector (photomultiplier tube) instead of a CCD pixel array. For this purpose, a number of optical elements were applicably designed. A prism is used to amplify the dispersion angle, and the design of the relay optics matches the signals to the diameter of the wavelength-scanning galvano-mirror. Also, a birefringent material known as calcite is used to offset the displacement error at the image plane depending on the polarization states. The proposed multi-color confocal microscopy with the unique detection body has many advantages in comparison with commercial devices. In terms of the detection method, it can be easily applied to other imaging modalities. PMID:22667622

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

    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.

  2. Structure and chemical composition of the dentin-enamel junction analyzed by Confocal Raman Microscopy

    Desoutter, A.; Salehi, H.; Slimani, A.; Marquet, P.; Jacquot, B.; Tassery, H.; Cuisinier, F. J. G.

    2014-02-01

    The structure and chemical composition of the human dentin-enamel junction (DEJ) was studied using confocal Raman microscopy - a chemical imaging technique. Slices of non-fixed, sound teeth were prepared with an Isomet diamond saw and scanned with Witec Alpha300R system. The combination of different characteristics peaks of phosphate, carbonate and organic matrix (respectively 960, 1072 and 1545 cm-1), generates images representing the chemical composition of the DEJ area. Images are also calculated using peak ratios enabling precise determination of the chemical composition across the DEJ. Then, with two characterized peaks, different pictures are calculated to show the ratio of two components. The images of the spatial distribution of mineral phosphate (960cm-1) to organic matrix (1545 cm-1) ratios, mineral carbonates (1072cm-1) to mineral phosphate ratios; and mineral carbonates to organic matrix ratios were reconstructed. Cross sectional and calculated graphic profile show the variations of the different chemical component ratios through the enamel and the dentin. Phosphate to organic ratio shows an accumulation of organic material under the enamel surface. The cross sectional profile of these pictures shows a high phosphate content compared to enamel in the vicinity of the DEJ. The Confocal Raman imaging technique can be used to further provide full chemical imaging of tooth, particularly of the whole DEJ and to study enamel and dentin decay.

  3. Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue

    Gaertner, Maria; Cimalla, Peter; Meissner, Sven; Kuebler, Wolfgang M.; Koch, Edmund

    2012-07-01

    Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

  4. Examination of thick-section confocal microscopy as a tool for the histomorphometric analysis of bone

    The authors have examined the potential utility of confocal microscopy as part of an effort to evaluate the deposition patterns of potentially interesting bone-seeking radiopharmaceuticals or toxic radioactive agents in Beagles and to characterize these patterns in a way that the radiation doses or toxic agent dosage delivered to the skeleton might be more easily modeled. In separate studies, they have looked at the deposition of 166Ho-labeled pharmaceuticals and found that their deposition patterns were amendable to description with the same models heretofore used for various bone-seeking radionuclides. In other work, they are examining the relationship in skeletal tissue between the apparent densities that can be obtained from computerized tomographic (CT) scanners and the direct measurement of mass via neutron activation analysis. The CT method has the potential advantage of reducing skeletal dissection effort and the capability of providing a record of macroscopic bone morphology down to a thickness of about 300-400 μm, and at the same time providing an index of surface-to-skeletal tissue mass ratios in bone. In this abstract, they describe efforts to employ a combination of confocal microscopy and ultra-milling technology to efficaciously address the biological issues and the technical difficulties that require acquisition of the multi-megabyte quantities of data and their storage

  5. Confocal Raman microscopy and fluorescent in situ hybridization - A complementary approach for biofilm analysis.

    Kniggendorf, Ann-Kathrin; Nogueira, Regina; Kelb, Christian; Schadzek, Patrik; Meinhardt-Wollweber, Merve; Ngezahayo, Anaclet; Roth, Bernhard

    2016-10-01

    We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples. PMID:27423128

  6. QUANTIFICATION OF BIOFILMS IN MULTI-SPECTRAL DIGITAL1 VOLUMES FROM CONFOCAL LASER-SCANNING MICROSCOPES

    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.

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

    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. PMID:25828656

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

    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.

  9. Combining microtomy and confocal laser scanning microscopy for structural analyses of plant-fungus associations.

    Rath, Magnus; Grolig, Franz; Haueisen, Janine; Imhof, Stephan

    2014-05-01

    The serious problem of extended tissue thickness in the analysis of plant-fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl™. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant-fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf-fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant-fungus symbioses. PMID:24249491

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

    MassimilianoCardinale

    2014-03-01

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

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

    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 μm. 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

  12. Photothermal confocal multicolor microscopy of nanoparticles and nanodrugs in live cells.

    Nedosekin, Dmitry A; Foster, Stephen; Nima, Zeid A; Biris, Alexandru S; Galanzha, Ekaterina I; Zharov, Vladimir P

    2015-08-01

    Growing biomedical applications of non-fluorescent nanoparticles (NPs) for molecular imaging, disease diagnosis, drug delivery, and theranostics require new tools for real-time detection of nanomaterials, drug nano-carriers, and NP-drug conjugates (nanodrugs) in complex biological environments without additional labeling. Photothermal (PT) microscopy (PTM) has enormous potential for absorption-based identification and quantification of non-fluorescent molecules and NPs at a single molecule and 1.4 nm gold NP level. Recently, we have developed confocal PTM providing three-dimensional (3D) mapping and spectral identification of multiple chromophores and fluorophores in live cells. Here, we summarize recent advances in the application of confocal multicolor PTM for 3D visualization of single and clustered NPs, alone and in individual cells. In particular, we demonstrate identification of functionalized magnetic and gold-silver NPs, as well as graphene and carbon nanotubes in cancer cells and among blood cells. The potential to use PTM for super-resolution imaging (down to 50 nm), real-time NP tracking, guidance of PT nanotherapy, and multiplex cancer markers targeting, as well as analysis of non-linear PT phenomena and amplification of nanodrug efficacy through NP clustering and nano-bubble formation are also discussed. PMID:26133539

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

    Vineeta Nikhil

    2013-01-01

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

  14. Two-photon fluorescence and confocal reflected light imaging of thick tissue structures

    Kim, Ki H.; So, Peter T. C.; Kochevar, Irene E.; Masters, Barry R.; Gratton, Enrico

    1998-04-01

    The technology of two-photon excitation has opened a window of opportunity for developing non-invasive medical diagnostic tools capable of monitoring thick tissue biochemical states. Using cellular endogenous chromophores, (beta) -nicotinamide- adenine dinucleotide phosphate [NAD(P)H], the cellular metabolic rates in living human skin were determined. Although important functional information can be obtained from the fluorescence spectroscopy of endogenous chromophores, these chromophores are rather poor contrast enhancing agent for mapping cellular morphology. First, most endogenous chromophores are confined to the cellular cytoplasm which prevents the visualization of other cellular organelles. Second, there is significant variability in the distribution and the quantum yield of endogenous chromophores which depends on tissue biochemistry but prevents consistent comparison of cellular morphology. On the other hand, the deep tissue cellular morphology has been imaged with excellent resolution using reflected light confocal microscopy. In reflected light microscopy, the image contrast originates from the index of refraction differences of the cellular structures. The organelle boundaries with significant index differences such as the plasma membrane and the nucleus envelope can be consistently visualized. A combination of morphological and functional information is required for a thorough tissue study. This presentation describes the development of a new microscope which is capable of simultaneously collecting both two-photon fluorescence and confocal reflected light signals. Promising biomedical applications include the non-invasive diagnosis of skin cancer and the study of wound healing.

  15. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  16. Confocal Raman spectroscopy to trace lipstick with their smudges on different surfaces.

    López-López, Maria; Özbek, Nil; García-Ruiz, Carmen

    2014-06-01

    Lipsticks are very popular cosmetic products that can be transferred by contact to different surfaces, being important forensic evidence with an intricate analysis if they are found in a crime scene. This study evaluates the use of confocal Raman microscopy at 780 nm excitation wavelength for the nondestructive identification of 49 lipsticks of different brands and colors, overcoming the lipstick fluorescence problem reported by previous works using other laser wavelengths. Although the lipsticks samples showed some fluorescence, this effect was not so intense to completely overwhelm the Raman spectra. Lipsticks smudges on twelve different surfaces commonly stained with these samples were also analyzed. In the case of the surfaces, some of them provided several bands to the smudge spectra compromising the identification of the lipstick. For these samples spectral subtraction of the interfering bands from the surface was performed. Finally, five different red lipsticks with very similar color were measured on different surfaces to evaluate the lipstick traceability with their smudges even on interfering surfaces. Although previous spectral subtraction was needed in some cases, all the smudged were linked to their corresponding lipsticks even when they are smeared on the interfering surfaces. As a consequence, confocal Raman microscopy using the 780 nm excitation laser is presented as a nondestructive powerful tool for the identification of these tricky samples. PMID:24725875

  17. Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source.

    Träutlein, D; Adler, F; Moutzouris, K; Jeromin, A; Leitenstorfer, A; Ferrando-May, E

    2008-03-01

    The performance of a confocal microscopy setup based on a single femtosecond fiber system is explored over a broad range of pump wavelengths for both linear and nonlinear imaging techniques. First, the benefits of a laser source in linear fluorescence excitation that is continuously tunable over most of the visible spectrum are demonstrated. The influences of subpicosecond pulse durations on the bleaching behavior of typical fluorophores are discussed. We then utilize the tunable near-infrared output of the femtosecond system in connection with a specially designed prism compressor for dispersion control. Pulses as short as 33 fs are measured in the confocal region. As a consequence, 2 mW of average power are sufficient for two-photon microscopy in an organotypic sample from the mouse brain. This result shows great prospect for deep-tissue imaging in the optimum transparency window around 1100 nm. In a third experiment, we prove that our compact setup is powerful enough to exploit even higher-order nonlinearities such as three-photon absorption that we use to induce spatially localized photodamage in DNA. PMID:19343635

  18. Multi-confocal Fluorescence Correlation Spectroscopy : experimental demonstration and potential applications for living cell measurements

    Galland, Rémi; Kloster, Meike; Herbomel, Gaetan; Destaing, Olivier; Balland, Martial; Souchier, Catherine; Usson, Yves; Derouard, Jacques; Wang, Irène; Delon, Antoine; 10.2741/e263

    2011-01-01

    We report, for the first time, a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique which allows parallel measurements at different locations, by combining a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM is calculated by using the spherical wave approximation and makes it possible to produce several diffraction limited laser spots, either aligned or spread over the field of view. To attain fast enough imaging rates, the camera has been used in different acquisition modes, the fastest of which leads to a time resolution of 100 $\\mu$s. We qualified the experimental set-up by using solutions of sulforhodamine G in glycerol and demonstrated that the observation volumes are similar to that of a standard confocal set-up. To demonstrate that our mFCS method is suitable for intracellular studies, experiments have...

  19. Biomimetic Coating on Porous Alumina for Tissue Engineering: Characterisation by Cell Culture and Confocal Microscopy

    Elizabeth Kolos

    2015-06-01

    Full Text Available In this study porous alumina samples were prepared and then coated using the biomimetic coating technique using a five times Simulated Body Fluid (5.0SBF as the growth solution. A coating was achieved after pre-treatment with concentrated acid. From elemental analysis, the coating contained calcium and phosphorous, but also sodium and chlorine. Halite was identified by XRD, a sodium chloride phase. Sintering was done to remove the halite phase. Once halite was burnt off, the calcium phosphate crystals were not covered with halite and, therefore, the apatite phases can be clearly observed. Cell culturing showed sufficient cell attachment to the less porous alumina, Sample B, that has more calcium phosphate growth, while the porous alumina, Sample A, with minimal calcium phosphate growth attained very little cell attachment. This is likely due to the contribution that calcium phosphate plays in the attachment of bone-like cells to a bioinert ceramic such as alumina. These results were repeated on both SEM and confocal microscopy analysis. Confocal microscopy was a novel characterisation approach which gave useful information and was a visual aid.

  20. Reflectance confocal microscopy of red blood cells: simulation and experiment (Conference Presentation)

    Zeidan, Adel; Yeheskely-Hayon, Daniella; Minai, Limor; Yelin, Dvir

    2016-03-01

    The properties of red blood cells are a remarkable indicator of the body's physiological condition; their density could indicate anemia or polycythemia, their absorption spectrum correlates with blood oxygenation, and their morphology is highly sensitive to various pathologic states including iron deficiency, ovalocytosis, and sickle cell disease. Therefore, measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient's health. In this work, we simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the cells' morphological parameters and the resulting characteristic interference patterns. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry (SEFC) that imaged the cells during linear flow and without artificial staining. By matching the simulated patterns to the SEFC images of the cells, the cells' three-dimensional shapes were evaluated and their volumes were calculated. Potential applications include measurement of the mean corpuscular volume, cell morphological abnormalities, cell stiffness under mechanical stimuli, and the detection of various hematological diseases.

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

    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. PMID:12558687

  2. The surface morphology analysis based on progressive approximation method using confocal three-dimensional micro X-ray fluorescence

    Yi, Longtao; Sun, Tianxi; Wang, Kai; Qin, Min; Yang, Kui; Wang, Jinbang; Liu, Zhiguo

    2016-08-01

    Confocal three-dimensional micro X-ray fluorescence (3D MXRF) is an excellent surface analysis technology. For a confocal structure, only the X-rays from the confocal volume can be detected. Confocal 3D MXRF has been widely used for analysing elements, the distribution of elements and 3D image of some special samples. However, it has rarely been applied to analysing surface topography by surface scanning. In this paper, a confocal 3D MXRF technology based on polycapillary X-ray optics was proposed for determining surface topography. A corresponding surface adaptive algorithm based on a progressive approximation method was designed to obtain surface topography. The surface topography of the letter "R" on a coin of the People's Republic of China and a small pit on painted pottery were obtained. The surface topography of the "R" and the pit are clearly shown in the two figures. Compared with the method in our previous study, it exhibits a higher scanning efficiency. This approach could be used for two-dimensional (2D) elemental mapping or 3D elemental voxel mapping measurements as an auxiliary method. It also could be used for analysing elemental mapping while obtaining the surface topography of a sample in 2D elemental mapping measurement.

  3. Evaluation of new morphometric parameters of neoangiogenesis in human colorectal cancer using confocal laser endomicroscopy (CLE) and targeted panendothelial markers

    Ciocâlteu, Adriana; Săftoiu, Adrian; Cârţână, Tatiana;

    2014-01-01

    vessels (i.e. antiangiogenesis therapy). The aim of this study was to evaluate new vascular morphometric parameters in colorectal cancer, difficult to achieve through conventional immunohistochemistry, by using the confocal laser endomicroscopy method. Fresh biopsies from tumor and normal tissue were...... collected during colonoscopy from five patients with T3 colorectal carcinoma without metastasis and were marked with fluorescently labeled anti-CD31 antibodies. A series of optical slices spanning 250 µm inside the tissue were immediately collected for each sample using a confocal laser endomicroscope. All.......7033). The daughter/mother ratio (ratio of the sum of the squares of daughter vessel radii over the square of the mother vessel radius) was 1.15±0.09 in normal tissue, and 1.21±0.08 in tumor tissue (p = 0.6531). The confocal laser endomicroscopy is feasible for measuring more vascular parameters from fresh...

  4. A 10-Hz positive-branch confocal unstable resonator Nd:YAG laser

    A positive-cobranch confocal unstabel resonator Nd:YAG laser is designed and constructed, and experimental investigation of its performance characteristics is presented. The corfocal condition is achieved by considering thermal lensina effect of the Nd:YAG rod combined with back mirror radius of curvature. The laser is operated at 10-Hz repetition rate producing Q-switched output energy of 60 mJ per pulse with 15 ns pulsewidth (FWHM). In a performance comparison with a conventional stable resonator laser using the same rod and power supply, the unstable resonator laser produced equally efficient operation with less than 0.5 milliradian beam divergence, about 1/5-1/10 times smaller than that from the stable one. And excellent transverse mode discrimination was also observed. (Author)

  5. Estimation of single cell volume from 3D confocal images using automatic data processing

    Chorvatova, A.; Cagalinec, M.; Mateasik, A.; Chorvat, D., Jr.

    2012-06-01

    Cardiac cells are highly structured with a non-uniform morphology. Although precise estimation of their volume is essential for correct evaluation of hypertrophic changes of the heart, simple and unified techniques that allow determination of the single cardiomyocyte volume with sufficient precision are still limited. Here, we describe a novel approach to assess the cell volume from confocal microscopy 3D images of living cardiac myocytes. We propose a fast procedure based on segementation using active deformable contours. This technique is independent on laser gain and/or pinhole settings and it is also applicable on images of cells stained with low fluorescence markers. Presented approach is a promising new tool to investigate changes in the cell volume during normal, as well as pathological growth, as we demonstrate in the case of cell enlargement during hypertension in rats.

  6. Smart microscope: an adaptive optics learning system for aberration correction in multiphoton confocal microscopy.

    Albert, O; Sherman, L; Mourou, G; Norris, T B; Vdovin, G

    2000-01-01

    Off-axis aberrations in a beam-scanning multiphoton confocal microscope are corrected with a deformable mirror. The optimal mirror shape for each pixel is determined by a genetic learning algorithm, in which the second-harmonic or two-photon fluorescence signal from a reference sample is maximized. The speed of the convergence is improved by use of a Zernike polynomial basis for the deformable mirror shape. This adaptive optical correction scheme is implemented in an all-reflective system by use of extremely short (10-fs) optical pulses, and it is shown that the scanning area of an f:1 off-axis parabola can be increased by nine times with this technique. PMID:18059779

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

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

    2015-01-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented...... to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis...... scanning microscopy images can be used to provide information on the protein microstructure in yogurt products. For large numbers of microscopy images, subjective evaluation becomes a difficult or even impossible approach, if the images should be incorporated in any form of statistical analysis alongside...

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

    Nappini, Silvia

    2011-04-07

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

  9. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J.; Rohrbach, Alexander

    2016-01-01

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

  10. A System for Automated Acquisition of 3D Biomedical Images Using Confocal Microscopy

    Čapek, Martin; Tomori, Z.; Michálek, Jan; Janáček, Jiří; Kubínová, Lucie; Hozman, J.

    Vol.2. Graz: Verlag der Technischen Universität, 2009 - (Pabst, M.; Zellnig, G.), s. 415-416 ISBN 978-3-85125-062-6. [MC 2009 - Joint Meeting of Dreiländertagung and Multinational Congress on Microscopy /9./. Graz (AT), 30.08.2009-04.09.2009] R&D Projects: GA MŠk(CZ) LC06063; GA ČR(CZ) GA102/08/0691; GA ČR(CZ) GA304/09/0733; GA AV ČR(CZ) IAA100110502; GA MŠk(CZ) MEB080831 Grant ostatní: VEGA(SK) 2-0164-07 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal laser scanning microscope * illumination inhomogeneity * automated acquisition system Subject RIV: JD - Computer Applications, Robotics

  11. Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies

    Optical visualization of nanoscale morphological changes taking place in living biological cells during such important processes as endo- and exocytosis is challenging due to the low refractive index of lipid membranes. In this paper we summarize and discuss advances in the powerful combination of two complementary live imaging techniques, ion conductance and fluorescence confocal microscopy, that allows cell membrane topography to be related with molecular-specific fluorescence at high spatial and temporal resolution. We demonstrate the feasibility of the use of ion conductance microscopy to image apical plasma membrane of mouse embryo trophoblast outgrowth cells at a resolution sufficient to depict single endocytic pits. This opens the possibility to study individual endocytic events in embryo trophoblast outgrowth cells where endocytosis plays a crucial role during early stages of embryo development. (special issue article)

  12. Parameter-free binarization and skeletonization of fiber networks from confocal image stacks.

    Patrick Krauss

    Full Text Available We present a method to reconstruct a disordered network of thin biopolymers, such as collagen gels, from three-dimensional (3D image stacks recorded with a confocal microscope. The method is based on a template matching algorithm that simultaneously performs a binarization and skeletonization of the network. The size and intensity pattern of the template is automatically adapted to the input data so that the method is scale invariant and generic. Furthermore, the template matching threshold is iteratively optimized to ensure that the final skeletonized network obeys a universal property of voxelized random line networks, namely, solid-phase voxels have most likely three solid-phase neighbors in a 3 x 3 x 3 neighborhood. This optimization criterion makes our method free of user-defined parameters and the output exceptionally robust against imaging noise.

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

    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......Biofilms are agglomerates of microorganisms surrounded by a self-produced extracellular matrix. During the last 10 years, there has been an increasing recognition of biofilms as a highly significant topic in microbiology with relevance for a variety of areas in our society including the environment...... formed in flow-chamber experimental systems by genetically color-coded bacteria have provided detailed knowledge about biofilm developmental processes, cell differentiations, spatial organization, and function of laboratory-grown biofilms, in some cases down to the single cell level. In addition, the...

  14. In vivo confocal Raman spectroscopy study of the vitamin A derivative perfusion through human skin

    dos Santos, Laurita; Téllez Soto, Claudio A.; Favero, Priscila P.; Martin, Airton A.

    2016-03-01

    In vivo confocal Raman spectroscopy is a powerful non-invasive technique able to analyse the skin constituents. This technique was applied to transdermal perfusion studies of the vitamin A derivative in human skin. The composition of the stratum corneum (lipid bilayer) is decisive for the affinity and transport of the vitamin through skin. The vitamin A is significantly absorbed by human skin when applied with water in oil emulsion or hydro-alcoholic gel. The purpose of this study is to elucidate the behaviour of vitamin A derivative into human skin without the presence of enhancers. The results showed that the intensity band of the derivative (around 1600 cm-1), which represents the -C=O vibrational mode, was detected in different stratum corneum depths (up to 20 μm). This Raman peak of vitamin A derivative has non-coincident band with the Raman spectra of the skin epidermis, demonstrating that compound penetrated in forearm skin.

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

    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. PMID:21528993

  16. Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies

    Shevchuk, A. I.; Novak, P.; Velazquez, M. A.; Fleming, T. P.; Korchev, Y. E.

    2013-09-01

    Optical visualization of nanoscale morphological changes taking place in living biological cells during such important processes as endo- and exocytosis is challenging due to the low refractive index of lipid membranes. In this paper we summarize and discuss advances in the powerful combination of two complementary live imaging techniques, ion conductance and fluorescence confocal microscopy, that allows cell membrane topography to be related with molecular-specific fluorescence at high spatial and temporal resolution. We demonstrate the feasibility of the use of ion conductance microscopy to image apical plasma membrane of mouse embryo trophoblast outgrowth cells at a resolution sufficient to depict single endocytic pits. This opens the possibility to study individual endocytic events in embryo trophoblast outgrowth cells where endocytosis plays a crucial role during early stages of embryo development.

  17. Mapping Li(+) Concentration and Transport via In Situ Confocal Raman Microscopy.

    Forster, Jason D; Harris, Stephen J; Urban, Jeffrey J

    2014-06-01

    We demonstrate confocal Raman microscopy as a general, nonperturbative tool to measure spatially resolved lithium ion concentrations in liquid electrolytes. By combining this high-spatial-resolution technique with a simple microfluidic device, we are able to measure the diffusion coefficient of lithium ions in dimethyl carbonate in two different concentration regimes. Because lithium ion transport plays a key role in the function of a variety of electrochemical devices, quantifying and visualizing this process is crucial for understanding device performance. This method for detecting lithium ions should be immediately useful in the study of lithium-ion-based devices, ion transport in porous media, and at electrode-electrolyte interfaces, and the analytical framework is useful for any system exhibiting a concentration-dependent Raman spectrum. PMID:26273887

  18. Spatial distribution of perylenequinones in lichens and extended quinones in quincyte using confocal fluorescence microscopy.

