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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

    OpenAIRE

    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

    International Nuclear Information System (INIS)

    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

    Energy Technology Data Exchange (ETDEWEB)

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

    International Nuclear Information System (INIS)

    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%)

  5. [Artefacts of confocal microscopy].

    Science.gov (United States)

    Vekshin, N L; Frolov, M S

    2014-01-01

    Typical artefacts caused by using confocal fluorescent microscopy while studying living cells are considered. The role of light scattering, mobility, staining, local concentrations, etc. is discussed.

  6. Virtual pinhole confocal microscope

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-06-01

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

  7. Spectrally encoded confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tearney, G.J.; Webb, R.H.; Bouma, B.E. [Wellman Laboratories of Photomedicine, Massachusetts General Hospital, 50 Blossom Street, BAR 703, Boston, Massachusetts 02114 (United States)

    1998-08-01

    An endoscope-compatible, submicrometer-resolution scanning confocal microscopy imaging system is presented. This approach, spectrally encoded confocal microscopy (SECM), uses a quasi-monochromatic light source and a transmission diffraction grating to detect the reflectivity simultaneously at multiple points along a transverse line within the sample. Since this method does not require fast spatial scanning within the probe, the equipment can be miniaturized and incorporated into a catheter or endoscope. Confocal images of an electron microscope grid were acquired with SECM to demonstrate the feasibility of this technique. {copyright} {ital 1998} {ital Optical Society of America}

  8. Basic confocal microscopy

    Directory of Open Access Journals (Sweden)

    Manuela Monti

    2012-03-01

    Full Text Available This is an eleven chapter’s effort done by a bunch of Authors coordinated by Prof. R.L. Price and W.G. Jerome (who have personally written almost half of the book that with great skills are revealing us the secrets of confocal microscopy. Considering the significant progresses in different fields of biology, confocal microscopy is extremely important to dynamically see all the different molecules involved in the controlling networks build up by gene expressions in time and space. Necessary prerequisites to accomplish such goals are some fundamental microscopic technologies well and clearly presented in the first chapters....

  9. Confocal scanning microscopy

    DEFF Research Database (Denmark)

    Bariani, Paolo

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

  10. Biological confocal microscopy

    Directory of Open Access Journals (Sweden)

    Guy Cox

    2002-04-01

    Full Text Available The first practical use of confocal optics was by Hiroto Naora1,1,2, who built a device based upon a theoretical concept devised by his supervisor Z. Koana3, over 50 years ago. His system did not form images, but was used in high resolution micro-spectrophotometry. Some 10 years later, Marvin Minsky4 added a scanning stage to construct a microscope capable of forming images. Despite these early advances, in was not until the 1970s that reasonably practical confocal microscopes were built, and the mid 1980s before commercial models became generally available.

  11. Confocal laser endomicroscopy

    DEFF Research Database (Denmark)

    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...

  12. Molecular confocal laser endomicroscopy

    DEFF Research Database (Denmark)

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

    2014-01-01

    While flexible endoscopy is essential for macroscopic evaluation, confocal laser endomicroscopy (CLE) has recently emerged as an endoscopic method enabling visualization at a cellular level. Two systems are currently available, one based on miniprobes that can be inserted via a conventional...

  13. Confocal Raman Microscopy

    CERN Document Server

    Dieing, Thomas; Toporski, Jan

    2011-01-01

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

  14. Design of CUSP Gun for Ka-Band Helical Waveguide Gyro-Traveling Wave Tube%Ka波段螺纹波导回旋行波管大回旋电子枪的研究与设计

    Institute of Scientific and Technical Information of China (English)

    唐勇; 罗勇; 徐勇

    2014-01-01

    从拉氏方程出发,推导了大回旋电子枪(CUSP)的理论,得到了用于Ka波段螺纹波导回旋行波管大回旋电子枪的基本参数.通过CS-Particle Stuido 3D粒子模拟软件,分析了不同参数对大回旋电子枪横纵速度比、速度离散的影响,理论计算与粒子模拟结果相互吻合.研究发现,影响大回旋电子注质量的主要因素是阴极所处的位置,所以文中重点分析了阴极装配误差对电子注质量带来的影响.通过将理论与仿真设计相结合,最终得到一种工作电压70kV,电流5A,速度比1.0的大回旋电子枪,满足Ka波段螺纹波导回旋行波管对高质量电子枪的要求.

  15. Hyperspectral confocal microscope.

    Science.gov (United States)

    Sinclair, Michael B; Haaland, David M; Timlin, Jerilyn A; Jones, Howland D T

    2006-08-20

    We have developed a new, high performance, hyperspectral microscope for biological and other applications. For each voxel within a three-dimensional specimen, the microscope simultaneously records the emission spectrum from 500 nm to 800 nm, with better than 3 nm spectral resolution. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical sectioning. Optical throughput and detection efficiency are maximized through the use of a custom prism spectrometer and a backside thinned electron multiplying charge coupled device (EMCCD) array. A custom readout mode and synchronization scheme enable 512-point spectra to be recorded at a rate of 8300 spectra per second. In addition, the EMCCD readout mode eliminates curvature and keystone artifacts that often plague spectral imaging systems. The architecture of the new microscope is described in detail, and hyperspectral images from several specimens are presented.

  16. Confocal microscopy of colloids

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, V; Semwogerere, D; Weeks, Eric R [Department of Physics, Emory University, Atlanta, GA 30322 (United States)

    2007-03-21

    Colloids have increasingly been used to characterize or mimic many aspects of atomic and molecular systems. With confocal microscopy these colloidal particles can be tracked spatially in three dimensions with great precision over large time scales. This review discusses equilibrium phases such as crystals and liquids, and non-equilibrium phases such as glasses and gels. The phases that form depend strongly on the type of particle interaction that dominates. Hard-sphere-like colloids are the simplest, and interactions such as the attractive depletion force and electrostatic repulsion result in more non-trivial phases which can better model molecular materials. Furthermore, shearing or otherwise externally forcing these colloids while under microscopic observation helps connect the microscopic particle dynamics to the macroscopic flow behaviour. Finally, directions of future research in this field are discussed. (topical review)

  17. Confocal coded aperture imaging

    Energy Technology Data Exchange (ETDEWEB)

    Tobin, Jr., Kenneth William (Harriman, TN); Thomas, Jr., Clarence E. (Knoxville, TN)

    2001-01-01

    A method for imaging a target volume comprises the steps of: radiating a small bandwidth of energy toward the target volume; focusing the small bandwidth of energy into a beam; moving the target volume through a plurality of positions within the focused beam; collecting a beam of energy scattered from the target volume with a non-diffractive confocal coded aperture; generating a shadow image of said aperture from every point source of radiation in the target volume; and, reconstructing the shadow image into a 3-dimensional image of the every point source by mathematically correlating the shadow image with a digital or analog version of the coded aperture. The method can comprise the step of collecting the beam of energy scattered from the target volume with a Fresnel zone plate.

  18. Laser differential confocal radius measurement.

    Science.gov (United States)

    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

  19. Twin-Photon Confocal Microscopy

    CERN Document Server

    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.

  20. Fungal keratitis - improving diagnostics by confocal microscopy

    DEFF Research Database (Denmark)

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

    2013-01-01

    Purpose: Introducing a simple image grading system to support the interpretation of in vivo confocal microscopy (IVCM) images in filamentous fungal keratitis. Setting: Clinical and confocal studies took place at the Department of Ophthalmology, Aarhus University Hospital, Denmark. Histopathological...

  1. DMD-enabled confocal microendoscopy

    Science.gov (United States)

    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.

  2. QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

    Directory of Open Access Journals (Sweden)

    Merete Krog Raarup

    2011-05-01

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

  3. Confocal Imaging of porous media

    Science.gov (United States)

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

    2012-12-01

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

  4. Confocal Annular Josephson Tunnel Junctions

    Science.gov (United States)

    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.

  5. Confocal Endomicroscopy of Colorectal Polyps

    Directory of Open Access Journals (Sweden)

    Vivian M. Ussui

    2012-01-01

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

  6. Confocal Annular Josephson Tunnel Junctions

    Science.gov (United States)

    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.

  7. Diffractive elements performance in chromatic confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-01-01

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

  8. Confocal Laser Endomicroscopy in Inflammatory Bowel Disease

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... of histological features such as colonic crypts, epithelial gaps and epithelial leakiness to fluorescein. CONCLUSIONS: Confocal laser endomicroscopy remains an experimental but emerging tool for assessment of inflammatory bowel disease. It is the only method that enables in vivo functional assessment...

