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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 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.

  15. 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.

  16. 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.

  17. 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...

  18. 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.

  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. 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...

  4. 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.

  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

    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.

  11. 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

  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. 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.

  15. 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.

  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. 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.

  19. 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

  20. 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

  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 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

  4. 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

  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. 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

  11. 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.

  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. 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.

  15. 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...

  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. 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.

  18. 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...

  19. 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...

  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. 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...

  8. 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...

  9. 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

  10. 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...

  11. 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 ...

  12. 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...

  13. 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...

  14. 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...

  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. 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.

  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. 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

  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 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.

  3. 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

  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

    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.

  6. 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.

  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. 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...

  12. 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,...

  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. 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.

  15. 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...

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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...

  3. 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

  4. 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

  5. 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.

  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. 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.

  8. 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

  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 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

  11. 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

  12. 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.

  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. 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 ...

  2. 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...

  3. 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...

  4. 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...

  5. 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

  6. 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

  7. 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,...

  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. 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)

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  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. 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.

  13. 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

  14. 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

  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. 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

  19. 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

  20. 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...

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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...

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  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. 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

  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. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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...

  10. 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

  11. 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.

  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. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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...

  19. 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. ...

  20. 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...

  1. 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

  2. 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

  3. 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...

  4. 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 ...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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

  11. 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....

  12. 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...

  13. 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...

  14. 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

  15. 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.

  16. 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

  17. 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...

  18. 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.

  19. 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....

  20. 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.

  1. 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 ...

  2. 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...

  3. 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...

  4. 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...

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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...

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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)

  20. 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)

  1. 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.

  2. 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

  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 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.

  8. 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

  9. 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

  10. 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