    Mathey, A; Lukins, P B

    2001-02-01

    The application of confocal fluorescence microscopy and microspectrofluorimetry to the characterization of the distribution of organic compounds in bulk lichens and mineral structures is demonstrated. Perylenequinones and extended quinones were chosen as both model compounds and as the naturally occurring fluorophores. These molecules occur, respectively, in corticolous microlichens and in a pink-colored mineral called quincyte. The structures of quincyte and of the lichens Cryptothelium rhodotitton and Graphis hematites are described, and the possibilities of energy dissipation and photoprotection mechanisms in these lichens are discussed. This study also illustrates how, for a wide range of specimens, naturally occurring quinone fluorophores in the specimen can be exploited directly to yield chemical and structural information without using fluorescent labelling. These intrinsic quinonoid compounds have molecular fluorescence yields and laser damage thresholds comparable or superior to common microscopy dyes, and can therefore be used to obtain high-contrast 3D fluorescence imaging without the complications introduced by dye labelling. PMID:10936454

  19. Shear bond strength, failure modes, and confocal microscopy of bonded amalgam restorations.

    Cianconi, Luigi; Conte, Gabriele; Mancini, Manuele

    2011-01-01

    This study evaluated the shear bond strength, failure modes, and confocal microscopy of two different amalgam alloy restorations lined with five adhesive systems. Two regular-set high-copper dental amalgam alloys, Amalcap Plus and Valiant Ph.D, and five commercially available adhesive systems were selected. One hundred and twenty freshly-extracted human third molars were used for the study. The results were statistically evaluated using two-factor analysis of variance (ANOVA). The shear bond strength (SBS) of amalgam to dentin was significantly affected by both the adhesive (pValiant Ph.D, 31 of 50 exhibited adhesive failure, and 19 displayed mixed failure. Laser optical microscopy (OM) of the bonded interface revealed the presence of a good hybrid layer was evident in all experimental groups. Higher bond strengths were measured for four of the five adhesives when used in combination with the spherical alloy. PMID:21383518

  20. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots.

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J; Rohrbach, Alexander

    2016-01-01

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

  1. Modulated alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

    Leigh, Steven Y.; Chen, Ye; Liu, Jonathan T. C.

    2014-05-01

    A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 mW) diode laser illumination. While the DAC architecture's intersecting illumination and collection beams significantly improves the spatial-filtering and opticalsectioning performance of confocal microscopy, we propose that modulating the spatial alignment of the dual-axis beams at a frequency f, such the focal volume signal of the microscope is modulated at 2f, further provides nearly an order-of-magnitude improvement in optical-sectioning contrast. Lock-in detection is used to remove the unmodulated background light, thereby enhancing our ability to image deeply within highly scattering tissues.

  2. Characterization of acoustic lenses with the Foucault test by confocal laser scanning microscopy

    Ahmed Mohamed, E. T.; Abdelrahman, A.; Pluta, M.; Grill, W.

    2010-03-01

    In this work, the Foucault knife-edge test, which has traditionally been known as the classic test for optical imaging devices, is used to characterize an acoustic lens for operation at 1.2 GHz. A confocal laser scanning microscope (CLSM) was used as the illumination and detection device utilizing its pinhole instead of the classical knife edge that is normally employed in the Foucault test. Information about the geometrical characteristics, such as the half opening angle of the acoustic lens, were determined as well as the quality of the calotte of the lens used for focusing. The smallest focal spot size that could be achieved with the examined lens employed as a spherical reflector was found to be about 1 μm. By comparison to the idealized resolution a degradation of about a factor of 2 can be deduced. This limits the actual quality of the acoustic focus.

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

    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.

  4. HIV detection by in-situ hybridization based on confocal reflected light microscopy

    Smith, Louis C.; Jericevic, Zeljko; Cuellar, Roland; Paddock, Stephen W.; Lewis, Dorothy E.

    1991-05-01

    Elucidation of the pathogenesis of AIDS is confounded by the finding that few actively infected CD4+ cells (1 in 104-105) can be detected in the peripheral blood, even though there is dramatic depletion (often >90%) of CD4+ cells as the disease progresses. A sensitive, 35S-based human immunodeficiency virus (HIV) mRNA in situ hybridization technique was coupled with a new detection method, confocal laser scanning microscopy, to examine transcriptionally active HIV-infected cells from individuals at different disease stages. An algorithm for image segmentation and analysis has been developed to determine the proportion of HIV-positive cells. Data obtained using this improved detection method suggest that there are more HIV mRNA-producing cells in HIV-infected individuals than previously thought, based on other detection methods.

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

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

    2007-01-01

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

  6. Fraunhofer-type absorption line splitting and polarization in confocal double-pulse laser induced plasma

    Strong line splitting and polarization are observed in Fraunhofer-type absorption lines in Pb, Sn, Si, Cd, In, and Zn in confocal double-pulse laser induced plasma (DP-LIP) experiments. This effect is detectable using medium laser power densities: (∼ 1–2) × 1013 W/m2 for the first laser pulse and 1 × 1014 W/m2 for the second laser pulse. Polarization and splitting effects exist only during the second laser pulse (∼ 7 ns). Absorption line polarization and splitting phenomena may be explained by a high overall magnetic field and motional Stark effect caused by the second laser pulse inside the laser plasma created by the first pulse. - Highlights: • Certain Fraunhofer absorption lines in DP LIBS are polarized. • Certain Fraunhofer absorption lines in DP LIBS are split. • Those effects exist during laser pulse. • Effects take place in elements with ns2np2 and ns2 electron ground state

  7. Confocal Raman data analysis enables identifying apoptosis of MCF-7 cells caused by anticancer drug paclitaxel

    Salehi, Hamideh; Middendorp, Elodie; Panayotov, Ivan; Dutilleul, Pierre-Yves Collard; Vegh, Attila-Gergely; Ramakrishnan, Sathish; Gergely, Csilla; Cuisinier, Frederic

    2013-05-01

    Confocal Raman microscopy is a noninvasive, label-free imaging technique used to study apoptosis of live MCF-7 cells. The images are based on Raman spectra of cells components, and their apoptosis is monitored through diffusion of cytochrome c in cytoplasm. K-mean clustering is used to identify mitochondria in cells, and correlation analysis provides the cytochrome c distribution inside the cells. Our results demonstrate that incubation of cells for 3 h with 10 μM of paclitaxel does not induce apoptosis in MCF-7 cells. On the contrary, incubation for 30 min at a higher concentration (100 μM) of paclitaxel induces gradual release of the cytochrome c into the cytoplasm, indicating cell apoptosis via a caspase independent pathway.

  8. Method and apparatus for a high-resolution three dimensional confocal scanning transmission electron microscope

    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.

  9. Optimising performance of a confocal fluorescence microscope with a differential pinhole

    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)

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

    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

  11. The Signal Detection and Control Circuit Design for Confocal Auto-Focus System

    Yin Liu

    2016-01-01

    Full Text Available Based on the demands of Confocal Auto-Focus system, the implementation method of signal measurement circuit and control circuit is given. Using the high performance instrumental amplifier AD620BN, low noise precision FET Op amplifier AD795JRZ and ultralow offset voltage Op amplifier OP07EP, a signal measurement circuit used to converse the two differential light intensity signal to electric signal is designed. And a control circuit which takes MCU MSP430F149 as core processes the former signal and generate a control signal driving the platform for auto-focusing. The experimental results proved the feasibility and correctness of circuits. And the system meets the design requirement.

  12. Is Seeing Really Believing? Probe-based Confocal Laser Endomicroscopy in the Evaluation of Pancreaticobiliary Disease.

    Storm, Andrew C; Lee, Linda S

    2016-01-01

    Confocal laser endomicroscopy for real-time diagnosis during endoscopic procedures has now been in the mainstream clinical arena for a decade. Indeterminate biliary strictures and pancreatic cysts remain 2 difficult diagnostic challenges for the gastroenterologist, and the role this technology will play in the approach to these problems is still evolving. There is now a body of literature to guide the endoscopist in the use of this imaging tool, including how it may be useful in excluding biliary malignancy, and how miniaturization has allowed for endoscopic ultrasound-guided application of the probe within cysts. Interobserver variability remains a weakness of the system. Tips for use of this tool and interpretation of the imaging data it provides are discussed. PMID:26927493

  13. Visualization of microcrack anisotropy in granite affected by afault zone, using confocal laser scanning microscope

    Onishi, Celia T.; Shimizu, Ichiko

    2004-01-02

    Brittle deformation in granite can generate a fracture system with different patterns. Detailed fracture analyses at both macroscopic and microscopic scales, together with physical property data from a drill-core, are used to classify the effects of reverse fault deformation in four domains: (1) undeformed granite, (2) fractured granite with cataclastic seams, (3) fractured granite from the damage zone, and (4) foliated cataclasite from the core of the fault. Intact samples from two orthogonal directions, horizontal (H) and vertical (V), from the four domains indicate a developing fracture anisotropy toward the fault, which is highly developed in the damage zone. As a specific illustration of this phenomenon, resin impregnation, using a confocal laser scanning microscope (CLSM) technique is applied to visualize the fracture anisotropy developed in the Toki Granite, Japan. As a result, microcrack networks have been observed to develop in H sections and elongate open cracks in V sections, suggesting that flow pathways can be determined by deformation.

  14. Identification of different bacterial species in biofilms using confocal Raman microscopy

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

    2010-11-01

    Confocal Raman microspectroscopy is used to discriminate between different species of bacteria grown in biofilms. Tests are performed using two bacterial species, Streptococcus sanguinis and Streptococcus mutans, which are major components of oral plaque and of particular interest due to their association with healthy and cariogenic plaque, respectively. Dehydrated biofilms of these species are studied as a simplified model of dental plaque. A prediction model based on principal component analysis and logistic regression is calibrated using pure biofilms of each species and validated on pure biofilms grown months later, achieving 96% accuracy in prospective classification. When biofilms of the two species are partially mixed together, Raman-based identifications are achieved within ~2 μm of the boundaries between species with 97% accuracy. This combination of spatial resolution and predication accuracy should be suitable for forming images of species distributions within intact two-species biofilms.

  15. Sub-micrometer anatomical models of the sarcolemma of cardiac myocytes based on confocal imaging.

    Sachse, Frank B; Savio-Galimberti, Eleonora; Goldhaber, Joshua I; Bridge, John H B

    2008-01-01

    We describe an approach to develop anatomical models of cardiac cells. The approach is based on confocal imaging of living ventricular myocytes with submicrometer resolution, digital image processing of three-dimensional stacks with high data volume, and generation of dense triangular surface meshes representing the sarcolemma including the transverse tubular system. The image processing includes methods for deconvolution, filtering and segmentation. We introduce and visualize models of the sarcolemma of whole ventricular myocytes and single transversal tubules. These models can be applied for computational studies of cell and sub-cellular physical behavior and physiology, in particular cell signaling. Furthermore, the approach is applicable for studying effects of cardiac development, aging and diseases, which are associated with changes of cell anatomy and protein distributions. PMID:18229702

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

    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 (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.

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

    Manual and semi-automatic analyses of images, acquired in vivo by confocal microscopy, are often used to determine the quality of corneal endothelium in the human eye. These procedures are highly time consuming. Here, we present two fully automatic methods to analyze and quantify corneal endothelium imaged by in vivo white light slit-scanning confocal microscopy. In the first approach, endothelial cell density is estimated with the help of spatial frequency analysis. We evaluate published methods, and propose a new, parameter-free method. In the second approach, based on the stochastic watershed, cells are automatically segmented and the result is used to estimate cell density, polymegathism (cell size variability) and pleomorphism (cell shape variation). We show how to determine optimal values for the three parameters of this algorithm, and compare its results to a semi-automatic delineation by a trained observer. The frequency analysis method proposed here is more precise than any published method. The segmentation method outperforms the fully automatic method in the NAVIS software (Nidek Technologies Srl, Padova, Italy), which significantly overestimates the number of cells for cell densities below approximately 1200 mm−2, as well as previously published methods. The methods presented here provide a significant improvement over the state of the art, and make in vivo, automated assessment of corneal endothelium more accessible. The segmentation method proposed paves the way to many possible new morphometric parameters, which can quickly and precisely be determined from the segmented image. The online version of this article (doi:10.1186/s12880-015-0054-3) contains supplementary material, which is available to authorized users

  18. Comparison of 3D orientation distribution functions measured with confocal microscopy and diffusion MRI.

    Schilling, Kurt; Janve, Vaibhav; Gao, Yurui; Stepniewska, Iwona; Landman, Bennett A; Anderson, Adam W

    2016-04-01

    The ability of diffusion MRI (dMRI) fiber tractography to non-invasively map three-dimensional (3D) anatomical networks in the human brain has made it a valuable tool in both clinical and research settings. However, there are many assumptions inherent to any tractography algorithm that can limit the accuracy of the reconstructed fiber tracts. Among them is the assumption that the diffusion-weighted images accurately reflect the underlying fiber orientation distribution (FOD) in the MRI voxel. Consequently, validating dMRI's ability to assess the underlying fiber orientation in each voxel is critical for its use as a biomedical tool. Here, using post-mortem histology and confocal microscopy, we present a method to perform histological validation of orientation functions in 3D, which has previously been limited to two-dimensional analysis of tissue sections. We demonstrate the ability to extract the 3D FOD from confocal z-stacks, and quantify the agreement between the MRI estimates of orientation information obtained using constrained spherical deconvolution (CSD) and the true geometry of the fibers. We find an orientation error of approximately 6° in voxels containing nearly parallel fibers, and 10-11° in crossing fiber regions, and note that CSD was unable to resolve fibers crossing at angles below 60° in our dataset. This is the first time that the 3D white matter orientation distribution is calculated from histology and compared to dMRI. Thus, this technique serves as a gold standard for dMRI validation studies - providing the ability to determine the extent to which the dMRI signal is consistent with the histological FOD, and to establish how well different dMRI models can predict the ground truth FOD. PMID:26804781

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

    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. PMID:27050040

  20. Local Delivery of Fluorescent Dye For Fiber-Optics Confocal Microscopy of the Living Heart

    Chao eHuang

    2014-09-01

    Full Text Available Fiber-optics confocal microscopy (FCM is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release versus foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  1. Optical Coherence Tomography Angiography in Mice: Comparison with Confocal Scanning Laser Microscopy and Fluorescein Angiography

    Giannakaki-Zimmermann, Helena; Kokona, Despina; Wolf, Sebastian; Ebneter, Andreas; Zinkernagel, Martin S.

    2016-01-01

    Purpose Optical coherence tomography angiography (OCT-A) allows noninvasive visualization of retinal vessels in vivo. OCT-A was used to characterize the vascular network of the mouse retina and was compared with fluorescein angiography (FA) and histology. Methods In the present study, OCT-A based on a Heidelberg Engineering Spectralis system was used to investigate the vascular network in mice. Data was compared with FA and confocal microscopy of flat-mount histology stained with isolectin IB4. For quantitative analysis the National Cancer Institute's AngioTool software was used. Vessel density, the number of vessel junctions, and endpoints were measured and compared between the imaging modalities. Results The configuration of the superficial capillary network was comparable with OCT-A and flat-mount histology in BALBc mice. However, vessel density and the number of vessel junctions per region of interest (P = 0.0161 and P = 0.0015, respectively) in the deep vascular network of BALBc mice measured by OCT-A was significantly higher than with flat-mount histology. In C3A.Cg-Pde6b+Prph2Rd2/J mice, where the deep capillary plexus is absent, analysis of the superficial network provided similar results for all three imaging modalities. Conclusion OCT-A is a helpful imaging tool for noninvasive, in vivo imaging of the vascular plexus in mice. It may offer advantages over FA and confocal microscopy especially for imaging the deep vascular plexus. Translational Relevance The present study shows that OCT-A can be employed for small animal imaging to assess the vascular network and offers advantages over flat-mount histology and FA.

  2. Detection of advanced glycation end products (AGEs) on human skin by in vivo confocal Raman spectroscopy

    Martin, A. A.; Pereira, L.; Ali, S. M.; Pizzol, C. D.; Tellez, C. A.; Favero, P. P.; Santos, L.; da Silva, V. V.; Praes, C. E. O.

    2016-03-01

    The aging process involves the reduction in the production of the major components of skin tissue. During intrinsic aging and photoaging processes, in dermis of human skin, fibroblasts become senescent and have decreased activity, which produce low levels of collagen. Moreover, there is accumulation of advanced glycation end products (AGEs). AGEs have incidence in the progression of age-related diseases, principally in diabetes mellitus and in Alzheimer's diseases. AGEs causes intracellular damage and/or apoptosis leading to an increase of the free radicals, generating a crosslink with skin proteins and oxidative stress. The aim of this study is to detect AGEs markers on human skin by in vivo Confocal Raman spectroscopy. Spectra were obtained by using a Rivers Diagnostic System, 785 nm laser excitation and a CCD detector from the skin surface down to 120 μm depth. We analyzed the confocal Raman spectra of the skin dermis of 30 women volunteers divided into 3 groups: 10 volunteers with diabetes mellitus type II, 65-80 years old (DEW); 10 young healthy women, 20-33 years old (HYW); and 10 elderly healthy women, 65-80 years old (HEW). Pentosidine and glucosepane were the principally identified AGEs in the hydroxyproline and proline Raman spectral region (1000-800 cm-1), in the 1.260-1.320 cm-1 region assignable to alpha-helical amide III modes, and in the Amide I region. Pentosidine and glucosepane calculated vibrational spectra were performed through Density Functional Theory using the B3LYP functional with 3-21G basis set. Difference between the Raman spectra of diabetic elderly women and healthy young women, and between healthy elderly women and healthy young women were also obtained with the purpose of identifying AGEs Raman bands markers. AGEs peaks and collagen changes have been identified and used to quantify the glycation process in human skin.

  3. Raman confocal microscopy and AFM combined studies of cancerous cells treated with Paclitaxel

    Derely, L.; Collart Dutilleul, P.-Y.; Michotte de Welle, Sylvain; Szabo, V.; Gergely, C.; Cuisinier, F. J. G.

    2011-03-01

    Paclitaxel interferes with the normal function of microtubule breakdown, induces apoptosis in cancer cells and sequesters free tubulin. As this drug acts also on other cell mechanisms it is important to monitor its accumulation in the cell compartments. The intracellular spreading of the drug was followed using a WITEC 300R confocal Raman microscope equipped with a CCD camera. Hence Atomic force microscopy (an MFP3D- Asylum Research AFM) in imaging and force mode was used to determine the morphological and mechanical modifications induced on living cells. These studies were performed on living epithelial MCF-7 breast cancer cells. Paclitaxel was added to cell culture media for 3, 6 and 9 hours. Among the specific paclitaxel Raman bands we selected the one at 1670 cm-1 because it is not superposed by the spectrum of the cells. Confocal Raman images are formed by monitoring this band, the NH2 and the PO4 band. Paclitaxel slightly accumulates in the nucleus forming patches. The drug is also concentrated in the vicinity of the cell membrane and in an area close to the nucleus where proteins accumulate. Our AFM images reveal that the treated cancerous MCF-7 cells keep the same size as the non treated ones, but their shape becomes more oval. Cell's elasticity is also modified: a difference of 2 kPa in the Young Modulus characterizes the treated MCF-7 mammary cancerous cell. Our observations demonstrate that paclitaxel acts not only on microtubules but accumulates also in other cell compartments (nucleus) where microtubules are absent.

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

    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.

  5. Analysis of cellular phosphatidylinositol (3,4,5)-trisphosphate levels and distribution using confocal fluorescent microscopy.

    Palmieri, Michelle; Nowell, Cameron J; Condron, Melanie; Gardiner, James; Holmes, Andrew B; Desai, Jayesh; Burgess, Antony W; Catimel, Bruno

    2010-11-01

    We have developed an immunocytochemistry method for the semiquantitative detection of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) at the cell plasma membrane. This protocol combines the use of a glutathione S-transferase-tagged pleckstrin homology (PH) domain of the general phosphoinositides-1 receptor (GST-GRP1PH) with fluorescence confocal microscopy and image segmentation using cell mask software analysis. This methodology allows the analysis of PI(3,4,5)P3 subcellular distribution in resting and epidermal growth factor (EGF)-stimulated HEK293T cells and in LIM1215 (wild-type phosphoinositide 3-kinase (PI3K)) and LIM2550 (H1047R mutation in PI3K catalytic domain) colonic carcinoma cells. Formation of PI(3,4,5)P3 was observed 5min following EGF stimulation and resulted in an increase of the membrane/cytoplasm fluorescence ratio from 1.03 to 1.53 for HEK293T cells and from 2.2 to 3.3 for LIM1215 cells. Resting LIM2550 cells stained with GST-GRP1PH had an elevated membrane/cytoplasm fluorescence ratio of 9.8, suggesting constitutive PI3K activation. The increase in the membrane/cytoplasm fluorescent ratio was inhibited in a concentration-dependent manner by the PI3K inhibitor LY294002. This cellular confocal imaging assay can be used to directly assess the effects of PI3K mutations in cancer cell lines and to determine the potential specificity and effectiveness of PI3K inhibitors in cancer cells. PMID:20599646

  6. Three dimensional microvascular measurements in human endometrium using optical slices from laser scanning confocal microscopy (LSCM).

    Manconi, Frank; Kable, Eleanor P; Dwarte, Dennis; Jones, Allan; Russell, Peter; Chullapram, Teerapat; Gange, Prasantha V; Obeysekara, Sunil; Thomas, Graham A; Fraser, Ian S

    2011-12-01

    There is increasing interest in the structure of the microvascular environment in human endometrium because of the recognition of the complexity and functional importance of this tissue. Endometrial microcirculatory networks and their relationships have rarely been studied in three-dimensions. Longitudinal uterine slices containing endometrial tissue were carefully selected from women undergoing a hysterectomy. Formalin-fixed endometrial sections (≤ 50 μm) representing the fundal and isthmic regions were immunofluorescently labeled with monoclonal antibody (CD34) to target the endothelium of microvessel and fluorescein isothiocyanate (FITC) labeled goat anti-mouse. Digital images were acquired using a Nikon Eclipse E800 microscope equipped with a Radiance 2000 confocal scanning laser attachment. ImarisBasic 4.1 visualization suite was utilized for qualitative interpretation. NeuronTracer 1.0 software was utilized to derive the length and numerical densities. There were significant changes across the phases of the menstrual cycle in functional and basal endometrial layers in vessel length density (LD(v)) and branch point density (ND(v)) within both fundal and isthmic regions of the uterus (P<0.001). There was also a significant effect of menstrual cycle phase on mean vessel segment length (SL(v)) within each region and within each of the layers (P<0.001). The capillary radial diffusion distance r(diff) was negatively correlated with LD(v). In general, within each of the menstrual cycle phases, LD(v), ND(v) were greater in the fundal than the isthmic regions while, in contrast, SL(v) was found to be greatest in the isthmic region. Utilization of immunofluorescence and laser scanning confocal microscopy has enabled us to demonstrate significant vascular changes in human endometrial layers illustrating that in general, within each of the menstrual cycle phases, vessel length and branch point densities were greater in the fundal than the isthmic regions, while vessel

  7. Optical polarization of m-plane In-GaN/GaN light-emitting diodes characterized via confocal microscope

    Masui, Hisashi; Yamada, Hisashi; Iso, Kenji; Hirasawa, Hirohiko; Fellows, Natalie N.; Speck, James S.; Nakamura, Shuji; DenBaars, Steven P. [Solid-State Lighting and Display Center, Materials Department, College of Engineering, University of California, Santa Barbara, California 93106-5055 (United States)

    2008-05-15

    A confocal microscope was applied to characterize near-field optical polarization of light emission from InGaN-based light-emitting diodes prepared on a nonpolar orientation. Detection of stray light was significantly reduced as a result of the confocal technique compared to broad-area measurements. The confocal measurement required no special sample preparation and obtained data was directly interpretable. A polarization ratio of 0.83 was measured on long-wavelength emitting devices and was sustained over two orders of magnitude of low current range. Energy shifts and separation of emission peaks for the two polarization components were evaluated to discuss the valence-band structure. Energy shifts indicated that the topmost band vertical stroke X right angle had a heavier effective mass than the lower band vertical stroke Z right angle. Splitting energy of InGaN was estimated to be 30 meV. Results were consistent with the past reports, thus it was confirmed that the confocal microscope is a valid technique to characterize near-field optical polarization. (copyright 2008 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  8. Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging.