  9. Confocal microlaparoscope for imaging the fallopian tube

    Science.gov (United States)

    Wu, Tzu-Yu; Schafer, Rachel; Rouse, Andrew R.; Gmitro, Arthur F.

    2012-02-01

    Recent evidence suggests that epithelial ovarian cancer may originate in the fimbriated end of the fallopian tube1. Unlike many other cancers, poor access to the ovary and fallopian tubes has limited the ability to study the progression of this deadly disease and to diagnosis it during the early stage when it is most amenable to therapy. We have previously reported on a rigid confocal microlaparoscope system that is currently undergoing a clinical trial to image the epithelial surface of the ovary2. In order to gain in vivo access to the fallopian tubes we have developed a new confocal microlaparoscope with an articulating distal tip. The new instrument builds upon the technology developed for the existing confocal microlaparoscope. It has an ergonomic handle fabricated by a rapid prototyping printer. While maintaining compatibility with a 5 mm trocar, the articulating distal tip of the instrument consists of a 2.2 mm diameter bare fiber bundle catheter with automated dye delivery for fluorescence imaging. This small and flexible catheter design should enable the confocal microlaparoscope to image early stage ovarian cancer arising inside the fallopian tube. Early ex vivo mages of human fallopian tube and in vivo imaging results from recent open surgeries using the rigid confocal microlaparoscope system are presented. Ex vivo images from animal models using the new articulating bare fiber system are also presented. These high quality images collected by the new flexible system are similar in quality to those obtained from the epithelial surface of ovaries with the rigid clinical confocal microlaparoscope.

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  11. TelePresence Confocal Laser Scanning Microscopy.

    Science.gov (United States)

    Youngblom, Janey H.; Youngblom, James J.; Wilkinson, Jerry

    2001-05-01

    The advent of the Internet has allowed the development of remote access capabilities to a growing variety and number of microscopy systems. To date, the confocal microscope has not been included among these systems. At the California State University (CSU) Confocal Microscopy Core Facility, we have established a remote access confocal laser scanning microscope facility that allows users with virtually any type of computer platform to connect to our system. Our Leica TCS NT confocal system is accessible to any authorized user via the Internet by using a free software program called VNC (Virtual Network Computing). Once connectivity is established, remote users are able to control virtually all the functions to conduct real-time image analysis and quantitative assessments of their specimen. They can also move the motorized stage to view different regions of their specimen by using a software program associated with the stage. At the end of the session, all files generated during the session can be downloaded to the user's computer from a link on the CSU confocal website. A number of safeguard features have been developed to ensure security and privacy of data acquired during a remote session. PMID:12597815

  12. Confocal microlaparoscope for imaging the fallopian tube

    Science.gov (United States)

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

    2014-11-01

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

  13. Spatial heterodyne scanning laser confocal holographic microscopy

    CERN Document Server

    Liu, Changgeng

    2016-01-01

    Scanning laser confocal holographic microscopy using a spatial heterodyne detection method is presented. Spatial heterodyne detection technique employs a Mach-Zehnder interferometer with the reference beam frequency shifted by two acousto-optic modulators (AOM) relative to the object beam frequency. Different from the traditional temporal heterodyne detection technique in which hundreds temporal samples are taken at each scanning point to achieve the complex signal, the spatial heterodyne detection technique generates spatial interference fringes by use of a linear tempo-spatial relation provided by galvanometer scanning in a typical line-scanning confocal microscope or for the slow-scanning on one dimension in a point-scanning confocal microscope, thereby significantly reducing sampling rate and increasing the signal to noise ratio under the same illumination compared to the traditional temporal heterodyne counterpart. The proposed spatial heterodyne detection scheme applies to both line-scanning and point-s...

  14. Confocal Microscopy in Biopsy Proven Argyrosis

    Directory of Open Access Journals (Sweden)

    Melis Palamar

    2013-01-01

    Full Text Available Purpose. To evaluate the confocal microscopy findings of a 46-year-old male with bilateral biopsy proven argyrosis. Materials and Methods. Besides routine ophthalmologic examination, anterior segment photography and confocal microscopy with cornea Rostoch module attached to HRT II (Heidelberg Engineering GmbH, Heidelberg, Germany were performed. Findings. Squamous metaplastic changes on conjunctival epithelium and intense highly reflective extracellular punctiform deposits in conjunctival substantia propria were detected. Corneal epithelium was normal. Highly reflective punctiform deposits starting from anterior to mid-stroma and increasing through Descemet’s membrane were evident. Corneal endothelium could not be evaluated due to intense stromal deposits. Conclusion. Confocal microscopy not only supports diagnosis in ocular argyrosis, but also demonstrates the intensity of the deposition in these patients.

  15. Needle-based confocal laser endomicroscopy

    OpenAIRE

    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...

  16. Confocal microscopy via multimode fibers: fluorescence bandwidth

    Science.gov (United States)

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe

    2016-03-01

    We recently described a method for confocal reflection imaging through fibers, as a way to increase contrast when imaging unstained biological specimens. Using a transmission matrix, focused spots can be created at the distal end of a fiber. The backscattered field coming back from the sample can be filtered using optical correlation to obtain spatial selectivity in the detection. In this proceedings article, we briefly review the working principle of this method, and we discuss how the scheme could be adapted to confocal fluorescence imaging. In particular, we show simulations of the achievable detection bandwidth when using step-index multimode fibers as imaging devices.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Vibrometry using a chromatic confocal sensor

    Science.gov (United States)

    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.

  19. Confocal Microscopy Imaging of the Biofilm Matrix

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise

    2016-01-01

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

  20. Superresolution character of array confocal system

    Institute of Scientific and Technical Information of China (English)

    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.

  1. Digital confocal microscopy through a multimode fiber

    CERN Document Server

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

    2015-01-01

    Acquiring high-contrast optical images deep inside biological tissues is still a challenging issue. Confocal microscopy is an important tool for biomedical imaging since it improves image quality by rejecting background signals. On the other hand, it suffers from low sensitivities in deep tissues due to light scattering. Recently, multimode fibers have provided a new paradigm for minimally invasive endoscopic imaging by controlling light propagation through them. Here we introduce a combined imaging technique where confocal images of a human epithelial cell are acquired through a multimode fiber. We achieve this by digitally engineering the excitation wavefront and then applying a virtual digital pinhole on the collected signal. In this way, we are able to acquire images through the fiber with significantly increased contrast.

  2. Confocal Raman microscopy of frozen bread dough

    OpenAIRE

    Huen, Julien; Weikusat, Christian; Bayer-Giraldi, Maddalena; Weikusat, Ilka; Ringer, Linda; Lösche, Klaus

    2014-01-01

    The use of freezing technology is well established in industrial and craft bakeries and is still gaining importance. In order to optimize recipes and processes of frozen baked goods, it is essential to be able to investigate the products' microstructure. Especially ice crystals and their interaction with the other components of the frozen products are of interest. In this study, frozen wheat bread dough was investigated by confocal Raman microscopy. The Raman spectra measured within the dough...

  3. Pigmented eccrine poroma: dermoscopic and confocal features.

    Science.gov (United States)

    Bombonato, Caterina; Piana, Simonetta; Moscarella, Elvira; Lallas, Aimillios; Argenziano, Giuseppe; Longo, Caterina

    2016-07-01

    Eccrine poroma is a rare benign adnexal tumor of epithelial cells originating from the terminal ductal portion of the sweat glands that is typically located on palms and soles, although other cutaneous sites can be affected [1]. It is usually nonpigmented even if there is a pigmented variant that corresponds to 17% of cases and it is usually underdiagnosed, since it is mistakenly confused with other pigmented tumors [2,3]. Dermoscopy and reflectance confocal microscopy (RCM) may assist in the correct diagnosis of this tumor. Herein, we report one case of pigmented eccrine poroma (PEP) that simulated clinically a cutaneous melanoma or a basal cell carcinoma. Dermoscopy and RCM excluded the possibilities of those two diagnoses; the overall confocal findings were suggestive for a benign epithelial tumor. Histology was fundamental to diagnose this lesion as a pigmented eccrine poroma. Even if the diagnosis of eccrine poroma remains histopathological still, as in this case report, noninvasive tools such as dermoscopy and RCM examinations can be of help to rule out the diagnosis of melanoma. Larger studies on this rare pigmented variant of eccrine poroma could shed new light on the identification of specific diagnostic dermoscopic and confocal features. PMID:27648386

  4. Visualization of confocal microscopic biomolecular data

    Science.gov (United States)