    Wedekind, P; Kubitscheck, U; Peters, R

    1994-10-01

    The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the 'Scamper'. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result, almost any desired pattern could be bleached ('written') into fluorescent samples at high definition and then imaged ('read') at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems. PMID:7799426

  9. Selenium Preferentially Accumulates in the Eye Lens Following Embryonic Exposure: A Confocal X-ray Fluorescence Imaging Study

    Choudhury, Sanjukta; Thomas, Jith; Sylvain, Nicole J.; Ponomarenko, Olena; Gordon, Robert A.; Heald, Steve M.; Janz, David M.; Krone, Patrick H.; Coulthard, Ian; George, Graham N.; Pickering, Ingrid J.

    2015-02-17

    Maternal transfer of elevated selenium (Se) to offspring is an important route of Se exposure for fish in the natural environment. However, there is a lack of information on the tissue specific spatial distribution and speciation of Se in the early developmental stages of fish, which provide important information about Se toxicokinetics. The effect of maternal transfer of Se was studied by feeding adult zebrafish a Se-elevated or a control diet followed by collection of larvae from both groups. Novel confocal synchrotron-based techniques were used to investigate Se within intact preserved larvae. Confocal X-ray fluorescence imaging was used to compare Se distributions within specific planes of an intact larva from each of the two groups. The elevated Se treatment showed substantially higher Se levels than the control; Se preferentially accumulated to highest levels in the eye lens, with lower levels in the retina, yolk and other tissues. Confocal X-ray absorption spectroscopy was used to determine that the speciation of Se within the eye lens of the intact larva was a selenomethionine-like species. Preferential accumulation of Se in the eye lens may suggest a direct cause-and-effect relationship between exposure to elevated Se and Se-induced ocular impairments reported previously. This study illustrates the effectiveness of confocal X-ray fluorescence methods for investigating trace element distribution and speciation in intact biological specimens

  10. A method for the assessment of light-induced oxidative stress in embryos of fucoid algae via confocal laserscan microscopy

    Rijstenbil, J.W.; Coelho, S.; Eijsackers, M.

    2000-01-01

    A method was developed for measurement of active oxygen production in embryonic stages of the brown seaweed Fucus spiralis. using the label CM-DCFH-DA. Active oxygen species convert the label into the green fluorescent CM-DCF (exc/em 488/530 nm) that is detected via confocal laserscan microscopy and

  11. Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae

    Fischer-Parton, S; Parton, R M; Hickey, P C; Dijksterhuis, J; Atkinson, H A; Read, N D

    2000-01-01

    Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were

  12. Method of a Confocal Microscopy in Investigation of the Operational Wound Zone (in vivo) After a Cataract Phacoemulsification

    2011-01-01

    The results of the corneal wound zone investigations with a confocal microscope have been analyzed. 10 patients after a bimanual and 10 patients after a coaxial phacoemulsification of cataract took part in the study. It seems reasonable to conclude that the revealed alteration rate and character do not depend on a phacoemulsification method (bimanual or coaxial).

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

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

    2006-01-01

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

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

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

    2006-01-01

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

  15. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells.

    Yan, Yitang; Cummings, Connie A; Sutton, Deloris; Yu, Linda; Castro, Lysandra; Moore, Alicia B; Gao, Xiaohua; Dixon, Darlene

    2016-04-01

    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells. Published 2016. This article is a U.S. Government work and is in the public domain in the

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

    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.

  17. Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

    De Simoni, Maria-Grazia

    2013-01-01

    After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing. Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 μm z-axis and a 0.23 μm step size, corresponding to a 180 x 135 x 10 μm volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury. PMID:24056862

  18. Visualization of ferroelectric domain structures in lithium niobate by means of confocal nonlinear microscopy

    In the field of integrated optics nonlinear-optical effects play a central role. A typical example for the commercial use of such phenomena is the frequency conversion. A deciding parameter is here the phase matching, which determines the quantity of the constructive interaction range of contributing optical fields. In view of a high efficiency of such processes the dispersion of a crystal must be balanced for the contributing frequencies. In nonlinear components on the base of optical waveguides the principle of the ''quasi-phase matching'' is applied, which uses the microdomain inversion. Phase jumps occuring at the domain boundaries compensate in the mean the different phase velocities. The application range of such periodical structures depends essentially on sharpness, homogeneity, depth extent, and period of the domain structure. The nonlinear confocal laser scanning microscopy makes a mapping of this transferred ferroelectric domain structure possible. Primary aim of this thesis is the characterization and mapping of the transferred ferroelectric domain structure in lithium niobate. A modularly kept confocal microscope makes here a nonlinear analysis in reflection and transmission geometry possible. In both geometries systematic studies as function of important process parameters were performed. It was shown that because of the larger nonlinear coherence length in the transmission modus the SHG ensues above all in forward direction. By depth-resolved studies at Z-cut PPLN structured between the surface region and the volume crystal a flippling of the SHG contrast could be observed. In samples with circular pole structure additionally in the crystal a transition to a hexagonal structure took place. In the Ti:PPLN strip waveguide a strong and specific increasement of the nonlinear signal of the domain walls was discovered. Here also the usual SHG surface contrast between dhe domains and the boundaries is inverted. Also differently processed planar and ripple

  19. Blinking correlation in nanocrystal quantum dots probed with novel laser scanning confocal microscopy methods

    Hefti, Ryan Alf

    Semiconductor quantum dots have a vast array of applications: as fluorescent labels in biological systems, as physical or chemical sensors, as components in photovoltaic technology, and in display devices. An attribute of nearly every quantum dot is its blinking, or fluorescence intermittency, which tends to be a disadvantage in most applications. Despite the fact that blinking has been a nearly universal phenomenon among all types of fluorescent constructs, it is more prevalent in quantum dots than in traditional fluorophores. Furthermore, no unanimously accepted model of quantum dot blinking yet exists. The work encompassed by this dissertation began with an in-depth study of molecular motor protein dynamics in a variety of environments using two specially developed techniques, both of which feature applicability to live cell systems. Parked-beam confocal microscopy was utilized to increase temporal resolution of molecular motor motion dynamics by an order of magnitude over other popular methods. The second technique, fast-scanning confocal microscopy (FSCM), was used for long range observation of motor proteins. While using FSCM on motor protein assays, we discovered an unusual phenomenon. Single quantum dots seemingly communicated with neighboring quantum dots, indicated by a distinct correlation in their blinking patterns. In order to explain this novel correlation phenomenon, the majority of blinking models developed thus far would suggest a dipole-dipole interaction or a Coulomb interaction between singly charged quantum dots. However, our results indicate that the interaction energy is higher than supported by current models, thereby prompting a renewed examination. We propose that the blinking correlation we observed is due to a Coulomb interaction on the order of 3-4 elementary charges per quantum dot and that multiple charging of individual quantum dots may be required to plunge them into a non-emissive state. As a result of charging, charge carriers are

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

    Vyacheslav M Shkryl

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

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

    Kotlarchyk, M A; Botvinick, E L; Putnam, A J, E-mail: elliot.botvinick@uci.ed, E-mail: putnam@umich.ed [Department of Biomedical Engineering, University of California, Irvine, CA (United States)

    2010-05-19

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

  2. Adaptive Cell Segmentation and Tracking for Volumetric Confocal Microscopy Images of a Developing Plant Meristem

    Min Liu; Anirban Chakraborty; Damanpreet Singh; Ram Kishor Yadav; Gopi Meenakshisundaram; G. Venugopala Reddy; Amit Roy-Chowdhury

    2011-01-01

    Automated segmentation and tracking of cells in actively developing tissues can provide high-throughput and quantitative spatiotemporal measurements of a range of cell behaviors; cell expansion and cell-division kinetics leading to a better understanding of the underlying dynamics of morphogenesis.Here,we have studied the problem of constructing cell lineages in time-lapse volumetric image stacks obtained using Confocal Laser Scanning Microscopy (CLSM).The novel contribution of the work lies in its ability to segment and track cells in densely packed tissue,the shoot apical meristem (SAM),through the use of a close-loop,adaptive segmentation,and tracking approach.The tracking output acts as an indicator of the quality of segmentation and,in turn,the segmentation can be improved to obtain better tracking results.We construct an optimization function that minimizes the segmentation error,which is,in turn,estimated from the tracking results.This adaptive approach significantly improves both tracking and segmentation when compared to an open loop framework in which segmentation and tracking modules operate separately.

  3. Role of In vivo reflectance confocal microscopy in determining stability in vitiligo: A preliminary study

    Wei LI

    2013-01-01

    Full Text Available Background: Vitiligo is an acquired pigmentary disorder. In vivo reflectance confocal microscopy (RCM reproducible imaging technique has already been reported to be useful in the diagnosis of other skin diseases. Objective: To define RCM features of vitiligo on different clinical stages. Materials and Methods: A total of 125 patients with a clinical diagnosis of vitiligo were included in this study. After informed consent, lesional skins of those vitiligo patients were characterized by using RCM. Five patients with inflammatory cell infiltration observed at the edge of skin lesions and another 5 patients without inflammatory cell infiltration were selected. Biopsies were performed at same sites of the RCM examination areas for histological and immune-histological analysis. Results: In the active stage of vitiligo, the RCM examination revealed that the bright dermal papillary rings presented at the dermoepidermal junction level in normal skin lost their integrity or totally disappeared, border between vitiligo lesion and normal skin became unclear, and highly refractile cells that referred to infiltrated inflammatory cells could be seen within the papillary dermis at the edge of the lesions. In the stable stage of vitiligo, the RCM showed a complete loss of melanin in lesional skin and a clear border between lesional and normal skin. Conclusion: A simple clinical examination with RCM may reliably and efficiently allow evaluation of the stability status of vitiligo lesions.

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

    Hardman, P.; Spooner, B. S.

    1992-01-01

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

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

    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.

  6. Statistical strategies to reveal potential vibrational markers for in vivo analysis by confocal Raman spectroscopy

    Oliveira Mendes, Thiago de; Pinto, Liliane Pereira; Santos, Laurita dos; Tippavajhala, Vamshi Krishna; Téllez Soto, Claudio Alberto; Martin, Airton Abrahão

    2016-07-01

    The analysis of biological systems by spectroscopic techniques involves the evaluation of hundreds to thousands of variables. Hence, different statistical approaches are used to elucidate regions that discriminate classes of samples and to propose new vibrational markers for explaining various phenomena like disease monitoring, mechanisms of action of drugs, food, and so on. However, the technical statistics are not always widely discussed in applied sciences. In this context, this work presents a detailed discussion including the various steps necessary for proper statistical analysis. It includes univariate parametric and nonparametric tests, as well as multivariate unsupervised and supervised approaches. The main objective of this study is to promote proper understanding of the application of various statistical tools in these spectroscopic methods used for the analysis of biological samples. The discussion of these methods is performed on a set of in vivo confocal Raman spectra of human skin analysis that aims to identify skin aging markers. In the Appendix, a complete routine of data analysis is executed in a free software that can be used by the scientific community involved in these studies.

  7. New fluorogenic dyes for analysis of cellular processes by flow cytometry and confocal microscopy.

    Nikolova, Kalina; Kaloyanova, Stefka; Mihaylova, Nikolina; Stoitsova, Stoyanka; Chausheva, Stela; Vasilev, Aleksey; Lesev, Nedyalko; Dimitrova, Petya; Deligeorgiev, Todor; Tchorbanov, Andrey

    2013-12-01

    Fluorescent microscopy and fluorescent imaging by flow cytometry are two of the fastest growing areas in the medical and biological research. Innovations in fluorescent chemistry and synthesis of new dye probes are closely related to the development of service equipment such as light sources, and detection techniques. Among compounds known as fluorescent labels, the cyanine-based dyes have become widely used since they have high excitation coefficients, narrow emission bands and high fluorescence upon binding to nucleic acids. The key methods for evaluation of apoptosis and cell cycle allow measuring DNA content by several flow cytometric techniques. We have synthesized new monomethine cyanine dyes and have characterized their applicability for staining of live and/or apoptotic cells. Imaging experiments by flow cytometry and confocal laser scanning microscopy (CLSM) have been also performed. Two of the dyes have shown high-affinity binding to the nuclei at high dilutions, up to 10(-9)M. Flow cytometry and CLSM have confirmed that these dyes labeled selectively non-living, e.g. ethanol-fixed cells that makes them appropriate for estimations of cell viability and apoptosis. The novel structures proved to be appropriate also for analysis of the cell cycle. PMID:24231377

  8. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

    Fea, Antonio M.; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M.

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications. PMID:27200376

  9. A fully automated tortuosity quantification system with application to corneal nerve fibres in confocal microscopy images.

    Annunziata, Roberto; Kheirkhah, Ahmad; Aggarwal, Shruti; Hamrah, Pedram; Trucco, Emanuele

    2016-08-01

    Recent clinical research has highlighted important links between a number of diseases and the tortuosity of curvilinear anatomical structures like corneal nerve fibres, suggesting that tortuosity changes might detect early stages of specific conditions. Currently, clinical studies are mainly based on subjective, visual assessment, with limited repeatability and inter-observer agreement. To address these problems, we propose a fully automated framework for image-level tortuosity estimation, consisting of a hybrid segmentation method and a highly adaptable, definition-free tortuosity estimation algorithm. The former combines an appearance model, based on a Scale and Curvature-Invariant Ridge Detector (SCIRD), with a context model, including multi-range learned context filters. The latter is based on a novel tortuosity estimation paradigm in which discriminative, multi-scale features can be automatically learned for specific anatomical objects and diseases. Experimental results on 140 in vivo confocal microscopy images of corneal nerve fibres from healthy and unhealthy subjects demonstrate the excellent performance of our method compared to state-of-the-art approaches and ground truth annotations from 3 expert observers. PMID:27136674

  10. Confocal Analysis of Nuclear Lamina Behavior during Male Meiosis and Spermatogenesis in Drosophila melanogaster

    Fabbretti, Fabiana; Iannetti, Ilaria; Guglielmi, Loredana; Perconti, Susanna; Evangelistella, Chiara; Proietti De Santis, Luca; Bongiorni, Silvia; Prantera, Giorgio

    2016-01-01

    Lamin family proteins are structural components of a filamentous framework, the nuclear lamina (NL), underlying the inner membrane of nuclear envelope. The NL not only plays a role in nucleus mechanical support and nuclear shaping, but is also involved in many cellular processes including DNA replication, gene expression and chromatin positioning. Spermatogenesis is a very complex differentiation process in which each stage is characterized by nuclear architecture dramatic changes, from the early mitotic stage to the sperm differentiation final stage. Nevertheless, very few data are present in the literature on the NL behavior during this process. Here we show the first and complete description of NL behavior during meiosis and spermatogenesis in Drosophila melanogaster. By confocal imaging, we characterized the NL modifications from mitotic stages, through meiotic divisions to sperm differentiation with an anti-laminDm0 antibody against the major component of the Drosophila NL. We observed that continuous changes in the NL structure occurred in parallel with chromatin reorganization throughout the whole process and that meiotic divisions occurred in a closed context. Finally, we analyzed NL in solofuso meiotic mutant, where chromatin segregation is severely affected, and found the strict correlation between the presence of chromatin and that of NL. PMID:26963718

  11. Characterization of tumor microvascular structure and permeability: comparison between magnetic resonance imaging and intravital confocal imaging.

    Reitan, Nina Kristine; Thuen, Marte; Goa, Pål Erik; de Lange Davies, Catharina

    2010-01-01

    Solid tumors are characterized by abnormal blood vessel organization, structure, and function. These abnormalities give rise to enhanced vascular permeability and may predict therapeutic responses. The permeability and architecture of the microvasculature in human osteosarcoma tumors growing in dorsal window chambers in athymic mice were measured by confocal laser scanning microscopy (CLSM) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Dextran (40 kDa) and Gadomer were used as molecular tracers for CLSM and DCE-MRI, respectively. A significant correlation was found between permeability indicators. The extravasation rate K(i) as measured by CLSM correlated positively with DCE-MRI parameters, such as the volume transfer constant K(trans) and the initial slope of the contrast agent concentration-time curve. This demonstrates that these two techniques give complementary information. Extravasation was further related to microvascular structure and was found to correlate with the fractal dimension and vascular density. The structural parameter values that were obtained from CLSM images were higher for abnormal tumor vasculature than for normal vessels. PMID:20615006

  12. High resolution confocal imaging of vaginal epithelial microstructure as a means of detecting microbicide toxicity

    Vargas, Gracie; Shilagard, Tuya; Johnston, Rebecca; Bell, Brent; Stegal, Rachael; Vincent, Kathleen; Stanberry, Lawrence; Motamedi, Massoud; Bourne, Nigel

    2014-01-01

    High resolution optical imaging by confocal reflectance microscopy (CRM) was investigated for revealing epithelial microstructure and alterations which may occur from vaginal microbicides. The vaginal tracts of Swiss Webster mice treated with medroxyprogesterone acetate were exposed in vivo to a 4% nonoxynyl-9 containing gel or saline for 4, 16, or 48 hours. The vaginal tract was removed and imaged by CRM without staining and imaged regions were then biopsied and processed for histology. In control mice, CRM revealed columnar epithelium and lamina propria with features resembling that of histology. CRM revealed exfoliated epithelium following 4 and 16 hour N-9 treatments and quantitative measurement of epithelial thickness revealed a decrease from approximately 41.7 ± 1.7 (SEM) in controls to 14.9 ± 4.5 and 24.4 ± 2.1 after 4 and 16 hours, respectively. Indication of inflammation at 4 hours was given through the presence of inflammatory infiltrates. After 48 hours the epithelium was regenerating. The timecourse of change in structure and epithelial thickness detected by CRM closely resembled that observed and measured by histology. This study demonstrates CRM can reveal epithelial structure and indicators of inflammation following treatment with N-9 and may be a useful imaging tool for evaluating effects of microbicides. PMID:19355817

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

    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.

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

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

    2015-06-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented dairy products. When studying such networks, hundreds of images can be obtained, and here image analysis methods are essential for using the images in statistical analysis. Previously, methods including gray level co-occurrence matrix analysis and fractal analysis have been used with success. However, a range of other image texture characterization methods exists. These methods describe an image by a frequency distribution of predefined image features (denoted textons). Our contribution is an investigation of the choice of image analysis methods by performing a comparative study of 7 major approaches to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis, and cluster analysis. Our investigation suggests that the texton-based descriptors provide a fuller description of the images compared to gray-level co-occurrence matrix descriptors and fractal analysis, while still being as applicable and in some cases as easy to tune. PMID:25959794

  15. A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation.

    Lee, Jacqueline A; Collings, David A; Glover, Chris N

    2016-08-15

    The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na(+)]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na(+)]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na(+)]i, whereas no significant differences in branchial [Na(+)]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na(+)]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms. PMID:27235170

  16. Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology, calcium imaging and confocal microscopy.

    Galizia, C G; Kimmerle, B

    2004-01-01

    The insect antennal lobe is the first brain structure to process olfactory information. Like the vertebrate olfactory bulb the antennal lobe is substructured in olfactory glomeruli. In insects, glomeruli can be morphologically identified, and have characteristic olfactory response profiles. Local neurons interconnect glomeruli, and output (projection) neurons project to higher-order brain centres. The relationship between their elaborate morphology and their physiology is not understood. We recorded electrophysiologically from antennal lobe neurons, and iontophoretically injected a calcium-sensitive dye. We then measured their spatio-temporal calcium responses to a variety of odours. Finally, we confocally reconstructed the neurons, and identified the innervated glomeruli. An increase or decrease in spiking frequency corresponded to an intracellular calcium increase or decrease in the cell. While intracellular recordings generally lasted between 10 and 30 min, calcium imaging was stable for up to 2 h, allowing a more detailed physiological analysis. The responses indicate that heterogeneous local neurons get input in the glomerulus in which they branch most strongly. In many cases, the physiological response properties of the cells corresponded to the known response profile of the innervated glomerulus. In other words, the large variety of response profiles generally found when comparing antennal lobe neurons is reduced to a more predictable response profile when the innervated glomerulus is known. PMID:14639486

  17. Nanostructured transdermal hormone replacement therapy for relieving menopausal symptoms: a confocal Raman spectroscopy study

    Objective:to determine the safety and efficacy of a transdermal nanostructured formulation of progesterone (10%) combined with estriol (0.1%) + estradiol (0.25%) for relieving postmenopausal symptoms. Methods: a total of 66 postmenopausal Brazilian women with climacteric symptoms of natural menopause received transdermal nanostructured formulations of progesterone and estrogens in the forearm daily for 60 months to mimic the normal ovarian secretory pattern. Confocal Raman spectroscopy of hormones in skin layers was performed. Clinical parameters, serum concentrations of estradiol and follicle-stimulating hormone, blood pressure, BI-RADS classification from bilateral mammography, and symptomatic relief were compared between baseline and 60 months post-treatment. Clinicaltrials.gov: NCT02033512. Results: an improvement in climacteric symptoms was reported in 92.5% of women evaluated before and after 60 months of treatment. The serum concentrations of estradiol and follicle-stimulating hormone changed significantly (p<0.05) after treatment; the values of serum follicle-stimulating hormone decreased after 60 months from 82.04 ± 4.9 to 57.12 ± 4.1 IU/mL. A bilateral mammography assessment of the breasts revealed normal results in all women. No adverse health-related events were attributed to this hormone replacement therapy protocol. Conclusion: the nanostructured formulation is safe and effective in re-establishing optimal serum levels of estradiol and follicle-stimulating hormone and relieving the symptoms of menopause. This transdermal hormone replacement therapy may alleviate climacteric symptoms in postmenopausal women. (author)

  18. Modeling and simulation of protein uptake in cation exchanger visualized by confocal laser scanning microscopy.

    Yang, Kun; Shi, Qing-Hong; Sun, Yan

    2006-12-01

    Confocal laser scanning microscopy (CLSM) has been extensively applied in the area of protein chromatography to investigate the uptake mechanism of protein in adsorbents. However, due to the light attenuation in the deeper layers of a specimen, quantitative analysis using CLSM data is still far from reality. In this work, an attenuation equation for describing the darkening of the CLSM image in the deeper scanning layers was developed. Bovine serum albumin (BSA) adsorption to SP Sepharose FF was performed by batch adsorption and micro-column chromatography on which protein concentration in single absorbents were visualized by CLSM. The parameters in the equation were estimated by fitting it to the fluorescence intensity profiles obtained at adsorption equilibrium, and then the equation was used to simulate the effect caused by the light scattering and absorption. CLSM analysis demonstrated that BSA adsorption to SP Sepharose FF followed the shrinking core pattern and was predicted reasonably well by the pore diffusion model in combination with the attenuation equation. By comparison of the CLSM data with the simulations, it shows that the attenuation equation was useful to demonstrate the validity of an intraparticle mass transport model for the estimation of intraparticle protein concentration profiles. PMID:17034803

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

    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)

  20. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks.

    Hames, Samuel C; Ardigò, Marco; Soyer, H Peter; Bradley, Andrew P; Prow, Tarl W

    2016-01-01

    Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20-30 and 50-70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin. PMID:27088865

  1. Assessing strain mapping by electron backscatter diffraction and confocal Raman microscopy using wedge-indented Si.

    Friedman, Lawrence H; Vaudin, Mark D; Stranick, Stephan J; Stan, Gheorghe; Gerbig, Yvonne B; Osborn, William; Cook, Robert F

    2016-04-01

    The accuracy of electron backscatter diffraction (EBSD) and confocal Raman microscopy (CRM) for small-scale strain mapping are assessed using the multi-axial strain field surrounding a wedge indentation in Si as a test vehicle. The strain field is modeled using finite element analysis (FEA) that is adapted to the near-indentation surface profile measured by atomic force microscopy (AFM). The assessment consists of (1) direct experimental comparisons of strain and deformation and (2) comparisons in which the modeled strain field is used as an intermediate step. Direct experimental methods (1) consist of comparisons of surface elevation and gradient measured by AFM and EBSD and of Raman shifts measured and predicted by CRM and EBSD, respectively. Comparisons that utilize the combined FEA-AFM model (2) consist of predictions of distortion, strain, and rotation for comparison with EBSD measurements and predictions of Raman shift for comparison with CRM measurements. For both EBSD and CRM, convolution of measurements in depth-varying strain fields is considered. The interconnected comparisons suggest that EBSD was able to provide an accurate assessment of the wedge indentation deformation field to within the precision of the measurements, approximately 2×10(-4) in strain. CRM was similarly precise, but was limited in accuracy to several times this value. PMID:26939030

  2. Quantitative analyses of Streptococcus mutans biofilms with quartz crystal microbalance, microjet impingement and confocal microscopy.

    Kreth, J; Hagerman, E; Tam, K; Merritt, J; Wong, D T W; Wu, B M; Myung, N V; Shi, W; Qi, F

    2004-10-01

    Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm. PMID:16429589

  3. Confocal Raman study of aging process in diabetes mellitus human voluntaries

    Pereira, Liliane; Téllez Soto, Claudio Alberto; dos Santos, Laurita; Ali, Syed Mohammed; Fávero, Priscila Pereira; Martin, Airton A.

    2015-06-01

    Accumulation of AGEs [Advanced Glycation End - products] occurs slowly during the human aging process. However, its formation is accelerated in the presence of diabetes mellitus. In this paper, we perform a noninvasive analysis of glycation effect on human skin by in vivo confocal Raman spectroscopy. This technique uses a laser of 785 nm as excitation source and, by the inelastic scattering of light, it is possible to obtain information about the biochemical composition of the skin. Our aim in this work was to characterize the aging process resulting from the glycation process in a group of 10 Health Elderly Women (HEW) and 10 Diabetic Elderly Women (DEW). The Raman data were collected from the dermis at a depth of 70-130 microns. Through the theory of functional density (DFT) the bands positions of hydroxyproline, proline and AGEs (pentosidine and glucosepane) were calculated by using Gaussian 0.9 software. A molecular interpretation of changes in type I collagen was performed by the changes in the vibrational modes of the proline (P) and hydroxyproline (HP). The data analysis shows that the aging effects caused by glycation of proteins degrades type I collagen differently and leads to accelerated aging process.

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

    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)

  5. Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging.

    Kuznetsov, Andrey V; Hermann, Martin; Troppmair, Jakob; Margreiter, Raimund; Hengster, Paul

    2010-01-01

    Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission-fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1-0.3 mum/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 mum/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems. PMID:19382913

  6. Nanostructured transdermal hormone replacement therapy for relieving menopausal symptoms: a confocal Raman spectroscopy study

    Marco Antonio Botelho

    2014-02-01

    Full Text Available OBJECTIVE: To determine the safety and efficacy of a transdermal nanostructured formulation of progesterone (10% combined with estriol (0.1% + estradiol (0.25% for relieving postmenopausal symptoms. METHODS: A total of 66 postmenopausal Brazilian women with climacteric symptoms of natural menopause received transdermal nanostructured formulations of progesterone and estrogens in the forearm daily for 60 months to mimic the normal ovarian secretory pattern. Confocal Raman spectroscopy of hormones in skin layers was performed. Clinical parameters, serum concentrations of estradiol and follicle-stimulating hormone, blood pressure, BI-RADS classification from bilateral mammography, and symptomatic relief were compared between baseline and 60 months post-treatment. Clinicaltrials.gov: NCT02033512. RESULTS: An improvement in climacteric symptoms was reported in 92.5% of women evaluated before and after 60 months of treatment. The serum concentrations of estradiol and follicle-stimulating hormone changed significantly (p<0.05 after treatment; the values of serum follicle-stimulating hormone decreased after 60 months from 82.04±4.9 to 57.12±4.1 IU/mL. A bilateral mammography assessment of the breasts revealed normal results in all women. No adverse health-related events were attributed to this hormone replacement therapy protocol. CONCLUSION: The nanostructured formulation is safe and effective in re-establishing optimal serum levels of estradiol and follicle-stimulating hormone and relieving the symptoms of menopause. This transdermal hormone replacement therapy may alleviate climacteric symptoms in postmenopausal women.

  7. Measurement of diffusion of fluorescent compounds and autofluorescence in skin in vivo using a confocal instrument

    Buttenschoen, K. K.; Sutton, E. E.; Daly, D.; Girkin, J. M.

    2016-02-01

    Using compact and affordable instrumentation based upon fluorescent confocal imaging we have tracked the movement of autofluorescent compounds through skin in near real time with high temporal and spatial resolution and sensitivity. The ability to measure the diffusion of compounds through skin with such resolution plays an important role for applications such as monitoring the penetration of pharmaceuticals applied to skin and assessing the integrity of the skin barrier. Several measurement methods exist, but they suffer from a number of problems such as being slow, expensive, non-portable and lacking sensitivity. To address these issues, we adapted a technique that we previously developed for tracking fluorescent compounds in the eye to measure the autofluorescence and the diffusion of externally applied fluorescent compounds in skin in vivo. Results are presented that show the change in autofluorescence of the volar forearm over the course of a week. We furthermore demonstrate the ability of the instrument to measure the diffusion speed and depth of externally applied fluorescent compounds both in healthy skin and after the skin barrier function has been perturbed. The instrument is currently being developed further for increased sensitivity and multi-wavelength excitation. We believe that the presented instrument is suitable for a large number of applications in fields such as assessment of damage to the skin barrier, development of topical and systemic medication and tracking the diffusion of fluorescent compounds through skin constructs as well as monitoring effects of skin products and general consumer products which may come into contact with the skin.

  8. Comparison study of distinguishing cancerous and normal prostate epithelial cells by confocal and polarization diffraction imaging.

    Jiang, Wenhuan; Lu, Jun Qing; Yang, Li V; Sa, Yu; Feng, Yuanming; Ding, Junhua; Hu, Xin-Hua

    2016-07-01

    Accurate classification of malignant cells from benign ones can significantly enhance cancer diagnosis and prognosis by detection of circulating tumor cells (CTCs). We have investigated two approaches of quantitative morphology and polarization diffraction imaging on two prostate cell types to evaluate their feasibility as single-cell assay methods toward CTC detection after cell enrichment. The two cell types have been measured by a confocal imaging method to obtain their three-dimensional morphology parameters and by a polarization diffraction imaging flow cytometry (p-DIFC) method to obtain image texture parameters. The support vector machine algorithm was applied to examine the accuracy of cell classification with the morphology and diffraction image parameters. Despite larger mean values of cell and nuclear sizes of the cancerous prostate cells than the normal ones, it has been shown that the morphologic parameters cannot serve as effective classifiers. In contrast, accurate classification of the two prostate cell types can be achieved with high classification accuracies on measured data acquired separately in three measurements. These results provide strong evidence that the p-DIFC method has the potential to yield morphology-related “fingerprints” for accurate and label-free classification of the two prostate cell types. PMID:26616011

  9. Fluorescence confocal polarizing microscopy: Three-dimensional imaging of the director

    O D Lavrentovich

    2003-08-01

    Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by fluorescence confocal polarizing microscopy (FCPM). The technique employs the property of LC to orient the fluorescent dye molecules of anisometric shape, added in small quantities to the LC. In LC, smooth director deformations do not alter mass density of the material. Thus the density of dye is also uniform across the sample, except, perhaps, near the surfaces or at the cores of topological defects. In polarized light, the measured fluorescence signal is determined by the spatial orientation of the molecules rather than by dye concentration (as in regular biological samples stained with tissue-specific dyes). The contrast is enhanced when both excitation and detection of fluorescence light are performed in polarized light. This short review describes the essence of FCPM technique and illustrates some of its applications, including imaging of Frederiks electric-field induced effect in a nematic LC and defects such as dislocations in cholesteric LCs.

  10. Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

    Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres

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

    FedericoLuzzati

    2011-05-01

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

  12. Probe-Based Confocal Laser Endomicroscopy for Indeterminate Biliary Strictures: Refinement of the Image Interpretation Classification

    Michel Kahaleh

    2015-01-01

    Full Text Available Background. Accurate diagnosis and clinical management of indeterminate biliary strictures are often a challenge. Tissue confirmation modalities during Endoscopic Retrograde Cholangiopancreatography (ERCP suffer from low sensitivity and poor diagnostic accuracy. Probe-based confocal laser endomicroscopy (pCLE has been shown to be sensitive for malignant strictures characterization (98% but lacks specificity (67% due to inflammatory conditions inducing false positives. Methods. Six pCLE experts validated the Paris Classification, designed for diagnosing inflammatory biliary strictures, using a set of 40 pCLE sequences obtained during the prospective registry (19 inflammatory, 6 benign, and 15 malignant. The 4 criteria used included (1 multiple thin white bands, (2 dark granular pattern with scales, (3 increased space between scales, and (4 thickened reticular structures. Interobserver agreement was further calculated on a separate set of 18 pCLE sequences. Results. Overall accuracy was 82.5% (n=40 retrospectively diagnosed versus 81% (n=89 prospectively collected for the registry, resulting in a sensitivity of 81.2% (versus 98% for the prospective study and a specificity of 83.3% (versus 67% for the prospective study. The corresponding interobserver agreement for 18 pCLE clips was fair (k=0.37. Conclusion. Specificity of pCLE using the Paris Classification for the characterization of indeterminate bile duct stricture was increased, without impacting the overall accuracy.

  13. Progress in reflectance confocal microscopy for imaging oral tissues in vivo

    Peterson, Gary; Zanoni, Daniella K.; Migliacci, Jocelyn; Cordova, Miguel; Rajadhyaksha, Milind; Patel, Snehal

    2016-02-01

    We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and "video-mosaicing", extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.

  14. Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

    Lange-Asschenfeldt, Susanne; Bob, Adrienne; Terhorst, Dorothea; Ulrich, Martina; Fluhr, Joachim; Mendez, Gil; Roewert-Huber, Hans-Joachim; Stockfleth, Eggert; Lange-Asschenfeldt, Bernhard

    2012-07-01

    There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

  15. Confocal Analysis of Nuclear Lamina Behavior during Male Meiosis and Spermatogenesis in Drosophila melanogaster.

    Fabiana Fabbretti

    Full Text Available Lamin family proteins are structural components of a filamentous framework, the nuclear lamina (NL, underlying the inner membrane of nuclear envelope. The NL not only plays a role in nucleus mechanical support and nuclear shaping, but is also involved in many cellular processes including DNA replication, gene expression and chromatin positioning. Spermatogenesis is a very complex differentiation process in which each stage is characterized by nuclear architecture dramatic changes, from the early mitotic stage to the sperm differentiation final stage. Nevertheless, very few data are present in the literature on the NL behavior during this process. Here we show the first and complete description of NL behavior during meiosis and spermatogenesis in Drosophila melanogaster. By confocal imaging, we characterized the NL modifications from mitotic stages, through meiotic divisions to sperm differentiation with an anti-laminDm0 antibody against the major component of the Drosophila NL. We observed that continuous changes in the NL structure occurred in parallel with chromatin reorganization throughout the whole process and that meiotic divisions occurred in a closed context. Finally, we analyzed NL in solofuso meiotic mutant, where chromatin segregation is severely affected, and found the strict correlation between the presence of chromatin and that of NL.

  16. 3D optical sectioning with a new hyperspectral confocal fluorescence imaging system.

    Nieman, Linda T.; Sinclair, Michael B.; Davidson, George S.; Van Benthem, Mark Hilary; Haaland, David Michael; Timlin, Jerilyn Ann; Sasaki, Darryl Yoshio; Bachand, George David; Jones, Howland D. T.

    2007-02-01

    A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria.

  17. 3D Axon structure extraction and analysis in confocal fluorescence microscopy images.

    Zhang, Yong; Zhou, Xiaobo; Lu, Ju; Lichtman, Jeff; Adjeroh, Donald; Wong, Stephen T C

    2008-08-01

    The morphological properties of axons, such as their branching patterns and oriented structures, are of great interest for biologists in the study of the synaptic connectivity of neurons. In these studies, researchers use triple immunofluorescent confocal microscopy to record morphological changes of neuronal processes. Three-dimensional (3D) microscopy image analysis is then required to extract morphological features of the neuronal structures. In this article, we propose a highly automated 3D centerline extraction tool to assist in this task. For this project, the most difficult part is that some axons are overlapping such that the boundaries distinguishing them are barely visible. Our approach combines a 3D dynamic programming (DP) technique and marker-controlled watershed algorithm to solve this problem. The approach consists of tracking and updating along the navigation directions of multiple axons simultaneously. The experimental results show that the proposed method can rapidly and accurately extract multiple axon centerlines and can handle complicated axon structures such as cross-over sections and overlapping objects. PMID:18336075

  18. Confocal scanning laser microscopy on fluid-fluid demixing colloid-polymer mixtures

    We study gas-liquid phase separating colloid-polymer mixtures using a horizontally placed confocal scanning laser microscope. The phase separation proceeds via spinodal decomposition; first images immediately show sharp interfaces, which is explained in terms of the colloid diffusion time. The diffusion in both the liquid and gas phase is measured in a real space fluorescence recovery after a photo-bleaching experiment. The coarsening rate of the characteristic length in the system can be understood in terms of the capillary velocity. We observe that the spinodal structure collapses due to gravity at a typical size of the order of the capillary length, which is obtained from the static gas-liquid profile near a single wall and is accurately described by the interplay between hydrostatic and Laplace pressure. The present technique allows for precise contact angle measurements and the system shows complete wetting for all statepoints measured. Finally, we study the possibility of capillary condensation in colloid-polymer mixtures and show first indicative experimental results. The observed Kelvin length is surprisingly large, possibly because the system is not yet in complete equilibrium

  19. Evaluation of confocal laser scanning microscopy for enumeration of virus-like particles in aquatic systems

    Agis, Martin; Luef, Birgit

    2016-01-01

    Abstract Abundances of virus-like particles (VLPs, mostly bacteriophages) are high in aquatic environments; therefore, techniques for precise enumeration are essential in ecological monitoring. VLPs were determined after staining with SYBR Gold by conventional epifluorescence microscopy and compared to enumerations performed by confocal laser scanning microscopy (CLSM). In order to assess the potential of CLSM for viral direct counts (VDCs), we processed samples from different freshwater and marine systems. Optical sectioning by CLSM and production of an overlay picture of multiple scans enables the often uneven whole investigated filter area to be brought to the plane of focus. This allows for subsequent image analysis of digitally created high-quality images. Another advantage using the CLSM was that the short spot excitation of the stain via laser beam minimized fading of the stain. The VDC results show that there is no significant difference between the two methods. Regarding the known difficulties of viral abundance estimates on particulate material, CLSM was further applied to enumerate VLPs on a small set of marine transparent exopolymeric particles sampled from the Atlantic Ocean. Our data suggest that CLSM is a useful tool to count viruses in water samples as well as attached to certain types of aquatic aggregates. PMID:23108709

  20. Application of Laser Scanning Confocal Microscopy to Heat and Mass Transport Modeling in Porous Microstructures

    Marshall, Jochen; Milos, Frank; Fredrich, Joanne; Rasky, Daniel J. (Technical Monitor)

    1997-01-01

    Laser Scanning Confocal Microscopy (LSCM) has been used to obtain digital images of the complicated 3-D (three-dimensional) microstructures of rigid, fibrous thermal protection system (TPS) materials. These orthotropic materials are comprised of refractory ceramic fibers with diameters in the range of 1 to 10 microns and have open porosities of 0.8 or more. Algorithms are being constructed to extract quantitative microstructural information from the digital data so that it may be applied to specific heat and mass transport modeling efforts; such information includes, for example, the solid and pore volume fractions, the internal surface area per volume, fiber diameter distributions, and fiber orientation distributions. This type of information is difficult to obtain in general, yet it is directly relevant to many computational efforts which seek to model macroscopic thermophysical phenomena in terms of microscopic mechanisms or interactions. Two such computational efforts for fibrous TPS materials are: i) the calculation of radiative transport properties; ii) the modeling of gas permeabilities.

  1. Confocal scanning optical microscopy of a 3-million-year-old Australopithecus afarensis femur.

    Bromage, T G; Goldman, H M; McFarlin, S C; Perez Ochoa, A; Boyde, A

    2009-01-01

    Portable confocal scanning optical microscopy (PCSOM) has been specifically developed for the noncontact and nondestructive imaging of early human fossil hard tissues, which here we describe and apply to a 3-million-year-old femur from the celebrated Ethiopian skeleton, "Lucy," referred to Australopithecus afarensis. We examine two bone tissue parameters that demonstrate the potential of this technology. First, subsurface reflection images from intact bone reveal bone cell spaces, the osteocyte lacunae, whose density is demonstrated to scale negatively with body size, reflecting aspects of metabolism and organismal life history. Second, images of a naturally fractured cross section near to Lucy's femoral mid-shaft, which match in sign those of transmitted circularly polarized light, reveal relative collagen fiber orientation patterns that are an important indicator of femoral biomechanical efficacy. Preliminary results indicate that Lucy was characterized by metabolic constraints typical for a primate her body size and that in her femur she was adapted to habitual bipedalism. Limitations imposed by the transport and invasive histology of unique or rare fossils motivated development of the PCSOM so that specimens may be examined wherever and whenever nondestructive imaging is required. PMID:19191265

  2. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study.

    Fea, Antonio M; Aragno, Vittoria; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M

    2016-01-01

    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications. PMID:27200376

  3. In vivo assessment of the structure of skin microcirculation by reflectance confocal-laser-scanning microscopy

    Sugata, Keiichi; Osanai, Osamu; Kawada, Hiromitsu

    2012-02-01

    One of the major roles of the skin microcirculation is to supply oxygen and nutrition to the surrounding tissue. Regardless of the close relationship between the microcirculation and the surrounding tissue, there are few non-invasive methods that can evaluate both the microcirculation and its surrounding tissue at the same site. We visualized microcapillary plexus structures in human skin using in vivo reflectance confocal-laser-scanning microscopy (CLSM), Vivascope 3000® (Lucid Inc., USA) and Image J software (National Institutes of Health, USA) for video image processing. CLSM is a non-invasive technique that can visualize the internal structure of the skin at the cellular level. In addition to internal morphological information such as the extracellular matrix, our method reveals capillary structures up to the depth of the subpapillary plexus at the same site without the need for additional optical systems. Video images at specific depths of the inner forearm skin were recorded. By creating frame-to-frame difference images from the video images using off-line video image processing, we obtained images that emphasize the brightness depending on changes of intensity coming from the movement of blood cells. Merging images from different depths of the skin elucidates the 3-dimensional fine line-structure of the microcirculation. Overall our results show the feasibility of a non-invasive, high-resolution imaging technique to characterize the skin microcirculation and the surrounding tissue.

  4. Effect of Hypericin on Confocal Imaging of Ca2+ Signaling in Cultured Human Retinal Pigment Epithelium

    Qianying Gao; Yannian Hui; Yusheng Wang; Lin Wang

    2001-01-01

    Purpose: To investigate the mechanism of the Ca2 + signaling in cultured human retinal pigment epithelial(RPE) cells with the protein kinase C(PKC) specific inhibitor-hypericin stimulation.Methods: Cultured human RPE cells were analyzed using the fluorescence Ca2+ dye fluo-3 AM and laser scanning confocal microscope(LSCM) after stimulation with 100nM phorbol 12-myristate 13-acetate(PMA) and (or)5 concentrations of hypericin(1, 2, 3, 4 and 5 μM).Results: The normal fluorescence in RPE cells was strong and distributed throughout the cells. The nucleus appeared to be more fluorescent than the cytoplasm. After stimulation with PMA alone or 5 concentrations of hypericin, a rapid decrease in flurescence intensity was observed. There was no obvious difference in decreased curve among 5concentrations. However, after stimulation with a 24 hr preincubation of PMA and 5 concentrations of hypericin, a further decrease was not observed.Conclusion: Fluo-3 AM appears to be a good indicator of the change in Ca2+ occurring in RPE cells and hypericin is a strong inhibitor of Ca2 + influx channel. Hypericin has potential as a therapeutic drug for proliferative vitreoretinopathy(PVR), the inhibitory effect on PVR might be caused by blocking the PKC activity and inhibiting Ca2+ influxpathway.

  5. Handheld histology-equivalent sectioning laser-scanning confocal optical microscope for interventional imaging.

    Kumar, Karthik; Avritscher, Rony; Wang, Youmin; Lane, Nancy; Madoff, David C; Yu, Tse-Kuan; Uhr, Jonathan W; Zhang, Xiaojing

    2010-04-01

    A handheld, forward-imaging, laser-scanning confocal microscope (LSCM) demonstrating optical sectioning comparable with microtome slice thicknesses in conventional histology, targeted towards interventional imaging, is reported. Fast raster scanning (approximately 2.5 kHz line scan rate, 3.0-5.0 frames per second) was provided by a 2-axis microelectromechanical system (MEMS) scanning mirror fabricated by a method compatible with complementary metal-oxide-semiconductor (CMOS) processing. Cost-effective rapid-prototyped packaging combined the MEMS mirror with micro-optical components into a probe with 18 mm outer diameter and 54 mm rigid length. ZEMAX optical design simulations indicate the ability of the handheld optical system to obtain lateral resolution of 0.31 and axial resolution of 2.85 microm. Lateral and axial resolutions are experimentally measured at 0.5 microm and 4.2 microm respectively, with field of view of 200 x 125 microm. Results of reflectance imaging of ex vivo swine liver, and fluorescence imaging of the expression of cytokeratin and mammaglobin tumor biomarkers in epithelial human breast tissue from metastatic breast cancer patients are presented. The results indicate that inexpensive, portable handheld optical microscopy tools based on silicon micromirror technologies could be important in interventional imaging, complementing existing coarse-resolution techniques to improve the efficacy of disease diagnosis, image-guided excisional microsurgery, and monitored photodynamic therapy. PMID:20012209

  6. Assessment of starch-based wood adhesive quality by confocal Raman microscopic detection of reaction homogeneity.

    Wang, Panjun; Cheng, Li; Gu, Zhengbiao; Li, Zhaofeng; Hong, Yan

    2015-10-20

    Confocal Raman microscopy (CRM) was used to detect the reaction homogeneity of vinyl acetate grafted on starch granules and help to assess the quality of high solid content starch-based wood adhesive (HSSWA). Primarily, four experimental starch samples were investigated, and by analysis of band area ratio (carbonyl/carbohydrate) of each granule, information about reaction homogeneity was collected. The results showed that reaction extent and homogeneity were inconsistent for samples with different G values, and the distribution of ester groups on blend samples was much less uniform than grafted starch samples with the same G value, confirming that CRM was useful for determining the homogeneity of chemical modification. Afterwards, the technique was applied to research HSSWA prepared by two-stage seeded polymerization and traditional process. The distribution of ester groups was more uniform among starch granules prepared by former method, resulted in adhesive with much better performance, indicating that uniformity of polymerization was an important factor related to properties of starch-based wood adhesive. PMID:26256162

  7. Visualization of Golgia apparatus as an intracellular calcium store by laser scanning confocal microscope

    CUIJIE; YANLI; 等

    1995-01-01

    Using laser scanning confocal microscopy,we have found that the in cells loaded with fluo-3/AM,highest intracellular Ca2+ in the perinuclear region is associated with the Golgi apparatus.The spatiotemporal subcellular distribution of Ca2+ in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated.PDGF,which releases Ca2+ from intracellular stores by inositol(1,4,5)-trisphosphate pathway ,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the golgi apparatus.Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin,an inhibitor of the Ca2+ transport ATPase of intracellular calcium store,Permeablizing the plasma membrane by 10μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store.These results suggest that the Golgi apparatus plays a role in Ca2+ regulation in signal transduction.