    Liu, Zhanping; Moorhead, Robert J., II

    2005-04-01

    Biomolecular visualization facilitates insightful interpretation of molecular structures and complex mechanisms underlying bio-chemical processes. Effective visualization techniques are required to deal with confocal microscopic biomolecular data in which intricate structures, fine features, and obscure patterns might be overlooked without sophisticated data processing and image synthesis. This paper presents major challenges in visualizing confocal microscopic biomolecular data, followed by a survey of related work. We then introduce a case study conducted to investigate the interaction between two proteins contained in a budding yeast saccharomyces cerevisiae by embedding custom modules in Amira. The multi-channel confocal microscopic volume data was first processed using an exponential operator to correct z-drop artifacts introduced during data acquisition. Channel correlation was then exploited to extract the overlap between the proteins as a new channel to represent the interaction while a statistical method was employed to compute the intensity of interaction to locate hot spots. To take advantage of crisp surface representation of region boundaries by iso-surfaces and visually pleasing translucent delineation of dense volumes by volume rendering, we adopted hybrid rendering that incorporates these two methods to display clear-cut protein boundaries, amorphous interior materials, and the scattered interaction in the same view volume with suppressed and highlighted parts selected by the user. The highlighted overlap helped biologists learn where the interaction happens and how it spreads, particularly when the volume was investigated in an immersive Cave Automatic Virtual Environment (CAVE) for intuitive comprehension of the data.

  5. A near-infrared confocal scanner

    Science.gov (United States)

    Lee, Seungwoo; Yoo, Hongki

    2014-06-01

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

  6. Clinical applications of corneal confocal microscopy

    Directory of Open Access Journals (Sweden)

    Mitra Tavakoli

    2008-06-01

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

  7. High harmonic terahertz confocal gyrotron with nonuniform electron beam

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-01-15

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

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

    Science.gov (United States)

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

    2014-11-01

    Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three-dimensions. We describe a simple alternative based on spatial transformation of a through-focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen. PMID:25303106

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

    Science.gov (United States)

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

    2014-11-01

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

  10. ConfocalCheck--a software tool for the automated monitoring of confocal microscope performance.

    Directory of Open Access Journals (Sweden)

    Keng Imm Hng

    Full Text Available Laser scanning confocal microscopy has become an invaluable tool in biomedical research but regular quality testing is vital to maintain the system's performance for diagnostic and research purposes. Although many methods have been devised over the years to characterise specific aspects of a confocal microscope like measuring the optical point spread function or the field illumination, only very few analysis tools are available. Our aim was to develop a comprehensive quality assurance framework ranging from image acquisition to automated analysis and documentation. We created standardised test data to assess the performance of the lasers, the objective lenses and other key components required for optimum confocal operation. The ConfocalCheck software presented here analyses the data fully automatically. It creates numerous visual outputs indicating potential issues requiring further investigation. By storing results in a web browser compatible file format the software greatly simplifies record keeping allowing the operator to quickly compare old and new data and to spot developing trends. We demonstrate that the systematic monitoring of confocal performance is essential in a core facility environment and how the quantitative measurements obtained can be used for the detailed characterisation of system components as well as for comparisons across multiple instruments.

  11. Quantifying metarefraction with confocal lenslet arrays

    CERN Document Server

    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.

  12. Reflectance Confocal Microscopy for Inflammatory Skin Diseases.

    Science.gov (United States)

    Agozzino, M; Gonzalez, S; Ardigò, M

    2016-10-01

    In vivo reflectance confocal microscopy (RCM) is a relatively novel non-invasive tool for microscopic evaluation of the skin used prevalently for diagnosis and management of skin tumour. Its axial resolution, its non-invasive and easy clinical application represents the goals for a large diffusion of this technique. During the last 15 years, RCM has been demonstrated to be able to increase the sensibility and sensitivity of dermoscopy in the diagnosis of skin tumours integrating in real time clinic, dermoscopic and microscopic information useful for the definition of malignancy. Despite to date, no large comparative studies on inflammatory skin diseases has been published in the literature, several papers already showed that RCM has a potential for the evaluation of the descriptive features of the most common inflammatory skin diseases as psoriasis, lupus erythematosus, contact dermatitis and others. The aim of the application of this technique in non-neoplastic skin diseases has been prevalently focused on the possibility of clinical diagnosis confirmation, as well as therapeutic management. Moreover, the use of RCM as driver for an optimised skin biopsy has been also followed in order to reduce the number of unsuccessful histopathological examination. In this review article we describe the confocal features of the major groups of inflammatory skin disorders focusing on psoriasiform dermatitis, interface dermatitis and spongiotic dermatitis. PMID:26996333

  13. Line-scanning, stage scanning confocal microscope

    Science.gov (United States)

    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.

  14. Confocal Raman microspectroscopy of the skin.

    Science.gov (United States)

    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

  15. Confocal Raman microspectroscopy of the skin.

    Science.gov (United States)

    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.

  16. Quantitative confocal microscopy: beyond a pretty picture.

    Science.gov (United States)

    Jonkman, James; Brown, Claire M; Cole, Richard W

    2014-01-01

    Quantitative optical microscopy has become the norm, with the confocal laser-scanning microscope being the workhorse of many imaging laboratories. Generating quantitative data requires a greater emphasis on the accurate operation of the microscope itself, along with proper experimental design and adequate controls. The microscope, which is more accurately an imaging system, cannot be treated as a "black box" with the collected data viewed as infallible. There needs to be regularly scheduled performance testing that will ensure that quality data are being generated. This regular testing also allows for the tracking of metrics that can point to issues before they result in instrument malfunction and downtime. In turn, images must be collected in a manner that is quantitative with maximal signal to noise (which can be difficult depending on the application) without data clipping. Images must then be processed to correct for background intensities, fluorophore cross talk, and uneven field illumination. With advanced techniques such as spectral imaging, Förster resonance energy transfer, and fluorescence-lifetime imaging microscopy, experimental design needs to be carefully planned out and include all appropriate controls. Quantitative confocal imaging in all of these contexts and more will be explored within the chapter. PMID:24974025

  17. Reflectance Confocal Microscopy for Inflammatory Skin Diseases.

    Science.gov (United States)

    Agozzino, M; Gonzalez, S; Ardigò, M

    2016-10-01

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

  18. Fungal Keratitis - Improving Diagnostics by Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Esben Nielsen

    2013-12-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  20. Fluorescence confocal endomicroscopy in biological imaging

    Science.gov (United States)

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

    2007-02-01

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

  1. Biological applications of confocal fluorescence polarization microscopy

    Science.gov (United States)

    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

  2. Three-dimensional scanning confocal laser microscope

    Science.gov (United States)

    Anderson, R. Rox; Webb, Robert H.; Rajadhyaksha, Milind

    1999-01-01

    A confocal microscope for generating an image of a sample includes a first scanning element for scanning a light beam along a first axis, and a second scanning element for scanning the light beam at a predetermined amplitude along a second axis perpendicular to the first axis. A third scanning element scans the light beam at a predetermined amplitude along a third axis perpendicular to an imaging plane defined by the first and second axes. The second and third scanning element are synchronized to scan at the same frequency. The second and third predetermined amplitudes are percentages of their maximum amplitudes. A selector determines the second and third predetermined amplitudes such that the sum of the percentages is equal to one-hundred percent.

  3. Imaging white adipose tissue with confocal microscopy.

    Science.gov (United States)

    Martinez-Santibañez, Gabriel; Cho, Kae Won; Lumeng, Carey N

    2014-01-01

    Adipose tissue is composed of a variety of cell types that include mature adipocytes, endothelial cells, fibroblasts, adipocyte progenitors, and a range of inflammatory leukocytes. These cells work in concert to promote nutrient storage in adipose tissue depots and vary widely based on location. In addition, overnutrition and obesity impart significant changes in the architecture of adipose tissue that are strongly associated with metabolic dysfunction. Recent studies have called attention to the importance of adipose tissue microenvironments in regulating adipocyte function and therefore require techniques that preserve cellular interactions and permit detailed analysis of three-dimensional structures in fat. This chapter summarizes our experience with the use of laser scanning confocal microscopy for imaging adipose tissue in rodents.

  4. Reflectance confocal microscopy in infectious diseases.

    Science.gov (United States)

    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

  5. The First Olympus Confocal Micro Imaging Competition China Award Ceremony

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    On January 20, 2010, the award ceremony for the First Olympus Confocal Micro Imaging Competition China was held in Beijing. After rounds of judging and competition, 16 photos finally won the prize.The First Olympus Confocal MicroImaging Competition China Award Ceremony was organized by Sciencenet.cn

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

    NARCIS (Netherlands)

    G.M.R. De Luca; R.M.P. Breedijk; R.A.J. Brandt; C.H.C. Zeelenberg; B.E. de Jong; W. Timmermans; L.N. Azar; R.A. Hoebe; S. Stallinga; E.M.M. Manders

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity wh

  7. 4D confocal microscopy for visualisation of bone remodelling

    NARCIS (Netherlands)

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

    1996-01-01

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

  8. In Vivo Confocal Microscopy expanding horizons in corneal imaging

    NARCIS (Netherlands)

    T. Hillenaar (Toine)

    2012-01-01

    textabstractConfocal microscopy is an emerging optical technique that allows the living human cornea to be imaged on a cellular level. As such, confocal microscopy enables morphologic and quantitative analysis of corneal resident cells in health and disease and provides an exciting bridge between in

  9. Confocal Raman Microspectroscopy of Oral Streptococci

    Science.gov (United States)

    Beier, Brooke D.

    Raman spectroscopy has been used in a variety of applications throughout the field of biomedical optics. It has the ability to acquire chemically-specific information in a non-invasive manner, without the need for exogenous markers. This makes it useful in the identification of bacterial species, as well as in the study of tissues and other cells. In this work, a species identification model has been created in order to discriminate between the oral bacterial species Streptococcus sanguinis and Streptococcus mutans. These are two of the most prevalent species within the human mouth and their relative concentrations can be an indicator of a patient's oral health and risk of tooth decay. They are predominantly found within plaque on the tooth's surface. To study a simplified model for dental plaque, we have examined S. sanguinis and S. mutans grown in biofilm forms. Raman spectroscopy has been implemented here through a confocal microscope. The optical system has been equipped with computationally controlled stages to allow for automated scanning, including autofocusing to probe a consistent depth within a sample. A spectrum has been acquired from each position within a scan and sent for spectral preprocessing before being submitted for species identification. This preprocessing includes an algorithm that has been developed to remove fluorescence features from known contaminants within the confocal volume, to include signal from a fluorescent substrate. Species classification has been accomplished using a principal component score-fed logistic regression model constructed from a variety of biofilm samples that have been transferred and allowed to dry, as might occur with the study of plaque samples. This binary classification model has been validated on other samples with identical preparations. The model has also been transferred to determine the species of hydrated biofilms studied in situ. Artificially mixed biofilms have been examined to test the spatial

  10. Deep stroma investigation by confocal microscopy

    Science.gov (United States)

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

    2015-03-01

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

  11. THE PARALLEL CONFOCAL DETECTING SYSTEM USING OPTICAL FIBER PLATE

    Institute of Scientific and Technical Information of China (English)

    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.

  12. In vivo confocal microscopy in chloroquine-induced keratopathy

    Directory of Open Access Journals (Sweden)

    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.

  13. Confocal microscopy description of porosity defects in metallic composite alloys

    Directory of Open Access Journals (Sweden)

    K. Gawdzińska

    2008-03-01

    Full Text Available Possibilit ics of confocal microscopy applications for thc dcscripion of open porosity dcfccts in mctallic composirc alloys arcprcscntcd. This aniclc cbaractcrizcs rhc rncthnd and prcscnts its pssihle applications by describing a rcprcscntnr ivc nrcn of thc cxaminedvoid.

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

    International Nuclear Information System (INIS)

    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)

  15. A New Multichannel Spectral Imaging Laser Scanning Confocal Microscope

    Directory of Open Access Journals (Sweden)

    Yunhai Zhang

    2013-01-01

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

  16. Measuring Corneal Haze by Using Scheimpflug Photography and Confocal Microscopy

    Science.gov (United States)

    McLaren, Jay W.; Wacker, Katrin; Kane, Katrina M.; Patel, Sanjay V.

    2016-01-01

    Purpose We compared corneal backscatter estimated from a Scheimpflug camera with backscatter estimated from a clinical confocal microscope across a wide range of corneal haze. Methods A total of 59 corneas from 35 patients with a range of severity of Fuchs' endothelial corneal dystrophy and 15 corneas from 9 normal participants were examined using a Scheimpflug camera (Pentacam) and a confocal microscope (ConfoScan 4). The mean image brightness from the anterior 120 μm, midcornea, and posterior 60 μm of the cornea across the central 2 mm recorded by the Scheimpflug camera and analogous regions from the confocal microscope were measured and standardized. Differences between instruments and correlations between backscatter and disease severity were determined by using generalized estimating equation models. Results Backscatter measured by the two instruments in the anterior and midcornea were correlated (r = 0.67 and 0.43, respectively, P < 0.001), although in the posterior cornea they were not correlated (r = 0.13, P = 0.66). Measured with the Scheimpflug camera, mean backscatter from the anterior and midcornea were greater, whereas backscatter from the posterior cornea was lower (P < 0.001) than that measured by the confocal microscope. Backscatter from the anterior cornea was correlated with disease severity for both instruments (Scheimpflug, r = 0.55, P < 0.001; confocal, r = 0.49, P = 0.003). Conclusions The Scheimpflug camera and confocal microscope should not be used interchangeably to measure corneal haze. The ability to detect changes in backscatter with disease severity is superior with the Scheimpflug camera. However, the confocal microscope provides higher resolution of corneal structure. PMID:26803798

  17. Digital adaptive optics line-scanning confocal imaging system

    Science.gov (United States)

    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.

  18. Confocal and Two-Photon Microscopy: Foundations, Applications and Advances

    Science.gov (United States)

    Diaspro, Alberto

    2001-11-01

    Confocal and Two-Photon Microscopy Foundations, Applications, and Advances Edited by Alberto Diaspro Confocal and two-photon fluorescence microscopy has provided researchers with unique possibilities of three-dimensional imaging of biological cells and tissues and of other structures such as semiconductor integrated circuits. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances provides clear, comprehensive coverage of basic foundations, modern applications, and groundbreaking new research developments made in this important area of microscopy. Opening with a foreword by G. J. Brakenhoff, this reference gathers the work of an international group of renowned experts in chapters that are logically divided into balanced sections covering theory, techniques, applications, and advances, featuring: In-depth discussion of applications for biology, medicine, physics, engineering, and chemistry, including industrial applications Guidance on new and emerging imaging technology, developmental trends, and fluorescent molecules Uniform organization and review-style presentation of chapters, with an introduction, historical overview, methodology, practical tips, applications, future directions, chapter summary, and bibliographical references Companion FTP site with full-color photographs The significant experience of pioneers, leaders, and emerging scientists in the field of confocal and two-photon excitation microscopy Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances is invaluable to researchers in the biological sciences, tissue and cellular engineering, biophysics, bioengineering, physics of matter, and medicine, who use these techniques or are involved in developing new commercial instruments.

  19. Colloidal structural evolution of asphaltene studied by confocal microscopy

    Science.gov (United States)

    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.

  20. A Simple Model for Nonlinear Confocal Ultrasonic Beams

    Institute of Scientific and Technical Information of China (English)

    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.

  1. Image Restoration Phase-Filtering Lateral Superresolution Confocal Microscopy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Wei-Qian; QIU Li-Rong; CHEN Shan-Shan; FENG Zheng-De

    2006-01-01

    @@ Image restoration phase-filtering lateral superresolution confocal microscopy, a new approach, is proposed to achieve lateral superresolution using a confocal microscope. This approach uses a lateral superresolution pupil filter to preliminarily improve its lateral resolution and uses a single-image superresolution restoration technique based on a maximum likelihood estimate to further improve its lateral resolution. The new approach has the advantages of a low cost and the remarkable superresolution effect without excessive system complexity. Experiments indicate that the proposed approach can improve the lateral resolution of a confocal microscope from 0.3μm to less than 0.1 μm when λ = 632.8 nm and NA =0.85.