  8. Differentiating gastrointestinal stromal tumors from gastric adenocarcinomas and normal mucosae using confocal Raman microspectroscopy

    Hsu, Chih-Wei; Huang, Chia-Chi; Sheu, Jeng-Horng; Lin, Chia-Wen; Lin, Lien-Fu; Jin, Jong-Shiaw; Chen, Wenlung

    2016-07-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, and gastric adenocarcinomas are a common cancer worldwide. To differentiate GISTs from adenocarcinomas is important because the surgical processes for both are different; the former excises the tumor with negative margins, while the latter requires radical gastrectomy with lymph node dissection. Endoscopy with biopsy is used to distinguish GISTs from adenocarcinomas; however, it may cause tumor bleeding in GISTs. We reported here the confocal Raman microspectroscopy as an effective tool to differentiate GISTs, adenocarcinomas, and normal mucosae. Of 119 patients enrolled in this study, 102 patients underwent gastrectomy (40 GISTs and 62 adenocarcinomas), and 17 patients with benign lesions were obtained as normal mucosae. Raman signals were integrated for 100 s for each spot on the specimen, and 5 to 10 spots, depending on the sample size, were chosen for each specimen. There were significant differences among those tissues as evidenced by different Raman signal responding to phospholipids and protein structures. The spectral data were further processed and analyzed by using principal component analysis. A two-dimensional plot demonstrated that GISTs, adenocarcinomas, and normal gastric mucosae could be effectively differentiated from each other.

  9. Demeclocycline as a contrast agent for detecting brain neoplasms using confocal microscopy

    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.

  10. A light sheet confocal microscope for image cytometry with a variable linear slit detector

    Hutcheson, Joshua A.; Khan, Foysal Z.; Powless, Amy J.; Benson, Devin; Hunter, Courtney; Fritsch, Ingrid; Muldoon, Timothy J.

    2016-03-01

    We present a light sheet confocal microscope (LSCM) capable of high-resolution imaging of cell suspensions in a microfluidic environment. In lieu of conventional pressure-driven flow or mechanical translation of the samples, we have employed a novel method of fluid transport, redox-magnetohydrodynamics (redox-MHD). This method achieves fluid motion by inducing a small current into the suspension in the presence of a magnetic field via electrodes patterned onto a silicon chip. This on-chip transportation requires no moving parts, and is coupled to the remainder of the imaging system. The microscopy system comprises a 450 nm diode 20 mW laser coupled to a single mode fiber and a cylindrical lens that converges the light sheet into the back aperture of a 10x, 0.3 NA objective lens in an epi-illumination configuration. The emission pathway contains a 150 mm tube lens that focuses the light onto the linear sensor at the conjugate image plane. The linear sensor (ELiiXA+ 8k/4k) has three lateral binning modes which enables variable detection aperture widths between 5, 10, or 20 μm, which can be used to vary axial resolution. We have demonstrated redox-MHD-enabled light sheet microscopy in suspension of fluorescent polystyrene beads. This approach has potential as a high-throughput image cytometer with myriad cellular diagnostic applications.

  11. Modeling enzymatic hydrolysis of lignocellulosic substrates using fluorescent confocal microscopy II: pretreated biomass.

    Luterbacher, Jeremy S; Moran-Mirabal, Jose M; Burkholder, Eric W; Walker, Larry P

    2015-01-01

    In this study, we extend imaging and modeling work that was done in Part I of this report for a pure cellulose substrate (filter paper) to more industrially relevant substrates (untreated and pretreated hardwood and switchgrass). Using confocal fluorescence microscopy, we are able to track both the structure of the biomass particle via its autofluorescence, and bound enzyme from a commercial cellulase cocktail supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A. Imaging was performed throughout hydrolysis at temperatures relevant to industrial processing (50°C). Enzyme bound predominantly to areas with low autofluorescence, where structure loss and lignin removal had occurred during pretreatment; this confirms the importance of these processes for successful hydrolysis. The overall shape of both untreated and pretreated hardwood and switchgrass particles showed little change during enzymatic hydrolysis beyond a drop in autofluorescence intensity. The permanence of shape along with a relatively constant bound enzyme signal throughout hydrolysis was similar to observations previously made for filter paper, and was consistent with a modeling geometry of a hollowing out cylinder with widening pores represented as infinite slits. Modeling estimates of available surface areas for pretreated biomass were consistent with previously reported experimental results. PMID:25042048

  12. Nanostructured transdermal hormone replacement therapy for relieving menopausal symptoms: a confocal Raman spectroscopy study

    Botelho, Marco Antonio; Queiroz, Dinalva Brito; Barros, Gisele; Guerreiro, Stela; Umbelino, Sonia; Lyra, Arao; Borges, Boniek; Freitas, Allan, E-mail: marcobotelho@pq.cnpq.br [Universidade Potiguar, Natal, RN (Brazil). Lab. de Nanotecnologia; Fechine, Pierre [Universidade Federal do Ceara (GQMAT/UFCE), Fortaleza, CE (Brazil). Dept. de Quimica Analitica. Grupo Avancado de Biomateriais em Quimica; Queiroz, Danilo Caldas de [Instituto Federal de Ciencia e Tecnologia (IFCT), Fortaleza, CE (Brazil). Lab. de Biotecnologia; Ruela, Ronaldo [Instituto de Biotecnologia Aplicada (INBIOS), Fortaleza, CE (Brazil); Almeida, Jackson Guedes [Universidade Federal do Vale de Sao Francisco (UNIVALE), Petrolina, PE (Brazil). Fac. de Ciencias Farmaceuticas; Quintans Junior, Lucindo [Universidade Federal de Sergipe (UFSE), Sao Cristovao, SE (Brazil). Dept. de Fisiologia

    2014-06-01

    Objective:to determine the safety and efficacy of a transdermal nanostructured formulation of progesterone (10%) combined with estriol (0.1%) + estradiol (0.25%) for relieving postmenopausal symptoms. Methods: a total of 66 postmenopausal Brazilian women with climacteric symptoms of natural menopause received transdermal nanostructured formulations of progesterone and estrogens in the forearm daily for 60 months to mimic the normal ovarian secretory pattern. Confocal Raman spectroscopy of hormones in skin layers was performed. Clinical parameters, serum concentrations of estradiol and follicle-stimulating hormone, blood pressure, BI-RADS classification from bilateral mammography, and symptomatic relief were compared between baseline and 60 months post-treatment. Clinicaltrials.gov: NCT02033512. Results: an improvement in climacteric symptoms was reported in 92.5% of women evaluated before and after 60 months of treatment. The serum concentrations of estradiol and follicle-stimulating hormone changed significantly (p<0.05) after treatment; the values of serum follicle-stimulating hormone decreased after 60 months from 82.04 ± 4.9 to 57.12 ± 4.1 IU/mL. A bilateral mammography assessment of the breasts revealed normal results in all women. No adverse health-related events were attributed to this hormone replacement therapy protocol. Conclusion: the nanostructured formulation is safe and effective in re-establishing optimal serum levels of estradiol and follicle-stimulating hormone and relieving the symptoms of menopause. This transdermal hormone replacement therapy may alleviate climacteric symptoms in postmenopausal women. (author)

  13. Resolution and signal-to-noise ratio improvement in confocal fluorescence microscopy using array detection and maximum-likelihood processing

    Kakade, Rohan; Walker, John G.; Phillips, Andrew J.

    2016-08-01

    Confocal fluorescence microscopy (CFM) is widely used in biological sciences because of its enhanced 3D resolution that allows image sectioning and removal of out-of-focus blur. This is achieved by rejection of the light outside a detection pinhole in a plane confocal with the illuminated object. In this paper, an alternative detection arrangement is examined in which the entire detection/image plane is recorded using an array detector rather than a pinhole detector. Using this recorded data an attempt is then made to recover the object from the whole set of recorded photon array data; in this paper maximum-likelihood estimation has been applied. The recovered object estimates are shown (through computer simulation) to have good resolution, image sectioning and signal-to-noise ratio compared with conventional pinhole CFM images.

  14. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

    Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

    2013-12-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. PMID:23242824

  15. Confocal and Atomic Force Microscopies of Color Centers Produced by Ultrashort Laser Irradiation in LiF Crystals

    Courrol, Lilia Coronato; Martinez, Oscar; Samad, Ricardo Elgul; Gomes, Laércio; Ranieri, Izilda Márcia; Baldochi, Sonia Licia; de Freitas, Anderson Zanardi; Junior, Nilson Dias Vieira

    2008-04-01

    We report properties of the spatial and spectral distribution of color centers produced in LiF single crystals by ultrashort high intensity laser pulses (60 fs, 10 GW) using confocal spectral microscopy and atomic force microscopy. We could identify a large amount of F centers that gave rise to aggregates such as F2, F4, F2+ and F3+ distributed in cracked shape brownish areas. We have taken a 3D image using confocal microscopy of the sample (luminescent image) and no difference is observed in the different planes. The atomic force microscopy image clearly shows the presence of defects on the modified surface. The formation of micrometer or sub-micrometer voids, filaments and void strings was observed and related to filamentation process.

  16. Dermoscopy, confocal laser microscopy, and hi-tech evaluation of vascular skin lesions: diagnostic and therapeutic perspectives.

    Grazzini, Marta; Stanganelli, Ignazio; Rossari, Susanna; Gori, Alessia; Oranges, Teresa; Longo, Anna Sara; Lotti, Torello; Bencini, Pier Luca; De Giorgi, Vincenzo

    2012-01-01

    Vascular skin lesions comprise a wide and heterogeneous group of malformations and tumors that can be correctly diagnosed based on natural history and physical examination. However, considering the high incidence of such lesions, a great number of them can be misdiagnosed. In addition, it is not so rare that an aggressive amelanotic melanoma can be misdiagnosed as a vascular lesion. In this regard, dermoscopy and confocal laser microscopy examination can play a central role in increasing the specificity of the diagnosis of such lesions. In fact, the superiority of these tools over clinical examination has encouraged dermatologists to adopt these devices for routine clinical practice, with a progressive spread of their use. In this review, we will go through the dermoscopic and the confocal laser microscopy of diagnosis of most frequent vascular lesions (i.e., hemangiomas angiokeratoma, pyogenic granuloma, angiosarcoma) taking into particular consideration the differential diagnosis with amelanotic melanoma. PMID:22950556

  17. Computation of the lateral and axial point spread functions in confocal imaging systems using binary amplitude mask

    A M Hamed

    2006-06-01

    In this paper, a novel aperture based on Tolardo concept composed of a central clear disc surrounded by a series of black and white (B/W) concentric annuli of equal transmittance is presented. Different apodized apertures of different number of B/W annuli are suggested in order to improve further the three-dimensional resolving power of confocal imaging systems. Both the axial and lateral point spread functions (PSF) and the corresponding irradiances are computed in both cases of conventional and confocal scanning microscopes for the above-mentioned amplitude filters. These results of axial and lateral irradiances are graphically represented by constructing a computer program using MATLAB. The obtained results are compared with that obtained in case of circular, annular, and Martinez-Corral apodized aperture.

  18. Fast mapping of inhomogeneities in the popular metallic perovskite Nb:SrTiO3 by confocal Raman microscopy

    Confocal Raman microscopy was applied in order to investigate the homogeneity of donor doping in Nb:SrTiO3 single crystals. Measurements of local Raman spectra revealed a systematic relation between the intensity of the Raman signal and the donor content of the crystals. We successfully elaborated a correspondence between the electronic structure and the intensity of the Raman lines using a crystal with macroscopic inhomogeneity as a demonstration sample. By mapping the distribution of the intensity of the Raman signal, we identified a characteristic inhomogeneous structure related to the presence of clusters with sizes of 5 μm to 20 μm, indicating inhomogeneous donor distribution caused by flaws introduced during crystal growth. Hence, we propose confocal Raman microscopy as a convenient technique for investigating the homogeneity and quality of doped perovskite surfaces, which are needed for various technological applications. (copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  19. The technique of detecting the laser-ultrasonic vector displacement with a confocal Fabry-Perot interferometer waves in a plate

    PAN Yongdong; QIAN Menglu

    2003-01-01

    Generally, a confocal Fabry-Perot interferometer is only able to detect the out-of-plane component of a displacement field; while the in-plane component often has the information about the material which cannot be found in this out-of-plane component. In this paper, based on a confocal Fabry-Perot interferometer set-up for detecting the out-of-plane component of a laser generated acoustic field, a technique is developed to detect both the out-of-plane and inplane displacement components simultaneously with a novel two-channel confocal Fabry-Perot interferometer.

  20. In situ analysis of electrocrystallization process of metal electrodeposition with confocal energy dispersive X-ray diffraction based on polycapillary X-ray optics

    The confocal energy dispersive X-ray diffraction (EDXRD) based on a polycapillary focusing X-ray lens (PFXRL) in excitation channel and a polycapillary parallel X-ray lens (PPXRL) in detection channel was presented to study the electrocrystallization process of metal electrodeposition. The input focal spot of the PPXRL and the output focal spot of the PFXRL was adjusted in a confocal configuration, and only the X-rays from the volume overlapped by the two foci could be accordingly detected by the detector. The experimental results demonstrated the confocal EDXRD could be used to in situ real-time analysis of electrochemical crystal growth process

  1. In situ analysis of electrocrystallization process of metal electrodeposition with confocal energy dispersive X-ray diffraction based on polycapillary X-ray optics

    Li, Fangzuo; 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: stx@bnu.edu.cn [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); Yang, Chaolin; Sun, Weiyuan; Sun, Xuepeng [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); 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)

    2015-06-11

    The confocal energy dispersive X-ray diffraction (EDXRD) based on a polycapillary focusing X-ray lens (PFXRL) in excitation channel and a polycapillary parallel X-ray lens (PPXRL) in detection channel was presented to study the electrocrystallization process of metal electrodeposition. The input focal spot of the PPXRL and the output focal spot of the PFXRL was adjusted in a confocal configuration, and only the X-rays from the volume overlapped by the two foci could be accordingly detected by the detector. The experimental results demonstrated the confocal EDXRD could be used to in situ real-time analysis of electrochemical crystal growth process.

  2. Proteasome Particle-Rich Structures Are Widely Present in Human Epithelial Neoplasms: Correlative Light, Confocal and Electron Microscopy Study

    Necchi, Vittorio; Sommi, Patrizia; Vanoli, Alessandro; Manca, Rachele; Ricci, Vittorio; Solcia, Enrico

    2011-01-01

    A novel cytoplasmic structure has been recently characterized by confocal and electron microscopy in H. pylori-infected human gastric epithelium, as an accumulation of barrel-like proteasome reactive particles colocalized with polyubiquitinated proteins, H. pylori toxins and the NOD1 receptor. This proteasome particle-rich cytoplasmic structure (PaCS), a sort of focal proteasome hyperplasia, was also detected in dysplastic cells and was found to be enriched in SHP2 and ERK proteins, known to ...

  3. Analysis of a piezoelectric screw dislocation inside an elliptical inhomogeneity with confocal rigid line in piezoelectric material

    2011-01-01

    This paper deals with the electro-elastic coupling interaction between a piezoelectric screw dislocation which is located inside the elliptical inhomogeneity and an electrically conductive confocal rigid line under remote anti-plane shear stresses and in-plane electrical loads in piezoelectric composite material. The analytical-functions of the complex potentials, stress fields and the image force acting on the piezoelectric screw dislocation are obtained based on the principle of conformal mapping, the met...

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

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

    2009-01-01

    Liesegang rings are concentric noncellular lamellar structures, occasionally found in inflammatory tissues. They have been confused with various parasites, algas, calcification, and psammoma bodies. The authors examined Liesegang rings from oral inflammatory cysts by both optical and confocal laser scanning microscopy, and perfomed a three-dimensional reconstruction. These investigations indicate that Liesegang rings are composed of multiple birefringent concentric rings, resulting from a progressive deposition of organic substances, with an unclear pathogenesis. PMID:19274580

  5. Correlation of Nitrogen Sorption and Confocal Laser Scanning Microscopy for the Analysis of Amino Group Distributions on Mesoporous Silica

    Dominik Brühwiler; Nando Gartmann

    2011-01-01

    Aminopropylalkoxysilanes are frequently used for the functionalization of mesoporous silica. The analysis of amino group distributions on arrays of silica nanochannels by a combination of nitrogen sorption and confocal laser scanning microscopy provides valuable insight into the mechanisms underlying the interaction of these silanes with mesoporous silica surfaces. Tendencies towards external surface functionalization, non-uniform distribution in the pores, and hydrolysis of the silica framew...

  6. Correlation of nitrogen sorption and confocal laser scanning microscopy for the analysis of amino group distributions on mesoporous silica

    Gartmann, N.; Brühwiler, D.

    2011-01-01

    Aminopropylalkoxysilanes are frequently used for the functionalization of mesoporous silica. The analysis of amino group distributions on arrays of silica nanochannels by a combination of nitrogen sorption and confocal laser scanning microscopy provides valuable insight into the mechanisms underlying the interaction of these silanes with mesoporous silica surfaces. Tendencies towards external surface functionalization, non-uniform distribution in the pores, and hydrolysis of the silica framew...

  7. Large congenital nevus spilus—improved follow-up through the use of in vivo reflectance confocal microscopy

    Prodinger, Christine; Tatarski, Rafaella; Laimer, Martin; Ahlgrimm-Siess, Verena

    2013-01-01

    Background: Nevus spilus (NS) is a potential precursor of melanoma; the vast majority of cases reported in the literature were histologically classified as superficial spreading melanoma. Objective: To demonstrate the diagnostic value of reflectance confocal microscopy (RCM) in this subtype of congenital nevi. Methods: We report a case of a large congenital NS with equivocal clinical and dermoscopic findings in which RCM was applied for diagnosis and follow-up. Results: There was a good corre...

  8. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J.; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-01-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratiti...

  9. OBSERVATION OF THE ALTERNATION OF NUCLEIC ACID IN BRAIN SLICE AND NEURONS BY CONFOCAL LASER SCANNING MICROSCOPY

    2000-01-01

    @@Confocal laser scanning microscope is one of the most important biomedicine Altus instru ment〔1〕. It has the characteristics of high sensitivity for detecting the stereo structure, and can scan a few hundreds of micrometer-thick tissue. It may get graphs of intracyte or tissue with uninvading stage scan and is named "cell CT". In this study, the nucleic acid alterations of whole brain slice was investigated with this technique after the formation of LTP.

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

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

    2013-01-01

    International audience 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...

  11. Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope.

    Trepte, O; Rokahr, I; Andersson-Engels, S; Carlsson, K

    1994-12-01

    A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/delta lambda, ranges from 350 at lambda = 400 nm to 100 at lambda = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7869364

  12. Nanoscale residual stress-field mappingaround nanoindents in SiCby IR s-SNOM and confocal Raman microscopy.

    Gigler, Alexander M; Huber, Andreas J; Bauer, Michael; Ziegler, Alexander; Hillenbrand, Rainer; Stark, Robert W

    2009-12-01

    We map a nanoindent in a silicon carbide (SiC) crystal by infrared (IR) scattering-type scanning near-field optical microscopy (s-SNOM) and confocal Raman microscopy and interpret the resulting images in terms of local residual stress-fields. By comparing near-field IR and confocal Raman images, we find that the stress-induced shifts of the longitudinal optical phonon-frequencies (LO) and the related shift of the phonon-polariton near-field resonance give rise to Raman and s-SNOM image contrasts, respectively. We apply single-frequency IR s-SNOM for nanoscale resolved imaging of local stress-fields and confocal Raman microscopy to obtain the complete spectral information about stress-induced shifts of the phonon frequencies at diffraction limited spatial resolution. The spatial extension of the local stress-field around the nanoindent agrees well between both techniques. Our results demonstrate that both methods ideally complement each other, allowing for the detailed analysis of stress-fields at e.g. material and grain boundaries, in Micro-Electro-Mechanical-Systems (MEMS), or in engineered nanostructures. PMID:20052158

  13. Development of confocal 3D X-ray fluorescence instrument and its applications to micro depth profiling

    We have developed a confocal micro X-ray fluorescence instrument. Two independent X-ray tubes of Mo and Cr targets were installed to this instrument. Two polycapillary full X-ray lenses were attached to two X-ray tubes, and a polycapillary half X-ray lens was also attached to the X-ray detector (silicon drift detector, SDD). Finally, three focus spots of three lenses were adjusted at a common position. By using this confocal micro X-ray fluorescence instrument, depth profiling for layered samples were performed. It was found that depth resolution depended on energy of X-ray fluorescence that was measured. In addition, X-ray elemental maps were determined at different depths for an agar sample including metal fragments of Cu, Ti and Au. The elemental maps showed actual distributions of metal fragments in the agar, indicating that the confocal micro X-ray fluorescence is a feasible technique for non-destructive depth analysis and 3D X-ray fluorescence analysis. (author)

  14. Hybrid detectors improved time-lapse confocal microscopy of PML and 53BP1 nuclear body colocalization in DNA lesions.

    Foltánková, Veronika; Matula, Pavel; Sorokin, Dmitry; Kozubek, Stanislav; Bártová, Eva

    2013-04-01

    We used hybrid detectors (HyDs) to monitor the trajectories and interactions of promyelocytic leukemia (GFP-PML) nuclear bodies (NBs) and mCherry-53BP1-positive DNA lesions. 53BP1 protein accumulates in NBs that occur spontaneously in the genome or in γ-irradiation-induced foci. When we induced local DNA damage by ultraviolet irradiation, we also observed accumulation of 53BP1 proteins into discrete bodies, instead of the expected dispersed pattern. In comparison with photomultiplier tubes, which are used for standard analysis by confocal laser scanning microscopy, HyDs significantly eliminated photobleaching of GFP and mCherry fluorochromes during image acquisition. The low laser intensities used for HyD-based confocal analysis enabled us to observe NBs for the longer time periods, necessary for studies of the trajectories and interactions of PML and 53BP1 NBs. To further characterize protein interactions, we used resonance scanning and a novel bioinformatics approach to register and analyze the movements of individual PML and 53BP1 NBs. The combination of improved HyD-based confocal microscopy with a tailored bioinformatics approach enabled us to reveal damage-specific properties of PML and 53BP1 NBs. PMID:23410959

  15. Femtosecond laser subsurface scleral treatment in cadaver human sclera and evaluation using two-photon and confocal microscopy

    Sun, Hui; Fan, Zhongwei; Yan, Ying; Lian, Fuqiang; Kurtz, Ron; Juhasz, Tibor

    2016-03-01

    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial-thickness drainage channels can be created with femtosecond laser in the translucent sclera for the potential treatment of glaucoma. We demonstrate the creation of partial-thickness subsurface drainage channels with the femtosecond laser in the cadaver human eyeballs and describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. A femtosecond laser operating at a wavelength of 1700 nm was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of cadaver human eyes. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such partial thickness subsurface scleral channels. Our studies suggest that the confocal and two-photon microscopy can be used to investigate femtosecond-laser created partial-thickness drainage channels in the sclera of cadaver human eyes.