  2. Ex vivo laser confocal microscopy findings of cultured Acanthamoeba trophozoites

    Directory of Open Access Journals (Sweden)

    Yamazaki N

    2012-08-01

    Full Text Available Natsuko Yamazaki,1 Akira Kobayashi,1 Hideaki Yokogawa,1 Yasuhisa Ishibashi,2 Yosaburo Oikawa,3 Masaharu Tokoro,4 Kazuhisa Sugiyama11Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Department of Ophthalmology, East Washinomiya Hospital, Kuki, Japan; 3Department of Medical Zoology, Kanazawa Medical University, Kahoku, Japan; 4Department of Parasitology, Kanazawa University Graduate School of Medical Science, Kanazawa, JapanPurpose: The purpose of the current study was to investigate ex vivo laser confocal microscopic findings of cultured Acanthamoeba trophozoites obtained from Acanthamoeba keratitis patients.Methods: Eight cultured samples of Acanthamoeba trophozoites from eight eyes of seven patients (mean age, 26.9 years; age range, 18–52 years were used. Seven samples were from corneal scrapings of Acanthamoeba keratitis patients and one sample was from the solution in a soft contact lens case. Ex vivo laser confocal microscopy was performed to qualitatively evaluate the shape and degree of light reflection of the living Acanthamoeba trophozoites.Results: Ex vivo laser confocal microscopy demonstrated highly reflective, high-contrast Acanthamoeba trophozoites with no walls (mean size, 25.4 µm; range, 17.1–58.5 µm. The shapes of the trophozoites were highly pleomorphic, and some showed characteristic acanthopodia by laser confocal microscopy.Conclusion: Ex vivo laser confocal microscopy was effective in demonstrating cultured Acanthamoeba trophozoites of various shapes and sizes. The observations of the current study may be helpful when similar structures are identified under in vivo conditions.Keywords: Acanthamoeba, trophozoite, laser confocal microscopy

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

    Science.gov (United States)

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

    2011-03-01

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

  4. EUS-Guided Needle-Based Confocal Laser Endomicroscopy

    DEFF Research Database (Denmark)

    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...

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

    Science.gov (United States)

    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.

  6. Laser differential fitting confocal microscopy with high imaging efficiency.

    Science.gov (United States)

    Sheng, Zhong; Wang, Yun; Zhao, Weiqian; Qiu, Lirong; Sun, Yingbin

    2016-09-01

    Based on the optical arrangement of a bipolar differential confocal microscopy (BDCM), laser differential fitting confocal microscopy (DFCM) is proposed in this paper using the feature of BDCM that a zero-crossing point (ZCP) of the axial response curve precisely corresponds to the focus of the system objective. A linear segment of the DFCM axial response around the ZCP is used to fit a straight line. Focus can be determined by solving the equations of the fitting lines, and then, the sample surface could be measured and reconstructed with a high resolution. Compared with the curve-fitting peak detection, which is an algorithm for focus detection widely used in conventional confocal microscopy, the line-fitting zero solution method used in DFCM has several advantages, such as high precision and sensitivity. Most importantly, precise focus detection can be realized using less data, i.e., DFCM has a high measurement efficiency. Furthermore, DFCM can effectively eliminate common-mode noise in a confocal microscopy system and has good noise suppression and disturbance resistance capability.

  7. Confocal raman microspectroscopy : a novel diagnostic tool in medical microbiology

    NARCIS (Netherlands)

    K. Maquelin (Kees)

    2002-01-01

    textabstractThe aim of the research described in this thesis was to develop confocal Raman microspectroscopy techniques for the rapid identification and characterisation of clinically relevant microorganisms. Chapter 2 describes a study in which the accuracy of the identification of Enterococcus spp

  8. Study of liquid jet instability by confocal microscopy

    Science.gov (United States)

    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.

  9. Laser differential fitting confocal microscopy with high imaging efficiency.

    Science.gov (United States)

    Sheng, Zhong; Wang, Yun; Zhao, Weiqian; Qiu, Lirong; Sun, Yingbin

    2016-09-01

    Based on the optical arrangement of a bipolar differential confocal microscopy (BDCM), laser differential fitting confocal microscopy (DFCM) is proposed in this paper using the feature of BDCM that a zero-crossing point (ZCP) of the axial response curve precisely corresponds to the focus of the system objective. A linear segment of the DFCM axial response around the ZCP is used to fit a straight line. Focus can be determined by solving the equations of the fitting lines, and then, the sample surface could be measured and reconstructed with a high resolution. Compared with the curve-fitting peak detection, which is an algorithm for focus detection widely used in conventional confocal microscopy, the line-fitting zero solution method used in DFCM has several advantages, such as high precision and sensitivity. Most importantly, precise focus detection can be realized using less data, i.e., DFCM has a high measurement efficiency. Furthermore, DFCM can effectively eliminate common-mode noise in a confocal microscopy system and has good noise suppression and disturbance resistance capability. PMID:27607265

  10. Optomechatronics Design and Control for Confocal Laser Scanning Microscopy

    NARCIS (Netherlands)

    Yoo, H.W.

    2015-01-01

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

  11. Analysis of confocal microscopy under ultrashort light-pulse illumination

    Energy Technology Data Exchange (ETDEWEB)

    Kempe, M.; Rudolph, W. (Univ. of New Mexico, Albuquerque (United States))

    1993-02-01

    The resolution of confocal laser scanning microscopes is analyzed if they are used in measurements that are to combine high spatial and high temporal resoltuion. A generalized Fourier-optical treatment is developed in which the system characteristics contain all necessary information regarding the optical arrangement and the illuminating light pulses. Coherent and incoherent imaging are considered in detail. 10 refs., 8 figs.

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

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.

    1995-01-01

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

  13. Nuclear area measurement on viable cells, using confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Townsend, K.M.S.; Marsden, S.J. (Medical Research Council, Harwell (United Kingdom). Radiobiological Research Unit)

    1992-04-01

    The authors describe a rapid procedure for the accurate measurement of nuclear areas on unperturbed living cells as used in radiobiological experiments, using the confocal laser scanning microscope. The microdosimetric interpretation of radiobiological data requires precise information on the nuclear area of cells as irradiated with high-LET radiation. (author).

  14. Comprehensive confocal endomicroscopy of the esophagus in vivo

    Science.gov (United States)

    Kang, Dongkyun; Schlachter, Simon C.; Carruth, Robert W.; Kim, Minkyu; Wu, Tao; Tabatabaei, Nima; Vacas-Jacques, Paulino; Shishkov, Milen; Woods, Kevin; Sauk, Jenny S.; Leung, John; Nishioka, Norman S.; Tearney, Guillermo J.

    2014-01-01

    Background and study aims: Biopsy sampling error can be a problem for the diagnosis of certain gastrointestinal tract diseases. Spectrally-encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has the potential to overcome sampling error by imaging large regions of gastrointestinal tract tissues. The aim of this study was to test a recently developed SECM endoscopic probe for comprehensively imaging large segments of the esophagus at the microscopic level in vivo. Methods: Topical acetic acid was endoscopically applied to the esophagus of a normal living swine. The 7 mm diameter SECM endoscopic probe was transorally introduced into the esophagus over a wire. Optics within the SECM probe were helically scanned over a 5 cm length of the esophagus. Confocal microscopy data was displayed and stored in real time. Results: Very large confocal microscopy images (length = 5 cm; circumference = 2.2 cm) of swine esophagus from three imaging depths, spanning a total area of 33 cm2, were obtained in about 2 minutes. SECM images enabled the visualization of cellular morphology of the swine esophagus, including stratified squamous cell nuclei, basal cells, and collagen within the lamina propria. Conclusions: The results from this study suggest that the SECM technology can rapidly provide large, contiguous confocal microscopy images of the esophagus in vivo. When applied to human subjects, the unique comprehensive, microscopic imaging capabilities of this technology may be utilized for improving the screening and surveillance of various esophageal diseases. PMID:26134959

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

    NARCIS (Netherlands)

    Winogrodzka, A.; Valefi, M.; Rooij, de M.B.; Schipper, D.J.

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  18. ERROR ANALYSIS OF 3D DETECTING SYSTEM BASED ON WHOLE-FIELD PARALLEL CONFOCAL MICROSCOPE

    Institute of Scientific and Technical Information of China (English)

    Wang Yonghong; Yu Xiaofen

    2005-01-01

    Compared with the traditional scanning confocal microscopy, the effect of various factors on characteristic in multi-beam parallel confocal system is discussed, the error factors in multi-beam parallel confocal system are analyzed. The factors influencing the characteristics of the multi-beam parallel confocal system are discussed. The construction and working principle of the non-scanning 3D detecting system is introduced, and some experiment results prove the effect of various factors on the detecting system.

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

    Science.gov (United States)

    Shieh, Ian C.

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2014-08-01

    We present an interferometric confocal microscope using an array of 1200 vertical cavity surface emitting lasers (VCSELs) coupled to a multimode fiber. Spatial coherence gating provides ~18,000 continuous virtual pinholes, allowing an entire en face plane to be imaged in a snapshot. This approach maintains the same optical sectioning as a scanning confocal microscope without moving parts, while the high power of the VCSEL array (∼5  mW per laser) enables high-speed image acquisition with integration times as short as 100 μs. Interferometric detection also recovers the phase of the image, enabling quantitative phase measurements and improving the contrast when imaging phase objects. PMID:25078199

  2. A laser reflection confocal large-radius measurement

    International Nuclear Information System (INIS)

    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)

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

    Science.gov (United States)

    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

  4. Anabaena cell ageing monitored with confocal fluorescence spectroscopy.

    Science.gov (United States)

    Ke, Shan; Bindokas, Vytas; Haselkorn, Robert

    2015-01-01

    Cyanobacteria use a sophisticated system of pigments to collect light energy across the visible spectrum for photosynthesis. The pigments are assembled in structures called phycobilisomes, composed of phycoerythrocyanin, phycocyanin and allophycocyanin, which absorb energy and transfer it to chlorophyll in photosystem II reaction centres. All of the components of this system are fluorescent, allowing sensitive measurements of energy transfer using single cell confocal fluorescence microscopy. The native pigments can be interrogated without the use of reporters. Here, we use confocal fluorescence microscopy to monitor changes in the efficiency of energy transfer as single cells age, between the time they are born at cell division until they are ready to divide again. Alteration of fluorescence was demonstrated to change with the age of the cyanobacterial cell.

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

    Science.gov (United States)

    Rouse, Andrew Robert

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

  6. Corneal confocal microscopy in chronic inflammatory demyelinating polyneuropathy

    OpenAIRE

    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...

  7. IV Neuropathy: An In Vivo Confocal Microscopic Study

    OpenAIRE

    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 ...

  8. Confocal Imaging of Early Heart Development in Xenopus laevis

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  10. Application of Reflectance Confocal Microscopy in Dermatology Practice

    OpenAIRE

    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...

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

    OpenAIRE

    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,...

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

    Institute of Scientific and Technical Information of China (English)

    TANG Zhilie; XING Da; LIU Songhao

    2004-01-01

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

  13. In-vivo multi-spectral confocal microscopy

    Science.gov (United States)

    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.

  14. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    International Nuclear Information System (INIS)

    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

  15. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Adaptive optics parallel near-confocal scanning ophthalmoscopy.

    Science.gov (United States)

    Lu, Jing; Gu, Boyu; Wang, Xiaolin; Zhang, Yuhua

    2016-08-15

    We present an adaptive optics parallel near-confocal scanning ophthalmoscope (AOPCSO) using a digital micromirror device (DMD). The imaging light is modulated to be a line of point sources by the DMD, illuminating the retina simultaneously. By using a high-speed line camera to acquire the image and using adaptive optics to compensate the ocular wave aberration, the AOPCSO can image the living human eye with cellular level resolution at the frame rate of 100 Hz. AOPCSO has been demonstrated with improved spatial resolution in imaging of the living human retina compared with adaptive optics line scan ophthalmoscopy.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  18. Spatial resolution of confocal XRF technique using capillary optics

    OpenAIRE

    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...

  19. Atherosclerotic plaque detection by confocal Brillouin and Raman microscopies

    Science.gov (United States)

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

    2015-02-01

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

  20. Adaptive optics parallel near-confocal scanning ophthalmoscopy.

    Science.gov (United States)

    Lu, Jing; Gu, Boyu; Wang, Xiaolin; Zhang, Yuhua

    2016-08-15

    We present an adaptive optics parallel near-confocal scanning ophthalmoscope (AOPCSO) using a digital micromirror device (DMD). The imaging light is modulated to be a line of point sources by the DMD, illuminating the retina simultaneously. By using a high-speed line camera to acquire the image and using adaptive optics to compensate the ocular wave aberration, the AOPCSO can image the living human eye with cellular level resolution at the frame rate of 100 Hz. AOPCSO has been demonstrated with improved spatial resolution in imaging of the living human retina compared with adaptive optics line scan ophthalmoscopy. PMID:27519106

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

    International Nuclear Information System (INIS)

    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

  2. UV laser mediated cell selective destruction by confocal microscopy

    Directory of Open Access Journals (Sweden)

    Giangrande Angela

    2008-04-01

    Full Text Available Abstract Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy. We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers. Coupling this technique with time-lapse enables the destruction and following of any cell(s in any pattern(s in living animals as well as in cell culture systems.

  3. Confocal microscopy through a multimode fiber using optical correlation

    CERN Document Server

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe

    2015-01-01

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

  4. Confocal Microscopy for Modeling Electron Microbeam Irradiation of Skin

    Energy Technology Data Exchange (ETDEWEB)

    Miller, John H.; Chrisler, William B.; Wang, Xihai; Sowa, Marianne B.

    2011-08-01

    For radiation exposures employing targeted sources such as particle microbeams, the deposition of energy and dose will depend on the spatial heterogeneity of the spample. Although cell structural variations are relatively minor for two-dimensional cell cultures, they can vary significantly for fully differential tissues. Employing high-resolution confocal microscopy, we have determined the spatial distribution, size, and shape of epidermal kerantinocyte nuclei for the full-thickness EpiDerm skin model (MatTek, Ashland, VA). Application of these data to claculate the microdosimetry and microdistribution of energy deposition by an electron microbeam is discussed.

  5. Confocal microscopy through a multimode fiber using optical correlation

    Science.gov (United States)

    Loterie, Damien; Goorden, Sebastianus A.; Psaltis, Demetri; Moser, Christophe

    2015-12-01

    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.

  6. Synchrotron radiation as a light source in confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    van der Oord, C.J.R.; Gerritsen, H.C.; Levine, Y.K. (University of Utrecht, P.O. Box 80.000, 3508 TA Utrecht (Netherlands)); Myring, W.J.; Jones, G.R.; Munro, I.H. (Daresbury Laboratory (United Kingdom))

    1992-01-01

    The optical properties of a confocal scanning microscope that was designed to utilize a synchrotron as light source are presented. The usable spectral range is from 200 nm up to 700 nm. Using 325-nm laser light, it is shown that the lateral resolution is about 125 nm, and the axial resolution better than 250 nm. After transport of the microscope from Utrecht to the Daresbury Synchrotron Source, 200-nm excitation can be applied, and the lateral resolution will drop to below 100 nm.

  7. [Confocal microscopy for the diagnostics of fungal keratitis].

    Science.gov (United States)

    Daas, L; Viestenz, A; Bischoff, M; Hasenfus, A; Seitz, B

    2016-09-01

    Fungal keratitis is a rare but very serious eye disease in industrial nations with a frequency of 1-5 % of all forms of keratitis from microbial causes. We present two patients with keratitis of primary unknown cause. Using confocal microscopy fungal filaments could be identified that partially showed a parallel configuration (like "railway tracks"). Thus, the correct diagnosis can often be made and suitable therapy can be non-invasively initiated even before the results of in vitro cultivation (fungal culture), polymerase chain reaction (PCR) and histological investigations are available.

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

    Directory of Open Access Journals (Sweden)

    Gustavo Victor

    2007-02-01

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

  9. The confocal plane grating spectrometer at BESSY II

    International Nuclear Information System (INIS)

    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

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

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    Confocal laser endomicroscopy permits in-vivo microscopyevaluation during endoscopy procedures. Itcan be used in all the parts of the gastrointestinaltract and includes Esophagus, stomach, small bowel,colon, biliary tract through and endoscopic retrogradecholangiopancreatography and pancreas through needlesduring endoscopic ultrasound procedures. Many researchesdemonstrated a high correlation of results betweenconfocal laser endomicroscopy and histopathology inthe diagnosis of gastrointestinal lesions; with accuracyin about 86% to 96%. Moreover, in spite that histopathologyremains the gold-standard technique for finaldiagnosis of any diseases; a considerable number ofmisdiagnosis rate could be present due to many factorssuch as interpretation mistakes, biopsy site inaccuracy,or number of biopsies. Theoretically; with the diagnosticaccuracy rates of confocal laser endomicroscopycould help in a daily practice to improve diagnosis andtreatment management of the patients. However, it isstill not routinely used in the clinical practice due to manyfactors such as cost of the procedure, lack of codificationand reimbursement in some countries, absence ofstandard of care indications, availability, physician imageinterpretationtraining, medico-legal problems, and therole of the pathologist. These limitations are relative,and solutions could be found based on new researchesfocused to solve these barriers.

  11. Statistical characterization of engineered tissues using confocal mosaic technology

    Science.gov (United States)

    Levitz, David; Ardeshiri, Ardalan; Ahmed, Jabeer; Gareau, Daniel S.; Jacques, Steven L.