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

    Alexandrov, Theodore; Lasch, Peter

    2013-06-18

    Over the past decade, confocal Raman microspectroscopic (CRM) imaging has matured into a useful analytical tool to obtain spatially resolved chemical information on the molecular composition of biological samples and has found its way into histopathology, cytology, and microbiology. A CRM imaging data set is a hyperspectral image in which Raman intensities are represented as a function of three coordinates: a spectral coordinate λ encoding the wavelength and two spatial coordinates x and y. Understanding CRM imaging data is challenging because of its complexity, size, and moderate signal-to-noise ratio. Spatial segmentation of CRM imaging data is a way to reveal regions of interest and is traditionally performed using nonsupervised clustering which relies on spectral domain-only information with the main drawback being the high sensitivity to noise. We present a new pipeline for spatial segmentation of CRM imaging data which combines preprocessing in the spectral and spatial domains with k-means clustering. Its core is the preprocessing routine in the spatial domain, edge-preserving denoising (EPD), which exploits the spatial relationships between Raman intensities acquired at neighboring pixels. Additionally, we propose to use both spatial correlation to identify Raman spectral features colocalized with defined spatial regions and confidence maps to assess the quality of spatial segmentation. For CRM data acquired from midsagittal Syrian hamster ( Mesocricetus auratus ) brain cryosections, we show how our pipeline benefits from the complex spatial-spectral relationships inherent in the CRM imaging data. EPD significantly improves the quality of spatial segmentation that allows us to extract the underlying structural and compositional information contained in the Raman microspectra. PMID:23701523

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

    Rodighiero, Simona

    2015-03-22

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

  18. In vivo probe-based confocal laser endomicroscopy in amiodarone-related pneumonia.

    Salaün, Mathieu; Roussel, Francis; Bourg-Heckly, Geneviève; Vever-Bizet, Christine; Dominique, Stéphane; Genevois, Anne; Jounieaux, Vincent; Zalcman, Gérard; Bergot, Emmanuel; Vergnon, Jean-Michel; Thiberville, Luc

    2013-12-01

    Probe-based confocal laser endomicroscopy (pCLE) allows microscopic imaging of the alveoli during bronchoscopy. The objective of the study was to assess the diagnostic accuracy of pCLE for amiodarone-related pneumonia (AMR-IP). Alveolar pCLE was performed in 36 nonsmoking patients, including 33 consecutive patients with acute or subacute interstitial lung disease (ILD), of which 17 were undergoing treatment with amiodarone, and three were amiodarone-treated patients without ILD. Nine out of 17 patients were diagnosed with high-probability AMR-IP (HP-AMR-IP) by four experts, and three separate observers. Bronchoalveolar lavage findings did not differ between HP-AMR-IP and low-probability AMR-IP (LP-AMR-IP) patients. In HP-AMR-IP patients, pCLE showed large (>20 μm) and strongly fluorescent cells in 32 out of 38 alveolar areas. In contrast, these cells were observed in only two out of 39 areas from LP-AMR-IP patients, in one out of 59 areas from ILD patients not receiving amiodarone and in none of the 10 areas from amiodarone-treated patients without ILD (p<0.001; HP-AMR-IP versus other groups). The presence of at least one alveolar area with large and fluorescent cells had a sensitivity, specificity, negative predictive value and positive predictive value for the diagnosis of AMR-IP of 100%, 88%, 100% and 90%, respectively. In conclusion, pCLE appears to be a valuable tool for the in vivo diagnosis of AMR-IP in subacute ILD patients. PMID:23018901

  19. Automatic classification of small bowel mucosa alterations in celiac disease for confocal laser endomicroscopy

    Boschetto, Davide; Di Claudio, Gianluca; Mirzaei, Hadis; Leong, Rupert; Grisan, Enrico

    2016-03-01

    Celiac disease (CD) is an immune-mediated enteropathy triggered by exposure to gluten and similar proteins, affecting genetically susceptible persons, increasing their risk of different complications. Small bowels mucosa damage due to CD involves various degrees of endoscopically relevant lesions, which are not easily recognized: their overall sensitivity and positive predictive values are poor even when zoom-endoscopy is used. Confocal Laser Endomicroscopy (CLE) allows skilled and trained experts to qualitative evaluate mucosa alteration such as a decrease in goblet cells density, presence of villous atrophy or crypt hypertrophy. We present a method for automatically classifying CLE images into three different classes: normal regions, villous atrophy and crypt hypertrophy. This classification is performed after a features selection process, in which four features are extracted from each image, through the application of homomorphic filtering and border identification through Canny and Sobel operators. Three different classifiers have been tested on a dataset of 67 different images labeled by experts in three classes (normal, VA and CH): linear approach, Naive-Bayes quadratic approach and a standard quadratic analysis, all validated with a ten-fold cross validation. Linear classification achieves 82.09% accuracy (class accuracies: 90.32% for normal villi, 82.35% for VA and 68.42% for CH, sensitivity: 0.68, specificity 1.00), Naive Bayes analysis returns 83.58% accuracy (90.32% for normal villi, 70.59% for VA and 84.21% for CH, sensitivity: 0.84 specificity: 0.92), while the quadratic analysis achieves a final accuracy of 94.03% (96.77% accuracy for normal villi, 94.12% for VA and 89.47% for CH, sensitivity: 0.89, specificity: 0.98).

  20. Waterproofing in Arabidopsis: Following phenolics and lipids in situ by Confocal Raman Microscopy

    Batirtze ePrats Mateu

    2016-02-01

    Full Text Available Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299 cm-1, 1600 cm-1 and 3400 cm-1 band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobising changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol and phenolic acids were acquired. Keywords: Arabidopsis thaliana, lignin, cutin, wax, Raman

  1. On the confocal images and the rheology of whey protein isolated and modified pectins associated complex.

    Lutz, Rachel; Aserin, Abraham; Portnoy, Yariv; Gottlieb, Moshe; Garti, Nissim

    2009-02-15

    The conditions necessary to form an associated complex between whey protein isolate (WPI) and enzymatically modified pectin in water, at pH values above the isoelectric point of the protein, have been documented. The existence of the complex is not easily verified and its characterization in solution is even more complicated, since the structure is an intermediate entity between the non-interacting, incompatible aqueous soluble mixture of the biopolymers, and a strongly interacting coacervated precipitating complex. Evidence for the formation of this associated complex is provided from confocal laser scanning microscope images and rheological behavior of the aqueous mixtures. The associated complex is characterized by small fluorescent "patches" interpreted as small aggregates. The viscosity of this solution is greater than that of its individual biopolymer constituents, indicating a synergy of attractive interactions that occurs in the solution. While individually, the pectin and the WPI solutions at the studied range of concentrations exhibit moderately non-Newtonian behavior, at specific weight ratios, mixtures of the two behave either as highly entangled polymeric structures or as weak gels. The values of the storage modulus G' are equal to or greater than those of the loss modulus G''. We conclude that the associated complexes are formed at pH 6, and at 4 wt% WPI with a pectin concentration ranging from 0.1 to 0.75 wt%. The influence of the charge distribution (degree of order of the carboxylic groups) of pectin on the associated complex was also investigated, and it was found that the more "ordered" pectin (U63) favors the formation of the associated soluble complex. PMID:19070469

  2. Waterproofing in Arabidopsis: Following phenolics and lipids in situ by Confocal Raman Microscopy

    Prats Mateu, Batirtze; Hauser, Marie-Theres; Heredia, Antonio; Gierlinger, Notburga

    2016-02-01

    Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm) at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299 cm-1, 1600 cm-1 and 3400 cm-1 band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobising changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum) as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol) and phenolic acids were acquired. Keywords: Arabidopsis thaliana, lignin, cutin, wax, Raman, cuticle, waterproofing

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

    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.

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

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

    2015-07-13

    The properties of paper sheets can be tuned by adjusting the surface or bulk chemistry using functional polymers that are applied during (online) or after (offline) papermaking processes. In particular, polymers are widely used to enhance the mechanical strength of the wet state of paper sheets. However, the mechanical strength depends not only on the chemical nature of the polymeric additives but also on the distribution of the polymer on and in the lignocellulosic paper. Here, we analyze the photochemical attachment and distribution of hydrophilic polydimethylacrylamide-co-methacrylate-benzophenone P(DMAA-co-MABP) copolymers with defined amounts of photoreactive benzophenone moieties in model paper sheets. Raman microscopy was used for the unambiguous identification of P(DMAA-co-MABP) and cellulose specific bands and thus the copolymer distribution within the cellulose matrix. Two-dimensional Raman spectral maps at the intersections of overlapping cellulose fibers document that the macromolecules only partially surround the cellulose fibers, favor to attach to the fiber surface, and connect the cellulose fibers at crossings. Moreover, the copolymer appears to accumulate preferentially in holes, vacancies, and dips on the cellulose fiber surface. Correlative brightfield, Raman, and confocal laser scanning microscopy finally reveal a reticular three-dimensional distribution of the polymer and show that the polymer is predominately deposited in regions of high capillarity (i.e., in proximity to fine cellulose fibrils). These data provide deeper insights into the effects of paper functionalization with a copolymer and aid in understanding how these agents ultimately influence the local and overall properties of paper. PMID:26101966

  5. Expression of GFP in tumor cells and fluorescent examination by confocal microscope

    Jin, Ying; Xing, Da; Xu, Chaoyang

    2002-04-01

    The green fluorescent protein (GFP), from the bioluminescent jellyfish Aequorea victoria, yields a bright green fluorescence when expressed in either eukaryotic or prokaryotic cells and illuminated by blue or UV light. The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the plasmid EGFP encoding GFP was used to transfect SWO cells (a cancer cell line of nerve gelatinous tissue) mediated by liposome: (1) The plasmid EGFP-C1, purchased from Clontech Co., propagated in suitable E. coli strain (JM 109), was extracted by Concert High Purity Plasmid Miniprep (Gibco). (2) SWO was cultured in RPMI 1640 (10% FCS and 25 mM HEPES), 37 degree(s)C, 5% CO2. Cancer cells were transfected in 6-cm tissue culture dishes by Lipofectin Reagent (Gibco) for 6-12 hr using 2 ug DNA. (3) Then, infected cells were collected in medium containing 800 ug/ml G418, and the resistant clones were harvested and subcloned in fresh culture medium maintaining 800 ug/ml G418. (4) The cells were examined by using Nikon fluorescent microscope (E600) and Bio-Rad confocal microscope (MRC 600). (5) Next step, the cancer cells, stably expressing GFP after in vivo transduction, were implanted by surgical orthotopic implantation (SOI) in nude mice. Tracking of these cancer cells will become more sensitive and rapid than the traditional procedure of histopathological examination or immunohistochemistry. This method demonstrates external, noninvasive, whole-body, real-time fluorescence optical imaging of internally growing tumors and metastases in transplanted animals.

  6. Confocal fluorescence anisotropy and FRAP imaging of α-synuclein amyloid aggregates in living cells.

    M Julia Roberti

    Full Text Available We assessed the intracellular association states of the Parkinson's disease related protein α-synuclein (AS in living cells by transfection with a functional recombinant mutant protein (AS-C4 bearing a tetracysteine tag binding the fluorogenic biarsenical ligands FlAsH and ReAsH, The aggregation states of AS-C4 were assessed by in situ microscopy of molecular translational mobility with FRAP (fluorescence recovery after photobleaching and of local molecular density with confocal fluorescence anisotropy (CFA. FRAP recovery was quantitative and rapid in regions of free protein, whereas AS in larger aggregates was>80% immobile. A small 16% recovery characterized by an apparent diffusion constant of 0.03-0.04 µm(2/s was attributed to the dynamics of smaller, associated forms of AS-C4 and the exchange of mobile species with the larger immobile aggregates. By CFA, the larger aggregates exhibited high brightness and very low anisotropy, consistent with homoFRET between closely packed AS, for which a Förster distance (R(o of 5.3 nm was calculated. Other bright regions had high anisotropy values, close to that of monomeric AS, and indicative of membrane-associated protein with both low mobility and low degree of association. The anisotropy-fluorescence intensity correlations also revealed regions of free protein or of small aggregates, undetectable by conventional fluorescence imaging alone. The combined strategy (FRAP+CFA provides a highly sensitive means for elucidating both the dynamics and structural features of protein aggregates and other intracellular complexes in living cells, and can be extended to other amyloid systems and to drug screening protocols.

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

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

    2015-06-01

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. PMID:25810353

  8. Confocal fluorometer for diffusion tracking in 3D engineered tissue constructs

    Daly, D.; Zilioli, A.; Tan, N.; Buttenschoen, K.; Chikkanna, B.; Reynolds, J.; Marsden, B.; Hughes, C.

    2016-03-01

    We present results of the development of a non-contacting instrument, called fScan, based on scanning confocal fluorometry for assessing the diffusion of materials through a tissue matrix. There are many areas in healthcare diagnostics and screening where it is now widely accepted that the need for new quantitative monitoring technologies is a major pinch point in patient diagnostics and in vitro testing. With the increasing need to interpret 3D responses this commonly involves the need to track the diffusion of compounds, pharma-active species and cells through a 3D matrix of tissue. Methods are available but to support the advances that are currently only promised, this monitoring needs to be real-time, non-invasive, and economical. At the moment commercial meters tend to be invasive and usually require a sample of the medium to be removed and processed prior to testing. This methodology clearly has a number of significant disadvantages. fScan combines a fiber based optical arrangement with a compact, free space optical front end that has been integrated so that the sample's diffusion can be measured without interference. This architecture is particularly important due to the "wet" nature of the samples. fScan is designed to measure constructs located within standard well plates and a 2-D motion stage locates the required sample with respect to the measurement system. Results are presented that show how the meter has been used to evaluate movements of samples through collagen constructs in situ without disturbing their kinetic characteristics. These kinetics were little understood prior to these measurements.

  9. Deep high-resolution fluorescence microscopy of full organs: the benefit of ultraminiature confocal miniprobes

    Schwarz, France; Le Nevez, Arnaud; Genet, Magalie; Osdoit, Anne; Lacombe, François

    2009-02-01

    Background: Confocal Laser Endomicroscopy (CLE) based on ultraminiature miniprobes (Cellvizio®, Mauna Kea Technologies, Paris, France) is able to image the inner microstructure of retroperitoneal full organs punctured during EUS-FNA procedures, such as pancreas, liver or lymph nodes. Therefore, pCLE can provide an easy-to-use and precise adjunct tool to ultrasonographic interventions in order to target suspicious areas for biopsies in EUS-FNA. Material and Methods: Probe-based CLE (pCLE) was performed on ex-vivo surgically resected specimens after topical application of fluorophores in standard 19G and 22G needles. Two prototype miniprobes ("S-probe" 300 microns diameter, field of view 400*280 microns, and "S-probe" 650 microns diameter, field of view 500*600 microns) were then inserted into the needles and enabled visualization of the inner microstructures of uterus, lung, kidney, stomach and esophagus, in both healthy and cancerous conditions. Then, pCLE was performed in-vivo on four pigs during three NOTES and one EUS-FNA procedures after intravenous injection of 2-7mL fluorescein 1-10% using the prototype "S-probe" 350 microns diameter inserted in 19G FNA needles. Liver, pancreas and spleen were imaged. Results: During the ex-vivo experiments, pCLE made it possible to distinguish microstructures, such as alveoli and macrophages in the lungs. During the in-vivo experiments, Cellvizio® video sequences showed hepatic lobules and the portal vein in the liver, and red and white pulp in the spleen. Conclusion: pCLE provides in vivo cellular information about full organs. It has the potential to help target biopsies during EUSFNA, which suffers from a high rate of false negatives, thus increasing its sensitivity.

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

    Montereali, R.M.; Bigotta, S.; Pace, A.; Piccinini, M. [ENEA, Divisione Fisica Applicata, Centro Ricerche Frascati, Frascati, RM (Italy); Burattini, E.; Grilli, A.; Raco, A. [Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Fisica, Frascati, Rome (Italy); Giammatteo, M. [Unita' Istituto Nazionale di Fisica Nucleare, Frascati, RM (Italy)]|[L' Aquila Univ., L' Aquila (Italy). Centro di Microscopia Elettronica; Picozzi, P.; Santucci, S. [Unita' Istituto Nazionale di Fisica Nucleare, Frascati, RM (Italy)]|[L' Aquila Univ., L' Aquila (Italy). Dipt. di Fisica

    2000-07-01

    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 F{sub 3}{sup +} and F{sub 2} aggregated defects appears restricted to the electron penetration and proportional to their energy depth profile, as obtained from Monte Carlo simulations. [Italian] L'irraggiamento con elettroni di bassa energia del fluoruro di litio (LiF), in forma di cristalli e film, induce la formazione di difetti primari F e centri di colore aggregati stabili in un sottile strato localizzato alla superficie del materiale investigato. Per la prima volta un microscopio confocale a scansione (CLSM) in modalita' fluorescenza e' stato usato per ricostruire la distribuzione di centri di colore laser attivi ad alta efficienza di emissione nel visibile, in strisce colorate ottenute con elettroni da 12 e 16 keV su film di LiF evaporati termicamente su vetro. La formazione dei difetti aggregati F2 e F3+ risulta ristretta spazialmente nella regione di penetrazione degli elettroni e proporzionale al profilo della distribuzione dell'energia da essi depositata, ricavata tramite simulazioni Monte Carlo.

  11. Integrated fingerprint and high wavenumber confocal Raman spectroscopy for in vivo diagnosis of cervical precancer

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

    2013-03-01

    Raman spectroscopy is a vibrational spectroscopic technique capable of optically probing the compositional, conformational, and structural changes in the tissue associated with disease progression. The main goal of this work is to develop an integrated fingerprint (FP) and high wavenumber (HW) in vivo confocal Raman spectroscopy for simultaneous FP/HW tissue Raman spectral measurements. This work further explores the potential of integrated FP/HW Raman spectroscopy developed as a diagnostic tool for in vivo detection of cervical precancer. A total of 473 in vivo integrated FP/HW Raman spectra (340 normal and 133 precancer) were acquired from 35 patients within 1 s during clinical colposcopy. The major tissue Raman peaks are noticed around 854, 937, 1001, 1095, 1253, 1313, 1445, 1654, 2946 and 3400 cm-1, related to the molecular changes (e.g., proteins, lipids, glycogen, nucleic acids, water, etc.) that accompany the dysplastic transformation of tissue. The FP (800 - 1800 cm-1), HW (2800 - 3800 cm-1) and the integrated FP/HW Raman spectra were analyzed using partial least squares-discriminant analysis (PLS-DA) together with the leave-one patient-out, cross-validation. The developed PLS-DA classification models and receiver operating characteristics (ROC) curves for the FP, HW and integrated FP/HW spectroscopy further discloses that the performance of integrated FP/HW Raman spectroscopy is superior to that of all others in discriminating the dysplastic cervix. The results of this work indicate that the co-contributions of underlying rich biochemical information revealed by the complementary spectral modalities (FP and HW Raman) can improve the in vivo early diagnosis of cervical precancer at clinical colposcopy

  12. In vivo detection of mucosal healing-involved histiocytes by confocal laser endomicroscopy

    Gheorghe Hundorfean; Abbas Agaimy; Mircea T Chiriac; Walter Geiβd(o)rfer; Jochen Wacker; Markus F Neurath; Jonas Mudter

    2012-01-01

    Histiocytes have a pivotal role in wound repair and intestinal epithelial recovery-the most important goal to sustain gut functionality.Yet,an in vivo description of colonic histiocytes by confocal laser endomicroscopy (CLE) is missing.Here,we report the case of a 45-yearsold male patient who was referred to our clinic with weight loss and a history of two consecutive Clostridium difficile colitis episodes,the latter cured 3 wk before present admission.Stool microbiology was negative.Conventional colonoscopy showed atrophy and a light mucosal oedema in the distal colon.During on-going endoscopy,we performed a fluorescein-aided CLE which revealed large polygonal (histiocytes-like) cells with copious cytoplasm and large nuclei in the lamina propria of the sigmoid colon as well as regenerative epithelial changes.Histopathological assessment of biopsies from the same areas confirmed the endomicroscopical findings:Periodic acid-Schiff-and CD68-positive foamy histiocytes in the colonic lamina propria and an advanced epithelial recovery.Since stool microbiology was repeatedly negative and polymerase chain reaction-analysis from colonic biopsies could not detect any mRNA for Thropheryma whippleii and common pathogens,we interpreted this particular setting as a mucosal healing process after consecutive Clostridium difficile infections.In conclusion,by describing these colonic histiocytes,we highlight the clinical usefulness of CLE in describing the entity of histiocytes in vivo and in real-time during the process of post-infectious mucosal healing in the colon.

  13. The reflectance confocal microscopy features of sebaceous adenoma in a case of Muir Torre syndrome

    Esma İnan Yüksel

    2015-03-01

    Full Text Available Muir-Torre syndrome (MTS is a rare autosomal dominant genodermatosis characterized by the occurrence of sebaceous gland neoplasms and/or keratoacanthomas associated with visceral malignancies. It is considered as a subtype of hereditary nonpolyposis colorectal cancer syndrome. Characteristic sebaceous gland neoplasms include sebaceous adenoma, sebaceous carcinoma, sebaceoma, and keratoacanthoma with sebaceous differentiation. The most common visceral malignancies are colorectal and genitourinary tumors. CASE: A 47year-old male patient admitted to our clinic complaining of two lesions on the nose. Dermatological examination revealed a plaque in 1 cm diameter consisting of bright yellowish-white coloured papules with slightly umblicated appearance and telangiectasias on the left site of the nose and had a dome shaped papule in 3 mm diameter with hyperkeratotic plug on the tip of the nose. He had personal history of partial colon resection because of colon cancer and familial Lynch 2 syndrome. On dermoscopic examination of sebaceous adenoma, a few yellow comedo-like globules and branching arborizing vessels were detected. Reflectance confocal microscopy (RCM revealed a good histopathologic correlation. Sebaceous lobules were composed by clusters of ovoid cells with hyporefractile dark nuclei and bright, hyperrefractile glistening cytoplasm. Numerous roundish to ovoid dark spaces corresponding to sebaceous ducts were detected. The diagnosis of MTS was established based on the personal and family history, dermoscopic, RCM and histopathologic findings. CONCLUSIONS: MTS evaluation is required in patients with biopsy-proven sebaceous adenoma. Early diagnosis may be lifesaving in patients with MTS. A better characterization of RCM features of sebaceous tumors will allow early diagnosis of the patients with MTS.

  14. A prospective cohort study: probe based confocal laser endomicroscopy for peripheral pulmonary lesions (Conference Presentation)

    Matsumoto, Yuji; Izumo, Takehiro; Hiraishi, Yoshihisa; Tsuchida, Takaaki

    2016-03-01

    Introduction: The diagnostic value of bronchoscopy for peripheral pulmonary lesions (PPLs) has improved since the application of radial endobronchial ultrasound (R-EBUS). Though R-EBUS indicates the position of the PPL, there is often a discrepancy between the obtained R-EBUS image and the diagnostic outcome. Meanwhile, probe based confocal laser endomicroscopy (pCLE) is a novel technique which provides in vivo real-time image of the contacted surface structures. However, its findings have not been established yet. Methods: Consecutive patients who have underwent bronchoscopy for PPLs were prospectively enrolled. R-EBUS with a guide sheath (GS) was inserted to the target PPL under X-ray fluoroscopic guidance. When an adequate R-EBUS image (within or adjacent to) was obtained, pCLE was sequentially inserted through the GS. Then pCLE image was scanned and biopsy was performed where an abnormal finding was estimated. The pCLE findings of PPLs and the background were recorded and analyzed exploratorily. Results: We analyzed 19 cases that we could get appropriate tissues. In all cases, bronchial walls showed longitudinal elastic fibers whereas alveolar walls formed grid-like elastic fiber networks. Conversely, discontinuous, crushed or aggregated alveolar structures accompanied by thickened and distorted fibers were detected in PPLs. Some cases showed dark hollow with fragmented or granular fluorescence. On the other hand, 11 cases (57.9%) indicated normal elastic fibers and needed the position change (3 cases; approached other bronchus, 6 cases; adjusted the position, 2 cases; penetrated the covered bronchial wall). Conclusion: The pCLE has a potential to improve the efficacy of diagnostic bronchoscopy for PPLs.

  15. A deep view in cultural heritage - confocal micro X-ray spectroscopy for depth resolved elemental analysis

    Quantitative X-ray fluorescence (XRF) and particle induced X-ray emission (PIXE) techniques have been developed mostly for the elemental analysis of homogeneous bulk or very simple layered materials. Further on, the microprobe version of both techniques is applied for 2D elemental mapping of surface heterogeneities. At typical XRF/PIXE fixed geometries and exciting energies (15-25 keV and 2-3 MeV, respectively), the analytical signal (characteristic X-ray radiation) emanates from a variable but rather extended depth within the analyzed material, according to the exciting probe energy, set-up geometry, specimen matrix composition and analyte. Consequently, the in-depth resolution offered by XRF and PIXE techniques is rather limited for the characterization of materials with micrometer-scale stratigraphy or 3D heterogeneous structures. This difficulty has been over-passed to some extent in the case of an X-ray or charged particle microprobe by creating the so-called confocal geometry. The field of view of the X-ray spectrometer is spatially restricted by a polycapillary X-ray lens within a sensitive microvolume formed by the two inter-sectioned focal regions. The precise scanning of the analyzed specimen through the confocal microvolume results in depth-sensitive measurements, whereas the additional 2D scanning microprobe possibilities render to element-specific 3D spatial resolution (3D micro-XRF and 3D micro-PIXE). These developments have contributed since 2003 to a variety of fields of applications in environmental, material and life sciences. In contrast to other elemental imaging methods, no size restriction of the objects investigated and the non-destructive character of analysis have been found indispensable for cultural heritage (CH) related applications. The review presents a summary of the experimental set-up developments at synchrotron radiation beamlines, particle accelerators and desktop spectrometers that have driven methodological developments and

  16. Design and initial development of 1 KW pulsed S-band solid state power amplifier

    We present design and initial developmental work for 1kW S-Band pulsed amplifier. This amplifier will be used for driving high power klystrons to be used in proposed accelerator test stand at RRCAT. The design is based on Class C pulsed power transistors to achieve higher efficiency. Proposed amplifier will provide ∼ 1kW of pulsed power with 10 us pulsed width and 300 Hz repetition rate. To achieve 1kW of output power, four 300W Class C transistors are combined using Wilkinson Power divider and combiners. Microstrip based Wilkinson power divider and combiners have been developed and tested, high power transistors have been ordered and are expected to arrive soon. (author)

  17. 1KW Power Transmission Using Wireless Acoustic-Electric Feed-Through (WAEF)

    Sherrit, S.; Bao, X.; Badescu, M.; Aldrich, J.; Bar-Cohen, Y.; Biederman, W.

    2008-01-01

    A variety of space applications require the delivery of power into sealed structures. Since the structural integrity can be degraded by holes for cabling we present an alternative method of delivering power and information using stress waves to the internal space of a sealed structure. One particular application of this technology is in sample return missions where it is critical to preserve the sample integrity and to prevent earth contamination. Therefore, the container has to be hermetically sealed and the integrity of the seal must be monitored in order to insure to a high degree of reliability the integrity of the sample return vessel. In this study we investigated the use of piezoelectric acoustic-electric power feed-through devices to transfer electric power wirelessly through a solid wall by using elastic or acoustic waves. The technology is applicable to a range of space and terrestrial applications where power is required by electronic equipment inside sealed containers, vacuum or pressure vessels, etc., where holes in the wall are prohibitive or may result in significant structural performance degradation or unnecessarily complex designs. To meet requirements of higher power applications, the feasibility to transfer kilowatts level power was investigated. Pre-stressed longitudinal piezoelectric feed-through devices were analyzed by finite element models and an equivalent circuit model was developed to predict the power transfer characteristics to different electric loads. Based on the results of the analysis a prototype device was designed, fabricated and a demonstration of the transmission of electric power up to 1.068-kW was successfully conducted. Efficiencies in the 80-90% range were also demonstrated and methods to increase the efficiency further are currently being considered.

  18. Confocal analysis of nervous system architecture in direct-developing juveniles of Neanthes arenaceodentata (Annelida, Nereididae

    Jacobs David K

    2010-06-01

    Full Text Available Abstract Background Members of Family Nereididae have complex neural morphology exemplary of errant polychaetes and are leading research models in the investigation of annelid nervous systems. However, few studies focus on the development of their nervous system morphology. Such data are particularly relevant today, as nereidids are the subjects of a growing body of "evo-devo" work concerning bilaterian nervous systems, and detailed knowledge of their developing neuroanatomy facilitates the interpretation of gene expression analyses. In addition, new data are needed to resolve discrepancies between classic studies of nereidid neuroanatomy. We present a neuroanatomical overview based on acetylated α-tubulin labeling and confocal microscopy for post-embryonic stages of Neanthes arenaceodentata, a direct-developing nereidid. Results At hatching (2-3 chaetigers, the nervous system has developed much of the complexity of the adult (large brain, circumesophageal connectives, nerve cords, segmental nerves, and the stomatogastric nervous system is partially formed. By the 5-chaetiger stage, the cephalic appendages and anal cirri are well innervated and have clear connections to the central nervous system. Within one week of hatching (9-chaetigers, cephalic sensory structures (e.g., nuchal organs, Langdon's organs and brain substructures (e.g., corpora pedunculata, stomatogastric ganglia are clearly differentiated. Additionally, the segmental-nerve architecture (including interconnections matches descriptions of other, adult nereidids, and the pharynx has developed longitudinal nerves, nerve rings, and ganglia. All central roots of the stomatogastric nervous system are distinguishable in 12-chaetiger juveniles. Evidence was also found for two previously undescribed peripheral nerve interconnections and aspects of parapodial muscle innervation. Conclusions N. arenaceodentata has apparently lost all essential trochophore characteristics typical of

  19. Computer Aided Diagnosis for Confocal Laser Endomicroscopy in Advanced Colorectal Adenocarcinoma

    Ştefănescu, Daniela; Streba, Costin; Cârţână, Elena Tatiana; Săftoiu, Adrian; Gruionu, Gabriel; Gruionu, Lucian Gheorghe

    2016-01-01

    Introduction Confocal laser endomicroscopy (CLE) is becoming a popular method for optical biopsy of digestive mucosa for both diagnostic and therapeutic procedures. Computer aided diagnosis of CLE images, using image processing and fractal analysis can be used to quantify the histological structures in the CLE generated images. The aim of this study is to develop an automatic diagnosis algorithm of colorectal cancer (CRC), based on fractal analysis and neural network modeling of the CLE-generated colon mucosa images. Materials and Methods We retrospectively analyzed a series of 1035 artifact-free endomicroscopy images, obtained during CLE examinations from normal mucosa (356 images) and tumor regions (679 images). The images were processed using a computer aided diagnosis (CAD) medical imaging system in order to obtain an automatic diagnosis. The CAD application includes image reading and processing functions, a module for fractal analysis, grey-level co-occurrence matrix (GLCM) computation module, and a feature identification module based on the Marching Squares and linear interpolation methods. A two-layer neural network was trained to automatically interpret the imaging data and diagnose the pathological samples based on the fractal dimension and the characteristic features of the biological tissues. Results Normal colon mucosa is characterized by regular polyhedral crypt structures whereas malignant colon mucosa is characterized by irregular and interrupted crypts, which can be diagnosed by CAD. For this purpose, seven geometric parameters were defined for each image: fractal dimension, lacunarity, contrast correlation, energy, homogeneity, and feature number. Of the seven parameters only contrast, homogeneity and feature number were significantly different between normal and cancer samples. Next, a two-layer feed forward neural network was used to train and automatically diagnose the malignant samples, based on the seven parameters tested. The neural network

  20. Proteasome particle-rich structures are widely present in human epithelial neoplasms: correlative light, confocal and electron microscopy study.

    Vittorio Necchi

    Full Text Available A novel cytoplasmic structure has been recently characterized by confocal and electron microscopy in H. pylori-infected human gastric epithelium, as an accumulation of barrel-like proteasome reactive particles colocalized with polyubiquitinated proteins, H. pylori toxins and the NOD1 receptor. This proteasome particle-rich cytoplasmic structure (PaCS, a sort of focal proteasome hyperplasia, was also detected in dysplastic cells and was found to be enriched in SHP2 and ERK proteins, known to play a role in H. pylori-mediated gastric carcinogenesis. However, no information is available on its occurrence in neoplastic growths. In this study, surgical specimens of gastric cancer and various other human epithelial neoplasms have been investigated for PaCSs by light, confocal and electron microscopy including correlative confocal and electron microscopy (CCEM. PaCSs were detected in gastric cohesive, pulmonary large cell and bronchioloalveolar, thyroid papillary, parotid gland, hepatocellular, ovarian serous papillary, uterine cervix and colon adenocarcinomas, as well as in pancreatic serous microcystic adenoma. H. pylori bodies, their virulence factors (VacA, CagA, urease, and outer membrane proteins and the NOD1 bacterial proteoglycan receptor were selectively concentrated inside gastric cancer PaCSs, but not in PaCSs from other neoplasms which did, however, retain proteasome and polyubiquitinated proteins reactivity. No evidence of actual microbial infection was obtained in most PaCS-positive neoplasms, except for H. pylori in gastric cancer and capsulated bacteria in a colon cancer case. Particle lysis and loss of proteasome distinctive immunoreactivities were seen in some tumour cell PaCSs, possibly ending in sequestosomes or autophagic bodies. It is concluded that PaCSs are widely represented in human neoplasms and that both non-infectious and infectious factors activating the ubiquitin-proteasome system are likely to be involved in their origin

  1. Latest advances in confocal microscopy of skin cancers toward guiding patient care: a Mohs surgeon's review and perspective (Conference Presentation)

    Nehal, Kishwer S.; Rajadhyaksha, Milind

    2016-02-01

    Latest advances in confocal microscopy of skin cancers toward guiding patient care: a Mohs surgeon's review and perspective About 350 publications worldwide have reported the ability of reflectance confocal microscopy (RCM) imaging to detect melanocytic skin lesions in vivo with specificity of 84-88% and sensitivity of 71-92%, and non-melanocytic skin lesions with specificity of 85-97% and sensitivity 100-92%. Lentigo maligna melanoma can be detected with sensitivity of 93% and specificity 82%. While the sensitivity is comparable to that of dermoscopy, the specificity is 2X superior, especially for lightly- and non-pigmented lesions. Dermoscopy combined with RCM imaging is proving to be both highly sensitive and highly specific. Recent studies have reported that the ratio of equivocal (i.e., would have been biopsied) lesions to detected melanomas dropped by ~2X when guided by dermoscopy and RCM imaging, compared to that with dermoscopy alone. Dermoscopy combined with RCM imaging is now being implemented to guide noninvasive diagnosis (to rule out malignancy and biopsy) and to also guide treatment, with promising initial impact: thus far, about 3,000 patients have been saved from biopsies of benign lesions. These are currently under follow-up monitoring. With fluorescence confocal microscopy (FCM) mosaicing, residual basal cell carcinomas can be detected in Mohs surgically excised fresh tissue ex vivo, with sensitivity of 94-97% and specificity 89-94%. FCM mosaicing is now being implemented for guiding Mohs surgery. To date, about 600 Mohs procedures have been performed, guided with mosaicing, and with pathology being performed in parallel to confirm the final outcome. These latest advances demonstrate the promising ability of RCM and FCM to guide patient care.

  2. Homonymous Hemianopic Hyporeflective Retinal Abnormality on Infrared Confocal Scanning Laser Photography: A Novel Sign of Optic Tract Lesion.

    Monteiro, Mario L R; Araújo, Rafael B; Suzuki, Ana C F; Cunha, Leonardo P; Preti, Rony C

    2016-03-01

    Infrared confocal scanning laser photography of a patient with long-standing optic tract lesion revealed a homonymous hemianopic hyporeflective image contralateral to the visual field defect. Spectral domain optical coherence tomography showed thinning of the retinal nerve fiber and retinal ganglion cell layer and thickening of the inner nuclear layer (with microcystic degeneration) in the macular area, matching the infrared image. Hyporeflective image on infrared laser photography is associated with retinal degeneration secondary to anterior visual pathway disease and, when located in homonymous hemianopic retinas, may represent a new sign of an optic tract lesion. PMID:26172159

  3. 3D Reconstruction of large tissue specimens using confocal microscopy data and correction of deformations by elastic registration

    Čapek, Martin; Brůža, Petr; Janáček, Jiří; Karen, Petr; Kubínová, Lucie; Vagnerová, R.; Hána, K.; Smrčka, P.

    2008-01-01

    Roč. 38, č. 2 (2008), s. 92-96. ISSN 0301-5491. [YBERC ´08:Biomedical engineering conference of young biomedical engineers and researches /3./. Ostrava, 08.07.2008-10.07.2008] R&D Projects: GA MŠk(CZ) LC06063; GA ČR(CZ) GA102/08/0691; GA AV ČR(CZ) IAA500200510; GA AV ČR(CZ) IAA100110502 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal microscopy * volume reconstruction Subject RIV: JD - Computer Applications, Robotics

  4. Development of a confocal laser scanning fluorescence microscope using two-photon excitation in combination with time-gated detection

    Sytsma, Joost; Vroom, Jurrien; Gerritsen, Hans C.; Levine, Yehudi K.

    1995-03-01

    Fluorescent molecules having single-photon absorption in the blue and the UV can be excited with infra-red light via a process known as two-photon excitation. The combination of this technique with scanning techniques can be exploited for 3D microscopic imaging. The two- photon process is confined to a restricted volume in the sample determined by the laser focus, resulting in inherent confocality. Other advantages are reduced photo-bleaching of the samples and a larger penetration depth of the excitation light. The implementation of time-gated detection techniques allows fluorescent lifetime imaging. This drastically improves the selectivity and contrast of the images.

  5. Relationship between Histological and Clinical Course of Psoriasis: A Pilot Investigation by Reflectance Confocal Microscopy during Goeckerman Treatment.

    Archid, Rami; Duerr, Hans Peter; Patzelt, Alexa; Philipp, Sandra; Röwert-Huber, Hans-Joachim; Ulrich, Martina; Meinke, Martina Claudia; Knorr, Fanny; Lademann, Jürgen

    2016-01-01

    Alterations of the skin microvasculature are known to play an important role in the development and maintenance of psoriatic skin lesions. In this study, we investigated lesional skin in 11 psoriatic patients during a modified Goeckerman treatment using reflectance confocal microscopy (RCM) to study the relationship between clinical clearance and histological normalization of psoriatic skin and the significance of histological abnormalities on the course of disease. The treatment regimen resulted in a significant reduction of the Psoriasis Area and Severity Index (PASI) as well as capillary and papillary diameters (p RCM findings as successor of the PASI in the monitoring of psoriasis. PMID:26841099

  6. Correlation of Nitrogen Sorption and Confocal Laser Scanning Microscopy for the Analysis of Amino Group Distributions on Mesoporous Silica

    Dominik Brühwiler

    2011-06-01

    Full Text Available Aminopropylalkoxysilanes are frequently used for the functionalization of mesoporous silica. The analysis of amino group distributions on arrays of silica nanochannels by a combination of nitrogen sorption and confocal laser scanning microscopy provides valuable insight into the mechanisms underlying the interaction of these silanes with mesoporous silica surfaces. Tendencies towards external surface functionalization, non-uniform distribution in the pores, and hydrolysis of the silica framework are shown to depend to a large extent on the mobility of the aminopropylalkoxysilane molecules, which can be adjusted by the number and type of alkoxy groups.

  7. Multiplex fluorescence in situ hybridization (M-FISH) and confocal laser scanning microscopy (CLSM) to analyze multispecies oral biofilms.

    Karygianni, Lamprini; Hellwig, Elmar; Al-Ahmad, Ali

    2014-01-01

    Multiplex fluorescence in situ hybridization (M-FISH) constitutes a favorable microbiological method for the analysis of spatial distribution of highly variable phenotypes found in multispecies oral biofilms. The combined use of confocal laser scanning microscopy (CLSM) produces high-resolution three-dimensional (3D) images of individual bacteria in their natural environment. Here, we describe the application of M-FISH on early (Streptococcus spp., Actinomyces naeslundii) and late colonizers (Fusobacterium nucleatum, Veillonella spp.) of in situ-formed oral biofilms, the acquisition of CLSM images, as well as the qualitative and quantitative analysis of these digitally obtained and processed images. PMID:24664826

  8. Combined reflectance confocal microscopy-optical coherence tomography for delineation of basal cell carcinoma margins: an ex vivo study

    Iftimia, Nicusor; Peterson, Gary; Chang, Ernest W.; Maguluri, Gopi; Fox, William; Rajadhyaksha, Milind

    2016-01-01

    We present a combined reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) approach, integrated within a single optical layout, for diagnosis of basal cell carcinomas (BCCs) and delineation of margins. While RCM imaging detects BCC presence (diagnoses) and its lateral spreading (margins) with measured resolution of ˜1 μm, OCT imaging delineates BCC depth spreading (margins) with resolution of ˜7 μm. When delineating margins in 20 specimens of superficial and nodular BCCs, depth could be reliably determined down to ˜600 μm, and agreement with histology was within about ±50 μm.

  9. Measuring Cellular-scale Nutrient Distribution in Algal Biofilms with Synchrotron Confocal Infrared Microspectroscopy

    J Murdock; W Dodds; J Reffner; D Wetzel

    2011-12-31

    The microscope and infrared spectrometer are two of the most useful tools for the study of biological materials, and their combined analytical power far exceeds the sum of the two. Performing molecular spectroscopy through a microscope superimposes chemical information onto the physical microstructure obtained from the optical microscope when visible and infrared information are collected under the same conditions. The instrument developments that enable current infrared microspectroscopic studies began with the introduction of the first research-grade infrared microscope, patented in 1989 (1). By 1993, published reports using this method to determine macroalgae (seaweed) cell-wall composition appeared (2-4). Since these initial reports, the use of infrared microspectroscopy (IMS) in microalgal (single cells or groups of cells) research has grown. Primarily, cultured algae have been used to hone IMS methodology and evaluate its capabilities in algal research (5-8). Studies involving natural, mixed species assemblages, which can utilize the spatial resolution potential of this technique fully are rare (9-11). For instance, in a recent review of IMS microalgal ecological research (12), only 3 of the 29 peer-reviewed publications investigated natural algal assemblages. Both thermal and synchrotron infrared sources provide a resolution capable of measuring individual algae in mixed species assemblages, and each has its advantages. For example, thermal source IMS is more accessible, allowing more samples to be analyzed than synchrotron IMS. However, synchrotron IMS with confocal masking provides superior resolution, which can be critical in isolating small or contiguous cells. Algal ecology is the study of the interaction between algae and their environment. Infrared microspectroscopy addresses a major logistical problem in this field, obtaining species-specific cellular biochemical information from natural, mixed-species assemblages (11,12). Benthic (bottom

  10. Velocity gradients in spatially resolved laser Doppler flowmetry and dynamic light scattering with confocal and coherence gating

    Uribe-Patarroyo, Néstor; Bouma, Brett E.

    2016-08-01

    Dynamic light scattering (DLS) is widely used to characterize diffusive motion to obtain precise information on colloidal suspensions by calculating the autocorrelation function of the signal from a heterodyne optical system. DLS can also be used to determine the flow velocity field in systems that exhibit mass transport by incorporating the effects of the deterministic motion of scatterers on the autocorrelation function, a technique commonly known as laser Doppler flowmetry. DLS measurements can be localized with confocal and coherence gating techniques such as confocal microscopy and optical coherence tomography, thereby enabling the determination of the spatially resolved velocity field in three dimensions. It has been thought that spatially resolved DLS can determine the axial velocity as well as the lateral speed in a single measurement. We demonstrate, however, that gradients in the axial velocity of scatterers exert a fundamental influence on the autocorrelation function even in well-behaved, nonturbulent flow. By obtaining the explicit functional relation between axial-velocity gradients and the autocorrelation function, we show that the velocity field and its derivatives are intimately related and their contributions cannot be separated. Therefore, a single DLS measurement cannot univocally determine the velocity field. Our extended theoretical model was found to be in good agreement with experimental measurements.

  11. Polycapillary based {mu}-XAS and confocal {mu}-XANES at a bending magnet source of the ESRF

    Silversmit, Geert; Vekemans, Bart; Vincze, Laszlo [X-ray Microspectroscopy and Imaging Research Group (XMI), Department of Analytical Chemistry, Ghent University, Krijgslaan 281 S12, B-9000 Gent (Belgium); Nikitenko, Sergey; Bras, Wim [Netherlands Organisation for Scientific Research (NWO), DUBBLE at ESRF, BP 220, 38043 Grenoble CEDEX 9 (France); Tirez, Kristof [VITO, Boeretang 200, 2400 Mol (Belgium); Brenker, Frank E, E-mail: Geert.Silversmit@UGent.b [Geoscience Institute - Mineralogy, Goethe University, Altenhoeferallee 1, 60438 Frankfurt am Main (Germany)

    2009-11-15

    Glass polycapillary optics are shown to be easy to use focusing optics for bending magnet XAS stations. These achromatic optics have acceptances of several mm horizontally and vertically, while their angular acceptances can be matched to the source divergence by the design of the polycapillary. A polycapillary half-lens based focusing system was successfully tested for transmission and fluorescence {mu}-XAS at the DUBBLE beamline (BM26A, ESRF) and the feasibility of confocal {mu}-XANES in fluorescence mode is presented. Transmission efficiencies of 25-45% with flux density gain factors of about 2000 and beam sizes of 10-20 {mu}m were obtained in the 7-14 keV energy range. Although the polycapillary optic has a smoothly changing energy dependent transmission efficiency, the amplitude and shape of the EXAFS oscillations are not influenced by this. The focusing properties of the polycapillary lens cancel slight vertical motions of the incoming X-ray beam, resulting in a fixed {mu}-beam spot in the focal plane, making polycapillaries also suitable optic in combination with a non-fixed exit monochromator. In addition, by mounting a second polycapillary half-lens in front of an energy dispersive detector, a confocal set-up is obtained, which restricts the part of the sample seen by the detector to a microscopic volume of about 20x20x15 {mu}m{sup 3} at the Fe K absorption edge for example.