    2010-02-01

    Characterization of engineered tissues using optical methods often involves tradeoff between the fraction of total volume that is imaged and the spatial resolution. The limitation is not technological but rather practical, having more to do with effective probe designs and computer memory storage for large datasets. In this paper, we propose using confocal mosaicing, a technique used to characterize large volumes of excisioned biopsies from Mohs surgeries, to characterizing collagen gels. This technique stitches together high-resolution 3D images of a series adjacent millimeter sized regions that collectively make up areas that are ~cm2. Image acquisition time is approximately 5 min. The resulting high-resolution images closely resemble hematoxylin and eosin histological sections, only obtained without the time-consuming embedding and sectioning steps. Disk-shaped collagen gels that are 1 ml volume and ~1.5 cm diameter were prepared with smooth muscle cells and imaged at days 1 and 5. Using the digital staining technique, we were able to survey the spatial distribution of cells in the hydrogel and assess spatial heterogeneity in 3D from the fluorescence data. The reflectance data provided information on collagen fibril structure and matrix remodeling by the cells. Digital staining presented the data in a way that is easily interpreted by tissue engineers. Altogether, we believe confocal mosaicing and digital staining represents an important technological novelty that significantly advances nondestructive optical evaluation of engineered tissues.

  12. Confocal Raman-AFM, A New Tool for Materials Research

    Science.gov (United States)

    Schmidt, Ute

    2005-03-01

    Characterization of heterogeneous systems, e.g. polymers, on the nanometer scale continues to grow in importance and to impact key applications in the field of materials science, nanotechnology and catalysis. The development of advanced polymeric materials for such applications requires detailed information about the physical and chemical properties of these materials on the nanometer scale. However, some details about the phase-separation process in polymers are difficult to study with conventional characterization techniques due to the inability of these methods to chemically differentiate materials with good spatial resolution, without damage, staining or preferential solvent washing. The CR-AFM is a breakthrough in microscopy. It combines three measuring techniques in one instrument: a high resolution confocal optical microscope, an extremely sensitive Raman spectroscopy system, and an Atomic Force Microscope. Using this instrument, the high spatial and topographical resolution obtained with an AFM can be directly linked to the chemical information gained by Confocal Raman spectroscopy. To demonstrate the capabilities of this unique combination of measuring techniques, polymer blend films, spin coated on glass substrates, have been characterized. AFM measurements reveal the structural and mechanical properties of the films, whereas Raman spectral images show the chemical composition of the blends.

  13. Dye-Enhanced Multimodal Confocal Imaging of Brain Cancers

    Science.gov (United States)

    Wirth, Dennis; Snuderl, Matija; Sheth, Sameer; Curry, William; Yaroslavsky, Anna

    2011-04-01

    Background and Significance: Accurate high resolution intraoperative detection of brain tumors may result in improved patient survival and better quality of life. The goal of this study was to evaluate dye enhanced multimodal confocal imaging for discriminating normal and cancerous brain tissue. Materials and Methods: Fresh thick brain specimens were obtained from the surgeries. Normal and cancer tissues were investigated. Samples were stained in methylene blue and imaged. Reflectance and fluorescence signals were excited at 658nm. Fluorescence emission and polarization were registered from 670 nm to 710 nm. The system provided lateral resolution of 0.6 μm and axial resolution of 7 μm. Normal and cancer specimens exhibited distinctively different characteristics. H&E histopathology was processed from each imaged sample. Results and Conclusions: The analysis of normal and cancerous tissues indicated clear differences in appearance in both the reflectance and fluorescence responses. These results confirm the feasibility of multimodal confocal imaging for intraoperative detection of small cancer nests and cells.

  14. Measurement of steep edges and undercuts in confocal microscopy.

    Science.gov (United States)

    Mueller, T; Jordan, M; Schneider, T; Poesch, A; Reithmeier, E

    2016-05-01

    Confocal microscopy is widely used to measure the surface topography of specimen with a precision in the micrometer range. The measurement uncertainty and quality of the acquired data of confocal microscopy depends on various effects, such as optical aberrations, vibrations of the measurement setup and variations in the surface reflectivity. In this article, the influence of steep edges and undercuts on measurement results is examined. Steep edges on the specimen's surface lead to a reduced detector signal which influences the measurement accuracy and undercuts cause surface regions, which cannot be captured in a measurement. The article describes a method to overcome the negative effects of steep edges and undercuts by capturing several measurements of the surface with different angles between the surface and the optical axis of the objective. An algorithm is introduced which stitches different angle measurements together without knowledge of the exact position and orientation of the rotation axis. Thus, the measurement uncertainty due to steep edges and undercuts can be avoided without expensive high-precision rotation stages and time consuming adjustment of the measurement setup.

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

    Directory of Open Access Journals (Sweden)

    Hongda Mao

    2013-01-01

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

  16. Emission characteristics of light-emitting diodes by confocal microscopy

    Science.gov (United States)

    Cheung, W. S.; Choi, H. W.

    2016-03-01

    The emission profiles of light-emitting diodes have typically be measured by goniophotometry. However this technique suffers from several drawbacks, including the inability to generate three-dimensional intensity profiles as well as poor spatial resolution. These limitations are particularly pronounced when the technique is used to compared devices whose emission patterns have been modified through surface texturing at the micrometer and nanometer scales,. In view of such limitations, confocal microscopy has been adopted for the study of emission characteristics of LEDs. This enables three-dimensional emission maps to be collected, from which two-dimensional cross-sectional emission profiles can be generated. Of course, there are limitations associated with confocal microscopy, including the range of emission angles that can be measured due to the limited acceptance angle of the objective. As an illustration, the technique has been adopted to compare the emission profiles of LEDs with different divergence angles using an objective with a numerical aperture of 0.8. It is found that the results are consistent with those obtained by goniophotometry when the divergence angle is less that the acceptance angle of the objective.

  17. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    Directory of Open Access Journals (Sweden)

    Beaudet A.

    1998-01-01

    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.

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

    OpenAIRE

    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,...

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

    Science.gov (United States)

    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.

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

    Data.gov (United States)

    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...

  1. 3D imaging of neutron tracks using confocal microscopy

    Science.gov (United States)

    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

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

    CERN Document Server

    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. Reflectance confocal microscopy for cutaneous infections and infestations.

    Science.gov (United States)

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

    2016-05-01

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

  4. Compact fiber optic dual-detection confocal displacement sensor.

    Science.gov (United States)

    Lee, Dong-Ryoung; Jang, Suin; Lee, Min Woo; Yoo, Hongki

    2016-09-20

    We propose a dual-detection confocal displacement sensor (DDCDS) with a compact fiber-based optical probe. This all-fiber-optic sensor probe is simple and robust, since it only requires simple alignment of a gradient refractive index lens and a double-clad fiber (DCF). The DDCDS is composed of two point detectors, one coupled to a single mode fiber and the other coupled to a multimode fiber, which are used to measure the light intensity from a core and an inner clad of a DCF, respectively. The ratio of the axial response curves, measured by the two detectors, can be used to obtain a linear relationship between the axial position of the object plane and the ratio of the intensity signals. We demonstrate the performance of the proposed method by measuring micromovement and fast vibration. PMID:27661592

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

    Science.gov (United States)

    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

  6. Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

    Science.gov (United States)

    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

  7. Compact fiber optic dual-detection confocal displacement sensor.

    Science.gov (United States)

    Lee, Dong-Ryoung; Jang, Suin; Lee, Min Woo; Yoo, Hongki

    2016-09-20

    We propose a dual-detection confocal displacement sensor (DDCDS) with a compact fiber-based optical probe. This all-fiber-optic sensor probe is simple and robust, since it only requires simple alignment of a gradient refractive index lens and a double-clad fiber (DCF). The DDCDS is composed of two point detectors, one coupled to a single mode fiber and the other coupled to a multimode fiber, which are used to measure the light intensity from a core and an inner clad of a DCF, respectively. The ratio of the axial response curves, measured by the two detectors, can be used to obtain a linear relationship between the axial position of the object plane and the ratio of the intensity signals. We demonstrate the performance of the proposed method by measuring micromovement and fast vibration.