  12. Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation

    Sandeau, N

    2006-01-01

    The main advantage of two-photon fluorescence confocal microscopy is the low absorption obtained with live tissues at the wavelengths of operation. However, the resolution of two-photon fluorescence confocal microscopes is lower than in the case of one-photon excitation. The 4Pi microscope type C working in two-photon regime, in which the excitation beams are coherently superimposed and, simultaneously, the emitted beams are also coherently added, has shown to be a good solution for increasing the resolution along the optical axis and for reducing the amplitude of the side lobes of the point spread function. However, the resolution in the transverse plane is poorer than in the case of one-photon excitation due to the larger wavelength involved in the two-photon fluorescence process. In this paper we show that a particular arrangement of the 4Pi microscope, referenced as 4Pi0 microscope, is a solution for obtaining a lateral resolution in the two-photon regime similar or even better to that obtained with 4Pi m...

  13. Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

    Mitra, Debasis; Boutchko, Rostyslav; Ray, Judhajeet; Nilsen-Hamilton, Marit

    2015-03-01

    In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.

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

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

    2013-03-01

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

  15. Development and verification of a novel device for dental intra-oral 3D scanning using chromatic confocal technology

    Zint, M.; Stock, K.; Graser, R.; Ertl, T.; Brauer, E.; Heyninck, J.; Vanbiervliet, J.; Dhondt, S.; De Ceuninck, P.; Hibst, R.

    2015-03-01

    The presented work describes the development and verification of a novel optical, powder-free intra-oral scanner based on chromatic confocal technology combined with a multifocal approach. The proof of concept for a chromatic confocal area scanner for intra-oral scanning is given. Several prototype scanners passed a verification process showing an average accuracy (distance deviation on flat surfaces) of less than 31μm +/- 21μm and a reproducibility of less than 4μm +/- 3μm. Compared to a tactile measurement on a full jaw model fitted with 4mm ceramic spheres the measured average distance deviation between the spheres was 49μm +/- 12μm for scans of up to 8 teeth (3- unit bridge, single Quadrant) and 104μm +/- 82μm for larger scans and full jaws. The average deviation of the measured sphere diameter compared to the tactile measurement was 27μm +/- 14μm. Compared to μCT scans of plaster models equipped with human teeth the average standard deviation on up to 3 units was less than 55μm +/- 49μm whereas the reproducibility of the scans was better than 22μm +/- 10μm.

  16. Investigation of metallurgical phenomena related to process and product development by means of High Temperature Confocal Scanning Laser Microscopy

    Diéguez-Salgado, U.; Michelic, S.; Bernhard, C.

    2016-03-01

    An increased interest for high temperature metallurgical processes appeared during the last decades, in order to achieve the high quality requirements in steel products. A defined steel cleanness and microstructure essentially influence the final product quality. The high temperatures involved in metallurgical processes and the lack of in situ observations do not only complicate the verification of simulation model predictions but also make significant conclusions regarding the industrial processes difficult. For that reason, new tools and techniques are necessary to develop. By combining the advances of a laser, confocal optics and an infrared image furnace, the High Temperature Confocal Scanning Laser Microscopy (HTCSLM) is a strong tool which enables high temperature in situ observations of different metallurgical phenomena. Next to solidification processes and phase transformations also the behavior of inclusions at different interfaces in the system steel-slag-refractory can be observed. The present study focuses on the aspects of inclusion agglomeration in the liquid steel and the inclusion behavior at the steel/refractory interface in two different steel grades. Out of the obtained experimental data, attraction forces are calculated and compared. This information provides an important basis for a better understanding of inclusion behavior in industrial processes and the therewith related process optimization, like for example the clogging phenomenon during continuous casting.

  17. Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells

    Rehman, Shagufta; Brown-Steinke, Kathleen; Palmer, Lisa; Periasamy, Ammasi

    2015-03-01

    Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.

  18. Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy.

    Michels, J; Gorb, S N

    2012-01-01

    Resilin is a rubber-like protein found in the exoskeleton of arthropods. It often contributes large proportions to the material of certain structures in movement systems. Accordingly, the knowledge of the presence and distribution of resilin is essential for the understanding of the functional morphology of these systems. Because of its specific autofluorescence, resilin can be effectively visualized using fluorescence microscopy. However, the respective excitation maximum is in the UV range, which is not covered by the lasers available in most of the modern commercial confocal laser scanning microscopes. The goal of this study was to test the potential of confocal laser scanning microscopy (CLSM) in combination with a 405 nm laser to visualize and analyse the presence and distribution of resilin in arthropod exoskeletons. The results clearly show that all resilin-dominated structures, which were visualized successfully using wide-field fluorescence microscopy (WFM) and a 'classical' UV excitation, could also be visualized efficiently with the proposed CLSM method. Furthermore, with the application of additional laser lines CLSM turned out to be very appropriate for studying differences in the material composition within arthropod exoskeletons in great detail. As CLSM has several advantages over WFM with respect to detailed morphological imaging, the application of the proposed CLSM method may reveal new information about the micromorphology and material composition of resilin-dominated exoskeleton structures leading to new insights into the functional morphology and biomechanics of arthropods. PMID:22142031

  19. Three-dimensional functional imaging of lung parenchyma using optical coherence tomography combined with confocal fluorescence microscopy

    Gaertner, Maria; Cimalla, Peter; Knels, Lilla; Meissner, Sven; Koch, Edmund

    2011-03-01

    Optical coherence tomography (OCT), as a non-invasive technique for studying tissue morphology, is widely used in in vivo studies, requiring high resolution and fast three-dimensional imaging. Based on light scattering it reveals micrometer sized substructures of the samples due to changes in their optical properties and therefore allows quantification of the specimen's geometry. Utilizing fluorescence microscopy further information can be obtained from molecular compositions embedded in the investigated object. Fluorescent markers, specifically binding to the substance of interest, reveal the sample's chemical structure and give rise to functional studies. This research presents the application of a combined OCT and laser scanning confocal microscopy (LSCM) system to investigate structural details in lung tissue. OCT reveals the three-dimensional morphology of the alveoli whereas fluorescence detection, arising from the fluorophore Sulforhodamin B (SRB) which is binding to elastin, shows the elastic meshwork of the organs extracellular matrix. Different plains of fluorescence can be obtained by using a piezo driven objective and exploiting the confocal functionality of the setup. Both techniques combined in one optical system not only ease the experimental procedure but also contribute to a thorough description of tissue's morphology and chemical composition.

  20. In-situ and elementally resolved determination of the thickness uniformity of multi-ply films by confocal micro XRF

    Confocal micro X-ray fluorescence (CM-XRF) with quasi-monochromatic excitation based on polycapillary X-ray optics was used to measure the thickness of multi-ply films. The relative errors of measuring an Fe film with a thickness of 16.3 μm and a Cu film with a thickness of 24.5 μm were 7.3% and 0.4%, respectively. The non-destructive and in-situ measurement of the thickness and uniformity of multi-ply films of Cu, Fe and Ni on a silicon surface was performed. CM-XRF was convenient in in-situ and elementally resolved analysis of the thickness of multi-ply films without a cumbersome theoretical correction model. - Highlights: • Confocal micro X-ray fluorescence based on polycapillary X-ray optics was used to measure the thickness of multi-ply films. • In-situ elemental resolved measurement of multi-ply films thickness was carried out. • The uniformity of multiply films was measured and spatially resolved

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

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

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

    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.

  3. Filtering, reconstruction, and measurement of the geometry of nuclei from hippocampal neurons based on confocal microscopy data.

    Queisser, Gillian; Wittmann, Malte; Bading, Hilmar; Wittum, Gabriel

    2008-01-01

    The cell nucleus is often considered a spherical structure. However, the visualization of proteins associated with the nuclear envelope in rat hippocampal neurons indicates that the geometry of nuclei is far more complex. The shape of cell nuclei is likely to influence the nucleo-cytoplasmic exchange of macromolecules and ions, in particular calcium, a key regulator of neuronal gene expression. We developed a tool to retrieve the 3-D view of cell nuclei from laser scanning confocal microscopy data. By applying an inertia-based filter, based on a special structure detection mechanism, the signal-to-noise ratio of the image is enhanced, the signal is smoothed, gaps in the membrane are closed, while at the same time the geometric properties, such as diameters of the membrane, are preserved. After segmentation of the image data, the microscopy data are sufficiently processed to extract surface information of the membrane by creating an isosurface with a marching tetrahedra algorithm combined with a modified Dijkstra graph-search algorithm. All methods are tested on artificial data, as well as on real data, which are recorded with a laser scanning confocal microscope. Significant advantages of the inertia-based filter can be observed when comparing it to other state of the art nonlinear diffusion filters. An additional program is written to calculate surface and volume of cell nuclei. These results represent the first step toward establishing a geometry-based model of the-dynamics of cytoplasmic and nuclear calcium. PMID:18315367

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

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

    2014-03-01

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

  5. Cellular-level near-wall unsteadiness of high-hematocrit erythrocyte flow using confocal μPIV

    Patrick, Michael J.; Chen, Chia-Yuan; Frakes, David H.; Dur, Onur; Pekkan, Kerem

    2011-04-01

    In hemodynamics, the inherent intermittency of two-phase cellular-level flow has received little attention. Unsteadiness is reported and quantified for the first time in the literature using a combination of fluorescent dye labeling, time-resolved scanning confocal microscopy, and micro-particle image velocimetry (μPIV). The near-wall red blood cell (RBC) motion of physiologic high-hematocrit blood in a rectangular microchannel was investigated under pressure-driven flow. Intermittent flow was associated with (1) the stretching of RBCs as they passed through RBC clusters with twisting motions; (2) external flow through local obstacles; and (3) transitionary rouleaux formations. Velocity profiles are presented for these cases. Unsteady flow clustered in local regions. Extra-cellular fluid flow generated by individual RBCs was examined using submicron fluorescent microspheres. The capabilities of confocal μPIV post-processing were verified using synthetic raw PIV data for validation. Cellular interactions and oscillating velocity profiles are presented, and 3D data are made available for computational model validation.

  6. Cellular-level near-wall unsteadiness of high-hematocrit erythrocyte flow using confocal {mu}PIV

    Patrick, Michael J. [Carnegie Mellon University, Molecular Biosensor and Imaging Center (MBIC), Pittsburgh, PA (United States); Chen, Chia-Yuan; Dur, Onur; Pekkan, Kerem [Carnegie Mellon University, Department of Biomedical and Mechanical Engineering, Pittsburgh, PA (United States); Frakes, David H. [Arizona State University, School of Biological and Health Systems Engineering and School of Electrical, Computer, and Energy Engineering, Tempe, AZ (United States)

    2011-04-15

    In hemodynamics, the inherent intermittency of two-phase cellular-level flow has received little attention. Unsteadiness is reported and quantified for the first time in the literature using a combination of fluorescent dye labeling, time-resolved scanning confocal microscopy, and micro-particle image velocimetry ({mu}PIV). The near-wall red blood cell (RBC) motion of physiologic high-hematocrit blood in a rectangular microchannel was investigated under pressure-driven flow. Intermittent flow was associated with (1) the stretching of RBCs as they passed through RBC clusters with twisting motions; (2) external flow through local obstacles; and (3) transitionary rouleaux formations. Velocity profiles are presented for these cases. Unsteady flow clustered in local regions. Extra-cellular fluid flow generated by individual RBCs was examined using submicron fluorescent microspheres. The capabilities of confocal {mu}PIV post-processing were verified using synthetic raw PIV data for validation. Cellular interactions and oscillating velocity profiles are presented, and 3D data are made available for computational model validation. (orig.)

  7. Development and Beam-Shape Analysis of an Integrated Fiber-Optic Confocal Probe for High-Precision Central Thickness Measurement of Small-Radius Lenses

    Boonsong Sutapun

    2015-04-01

    Full Text Available This work describes a new design of a fiber-optic confocal probe suitable for measuring the central thicknesses of small-radius optical lenses or similar objects. The proposed confocal probe utilizes an integrated camera that functions as a shape-encoded position-sensing device. The confocal signal for thickness measurement and beam-shape data for off-axis measurement can be simultaneously acquired using the proposed probe. Placing the probe’s focal point off-center relative to a sample’s vertex produces a non-circular image at the camera’s image plane that closely resembles an ellipse for small displacements. We were able to precisely position the confocal probe’s focal point relative to the vertex point of a ball lens with a radius of 2.5 mm, with a lateral resolution of 1.2 µm. The reflected beam shape based on partial blocking by an aperture was analyzed and verified experimentally. The proposed confocal probe offers a low-cost, high-precision technique, an alternative to a high-cost three-dimensional surface profiler, for tight quality control of small optical lenses during the manufacturing process.

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

    Wan, Yong

    2012-02-01

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

  9. Case Report: melanoma and melanocytic nevus differentiation with reflectance confocal microscopy. [v1; ref status: indexed, http://f1000r.es/5mr

    Joanna Łudzik

    2015-07-01

    Full Text Available Historically, melanoma has been typically diagnosed by naked-eye examination and confirmed with invasive biopsy. However, recently the use of reflectance confocal microscopy enables non-invasive bedside diagnosis of clinically equivocal lesions. We present a case in which reflectance confocal microscopy was used to evaluate two skin lesions in the same patient confirming the diagnosis of a melanoma and potentially avoiding invasive biopsy in the second benign melanocytic lesion.  Clinicians should be aware of the availability of new non-invasive technologies that can aid in early diagnosis of malignant skin tumors and potentially reduce the number of benign lesion excisions.

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

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

    2014-01-01

    In the present study, ethosomal and liposomal formulations containing tamoxifen citrate were prepared and evaluated for their penetration properties in human cadaver skin using Franz diffusion cell and confocal laser scanning microscope (CLSM). The results clearly revealed that ethosomal vesicles showed a better drug permeation profile than that of liposomal vesicles. In addition, low fluorescence intensity in CLSM was recorded with liposomes as compared to ethosomes, indicating lower cumulative amount of drug permeation from liposomal vesicles. Furthermore, CLSM showed uniform fluorescence intensity across the entire depth of skin in ethosomal treatment, indicating high penetrability of ethosomal vesicles through human cadaver skin. In contrast, low penetrability of conventional liposomal vesicles was recorded as penetration was limited to the 7(th) section (i.e. upper epidermis layer) of skin as evident from visualization of intact liposomal vesicles in CLSM. PMID:24428443

  11. In vivo confocal microscopic characteristics of crystalline keratopathy in patients with monoclonal gammopathy: Report of two cases

    Sibel Kocabeyoglu

    2014-01-01

    Full Text Available In this paper, we report two cases of a 62-year-old patient presented with blurred vision and a 45-year-old male diagnosed with multiple myeloma who was referred from the Department of Oncology. Slit-lamp examination, in vivo confocal microscopy (IVCM, systemic work-up and serum protein electrophoresis were obtained. In both patients, slit-lamp findings revealed bilateral diffuse subepithelial and anterior stromal crystals and IVCM showed highly reflective deposits in the corneal epithelium and stroma. The first patient was eventually diagnosed with monoclonal gammopathy of undetermined significance following bone marrow biopsy and systemic evaluation. Unusual corneal deposits may constitute the first sign of monoclonal gammopathies. IVCM may be helpful in showing the crystalline nature of the corneal deposits and guiding the clinician to the diagnosis of gammopathies. Both ophthalmologists and oncologists should be aware that corneal deposits may herald a life-threatening hematologic disease.

  12. Structural analysis of biofilms and pellets of Aspergillus niger by confocal laser scanning microscopy and cryo scanning electron microscopy.

    Villena, G K; Fujikawa, T; Tsuyumu, S; Gutiérrez-Correa, M

    2010-03-01

    Biomass organization of Aspergillus niger biofilms and pellets stained with fluorescein isothiocyanate were analyzed by means of confocal laser scanning microscopy and detectable differences between both types of growth were found. Three-dimensional surface plot analysis of biofilm structure revealed interstitial voids and vertical growth compared with pellets. Growth was lower in biofilm and according to fluorescence profile obtained, biomass density increased at the surface (0-20 microm). However, a decrease in fluorescence intensity was observed through optical sections of pellets even though growth was significantly higher than biofilms. Cryo scanning electron microscopy also showed structural differences. While biofilms showed a spatially ordered mycelium and well structured hyphal channels, pellets were characterized by an entangled and notoriously compacted mycelium. These findings revealed common structural characteristics between A. niger biofilms and those found in other microbial biofilms. Thus, biofilm microstructure may represent a key determinant of biofilm growth and physiology of filamentous fungi. PMID:19919894

  13. In-Situ Observation of Crystallization and Growth in High-Temperature Melts Using the Confocal Laser Microscope

    Sohn, Il; Dippenaar, Rian

    2016-08-01

    This review discusses the innovative efforts initiated by Emi and co-workers for in-situ observation of phase transformations at high temperatures for materials. By using the high-temperature confocal laser-scanning microscope (CLSM), a robust database of the phase transformation behavior during heating and cooling of slags, fluxes, and steel can be developed. The rate of solidification and the progression of solid-state phase transformations can be readily investigated under a variety of atmospheric conditions and be correlated with theoretical predictions. The various research efforts following the work of Emi and co-workers have allowed a deeper fundamental understanding of the elusive solidification and phase transformation mechanisms in materials beyond the ambit of steels. This technique continues to evolve in terms of its methodology, application to other materials, and its contribution to technology.

  14. Assessment of possibilities of ceramic biomaterial fracture surface reconstruction using laser confocal microscopy and long working distance objective lenses.

    Stach, Sebastian; Sapota, Wiktoria; Wróbel, Zygmunt; Ţălu, Ştefan

    2016-05-01

    A numerical description of fracture is an important step in the search of the correlation between specific micromechanisms of decohesion and material characteristics designated with the use of fracture mechanics methods. This issue is essential for the proper orientation of the search for basic relationships between chemical composition, technology, structure, and properties of materials. It often happens that fracture surfaces are well developed, which can significantly hinder or even prevent the measurement and reconstruction of the tested material surface geometry. In this article, comparative measurements of a biomaterial surface were performed using laser confocal microscopy. To this end, short working distance lenses dedicated to a focused UV laser beam and long working distance objective lenses were used. The article includes a quantitative comparative analysis and interpretation of the obtained results. Microsc. Res. Tech. 79:385-392, 2016. © 2016 Wiley Periodicals, Inc. PMID:26918261

  15. In-Situ Observation of Crystallization and Growth in High-Temperature Melts Using the Confocal Laser Microscope

    Sohn, Il; Dippenaar, Rian

    2016-04-01

    This review discusses the innovative efforts initiated by Emi and co-workers for in-situ observation of phase transformations at high temperatures for materials. By using the high-temperature confocal laser-scanning microscope (CLSM), a robust database of the phase transformation behavior during heating and cooling of slags, fluxes, and steel can be developed. The rate of solidification and the progression of solid-state phase transformations can be readily investigated under a variety of atmospheric conditions and be correlated with theoretical predictions. The various research efforts following the work of Emi and co-workers have allowed a deeper fundamental understanding of the elusive solidification and phase transformation mechanisms in materials beyond the ambit of steels. This technique continues to evolve in terms of its methodology, application to other materials, and its contribution to technology.

  16. Authentication of vegetable oils by confocal X-ray scattering analysis with coherent/incoherent scattered X-rays.

    Li, Fangzuo; Liu, Zhiguo; Sun, Tianxi

    2016-11-01

    This paper presents an alternative analytical method based on the Rayleigh to Compton scattering intensity ratio and effective atomic number for non-destructive identification of vegetable oils using confocal energy dispersive X-ray fluorescence and scattering spectrometry. A calibration curve for the Rayleigh to Compton scattering intensity ratio and effective atomic number was constructed on the basis of a reliable physical model for X-ray scattering. The content of light elements, which are "invisible" using X-ray fluorescence, can be calculated "by difference" from the calibration curve. In this work, we demonstrated the use of this proposed approach to identify complex organic matrices in different vegetable oils with high precision and accuracy. PMID:27211668

  17. Micro- and nanodomain imaging in uniaxial ferroelectrics: Joint application of optical, confocal Raman, and piezoelectric force microscopy

    The application of the most effective methods of the domain visualization in model uniaxial ferroelectrics of lithium niobate (LN) and lithium tantalate (LT) family, and relaxor strontium-barium niobate (SBN) have been reviewed in this paper. We have demonstrated the synergetic effect of joint usage of optical, confocal Raman, and piezoelectric force microscopies which provide extracting of the unique information about formation of the micro- and nanodomain structures. The methods have been applied for investigation of various types of domain structures with increasing complexity: (1) periodical domain structure in LN and LT, (2) nanodomain structures in LN, LT, and SBN, (3) nanodomain structures in LN with modified surface layer, (4) dendrite domain structure in LN. The self-assembled appearance of quasi-regular nanodomain structures in highly non-equilibrium switching conditions has been considered

  18. Evaluation through in vivo reflectance confocal microscopy of the cutaneous neurogenic inflammatory reaction induced by capsaicin in human subjects

    Căruntu, Constantin; Boda, Daniel

    2012-08-01

    We perform an in vivo analysis of the effects of capsaicin on cutaneous microvascularization. A total of 29 healthy subjects are administered a solution of capsaicin (CAP group) or a vehicle solution (nonCAP group) on the dorsal side of the nondominant hand. The evaluation is performed using in vivo reflectance confocal microscopy (RCM). Ten minutes after administration, the area of the section, the perimeter, and the Feret's diameter of the capillaries in the dermal papillae become significantly larger in the CAP group as against the nonCAP group, and this difference is maintained until the conclusion of the experiment. In vivo RCM allows the investigation of cutaneous vascular reactions induced by capsaicin. As such, this method may constitute an useful technique both for research and clinical practice.

  19. A confocal set-up for micro-XRF and XAFS experiments using diamond-anvil cells.

    Wilke, Max; Appel, Karen; Vincze, Laszlo; Schmidt, Christian; Borchert, Manuela; Pascarelli, Sakura

    2010-09-01

    A confocal set-up is presented that improves micro-XRF and XAFS experiments with high-pressure diamond-anvil cells (DACs). In this experiment a probing volume is defined by the focus of the incoming synchrotron radiation beam and that of a polycapillary X-ray half-lens with a very long working distance, which is placed in front of the fluorescence detector. This set-up enhances the quality of the fluorescence and XAFS spectra, and thus the sensitivity for detecting elements at low concentrations. It efficiently suppresses signal from outside the sample chamber, which stems from elastic and inelastic scattering of the incoming beam by the diamond anvils as well as from excitation of fluorescence from the body of the DAC. PMID:20724788

  20. In-situ and elementally resolved determination of the thickness uniformity of multi-ply films by confocal micro XRF.

    Peng, Song; Liu, Zhiguo; Sun, Tianxi; Wang, Guangfu; Ma, Yongzhong; Ding, Xunliang

    2014-08-01

    Confocal micro X-ray fluorescence (CM-XRF) with quasi-monochromatic excitation based on polycapillary X-ray optics was used to measure the thickness of multi-ply films. The relative errors of measuring an Fe film with a thickness of 16.3 μm and a Cu film with a thickness of 24.5 μm were 7.3% and 0.4%, respectively. The non-destructive and in-situ measurement of the thickness and uniformity of multi-ply films of Cu, Fe and Ni on a silicon surface was performed. CM-XRF was convenient in in-situ and elementally resolved analysis of the thickness of multi-ply films without a cumbersome theoretical correction model. PMID:24705010