  8. Confocal spectroscopy of InGaN LED structures

    Energy Technology Data Exchange (ETDEWEB)

    Dobrovolskas, D; Mickevicius, J; Kuokstis, E; Tamulaitis, G [Semiconductor Physics Department and Institute of Applied Research, Vilnius University, Sauletekio 9-III, LT-10222 Vilnius (Lithuania); Shur, M [Department of Electrical, Computer, and Systems Engineering, and Center of Integrated Electronics, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, NY 12180 (United States); Shatalov, M; Yang, J; Gaska, R, E-mail: darius.dobrovolskas@ff.stud.vu.l [Sensor Electronic Technology, Inc., 1195 Atlas Road, Columbia, SC (United States)

    2011-04-06

    Photoluminescence of InGaN structures for green light-emitting diodes (LEDs) with multiple quantum wells as an active medium was studied with spatial and spectral resolution using confocal microscopy. Bright spots of {approx}200 nm diameter were observed. Emission from these bright areas was up to 8 times more intense than from the rest of the sample surface and the band peak position in these areas was blueshifted with respect to the band position in the background surface of lower photoluminescence intensity. The data on emission properties in bright and dark areas and the dependence of these properties on the excitation power density were interpreted by assuming inhomogeneous distribution of defects acting as nonradiative recombination centres.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Jaikrit Bhutani

    2015-01-01

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

  11. ANALYSIS OF ENDOPLASMIC RETICULUM OF TOBACCO CELLS USING CONFOCAL MICROSCOPY

    Directory of Open Access Journals (Sweden)

    Barbora Radochová

    2011-05-01

    Full Text Available Image analysis techniques for preprocessing, segmentation and estimation of geometrical characteristics of fiber-like structures from 2-D or 3-D images captured by a confocal microscope are presented. Methods are demonstrated on fiber-like biological structure: endoplasmic reticulum (ER of tobacco cells. In the presented analysis of 2-D images of ER before and after the treatment of latrunculin B, ER and ER tubules were segmented and the area density of ER as well as the length density of ER tubules in the cell cortical layer were estimated by automatic image analysis algorithms. Images of 3-D arrangement of ER were reconstructed and rendered by various visualization techniques.

  12. High precision radially-polarized-light pupil-filtering differential confocal measurement

    Science.gov (United States)

    Wang, Yun; Qiu, Lirong; Zhao, Weiqian

    2016-08-01

    A new method, high precision radially-polarized light pupil-filtering differential confocal measurement (RPDCM), is proposed to improve the 3D measurement resolution of confocal system. SPDCM uses the property that the radially-polarized-light can produce a strong longitudinal field component after being focused by a high numerical aperture objective to reduce the lateral size of the focus spot, and relies on the pupil-filtering technique to optimize the pupil function of the optical system by the designed pupil filter, which therefore improves the lateral resolution of confocal system, and it uses the differential confocal technology to improve the axial measurement resolution of the confocal system, thereby improves the 3D measurement resolution of the confocal system. Based on RPDCM, we developed a high precision radially-polarized light pupil-filtering differential confocal setup, and use it to verify the effectiveness of RPDCM by experiments. The theoretical analysis and experimental results show that the RPDCM can reach the lateral and axial measurement resolutions of 150 nm and 1 nm, respectively, which are an improvement of 20-32% and 3.7 times compared with a confocal system.

  13. CONFOCAL SECTIONING OF THICK, OTHERWISE UNDIAGNOSABLE CELL GROUPINGS IN CERVICAL SMEARS

    NARCIS (Netherlands)

    BOON, ME; KOK, LP; SUTEDJA, G; DUTRIEUX, RP

    1993-01-01

    Confocal sectioning of thick, otherwise undiagnosable cell groupings in cervical smears results in detailed visualization of the diagnostic features of neoplastic fragments in the original Cytobrush smear. We argue that the large step forward due to confocal microscopy is the dramatic improvement of

  14. Characterization of Polymer Blends: Optical Microscopy (*Polarized, Interference and Phase Contrast Microscopy*) and Confocal Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ramanathan, Nathan Muruganathan [ORNL; Darling, Seth B. [Argonne National Laboratory (ANL)

    2015-01-01

    Chapter 15 surveys the characterization of macro, micro and meso morphologies of polymer blends by optical microscopy. Confocal Microscopy offers the ability to view the three dimensional morphology of polymer blends, popular in characterization of biological systems. Confocal microscopy uses point illumination and a spatial pinhole to eliminate out-of focus light in samples that are thicker than the focal plane.

  15. Improving spatial resolution of confocal Raman microscopy by super-resolution image restoration.

    Science.gov (United States)

    Cui, Han; Zhao, Weiqian; Wang, Yun; Fan, Ying; Qiu, Lirong; Zhu, Ke

    2016-05-16

    A new super-resolution image restoration confocal Raman microscopy method (SRIR-RAMAN) is proposed for improving the spatial resolution of confocal Raman microscopy. This method can recover the lost high spatial frequency of the confocal Raman microscopy by using Poisson-MAP super-resolution imaging restoration, thereby improving the spatial resolution of confocal Raman microscopy and realizing its super-resolution imaging. Simulation analyses and experimental results indicate that the spatial resolution of SRIR-RAMAN can be improved by 65% to achieve 200 nm with the same confocal Raman microscopy system. This method can provide a new tool for high spatial resolution micro-probe structure detection in physical chemistry, materials science, biomedical science and other areas.

  16. Corneal Confocal Microscopy Anomalies Associated with Cowden Syndrome: A Case Report

    Directory of Open Access Journals (Sweden)

    Sandro Sbordone

    2013-08-01

    Full Text Available Purpose: To describe bilateral corneal alterations through confocal microscopy in a patient affected by Cowden syndrome (CS. Methods: Evaluation of Schirmer's, fluorescein, and lissamine green dye tests. Confocal microscopy was performed in both eyes to investigate corneal abnormalities. Results: Slit lamp observation showed the focal involvement of anterior stromal and epithelial layers. Schirmer's, fluorescein, and lissamine green dye test results were regular, while corneal confocal examination confirmed the disorganization of anterior stromal and epithelial layers in both eyes. Conclusion: CS is a rare autosomal-dominant systemic disorder. In our case, confocal analysis revealed predominance of alterations in the anterior stromal corneal layer, showing an increase of reflectivity, and a totally unstructured architecture in the epithelium layer. Even though the main purpose remains the prevention and the early diagnosis of different systemic tumors that could occur in affected patients, corneal confocal evaluation could play an important role in the early diagnosis of this rare disease.

  17. Propagating Characteristics of Confocal Elliptical Waveguide Filled with Multilayered Dielectrics

    Institute of Scientific and Technical Information of China (English)

    熊天信; 杨儒贵

    2004-01-01

    Using the method of separation of variables in the elliptical coordinate system, a recursive formula for the electromagnetic fields in a confocal elliptical waveguide filled with multi-layered homogeneous isotropic media is derived; then the eigenequation for it is given. When an elliptical waveguide becomes a circular waveguide, the electromagnetic fields and the eigenequation of the circular waveguide can be obtained from the eigenequation of the elliptical waveguide using the asymptotic formulae of Mathieu and modified Mathieu functions for a large radial coordinate in the elliptical coordinate system, and the eigenequation of a circular waveguide filled with multilayered dielectrics can be treated as a special case of an elliptical waveguide.In addition, some numerical examples are presented to analyze the propagating characteristics influenced by the permittivity, permeability of dielectrics filled in the elliptical waveguide, etc. The results show that changing the permittivity or permeability of the dielectrics filled in the waveguide and the major semiaxis value of the i-th layer can change the propagating characteristics of an elliptical waveguide.

  18. Quantitative analysis of in vivo confocal microscopy images: a review.

    Science.gov (United States)

    Patel, Dipika V; McGhee, Charles N

    2013-01-01

    In vivo confocal microscopy (IVCM) is a non-invasive method of examining the living human cornea. The recent trend towards quantitative studies using IVCM has led to the development of a variety of methods for quantifying image parameters. When selecting IVCM images for quantitative analysis, it is important to be consistent regarding the location, depth, and quality of images. All images should be de-identified, randomized, and calibrated prior to analysis. Numerous image analysis software are available, each with their own advantages and disadvantages. Criteria for analyzing corneal epithelium, sub-basal nerves, keratocytes, endothelium, and immune/inflammatory cells have been developed, although there is inconsistency among research groups regarding parameter definition. The quantification of stromal nerve parameters, however, remains a challenge. Most studies report lower inter-observer repeatability compared with intra-observer repeatability, and observer experience is known to be an important factor. Standardization of IVCM image analysis through the use of a reading center would be crucial for any future large, multi-centre clinical trials using IVCM.

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

    Science.gov (United States)

    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

  20. Confocal laser endomicroscopy in gastrointestinal and pancreatobiliary diseases.

    Science.gov (United States)

    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

  1. Application of Reflectance Confocal Microscopy in Dermatology Practice

    Directory of Open Access Journals (Sweden)

    Ayşe Esra Koku Aksu

    2015-03-01

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

  2. Adaptive optics in digital micromirror based confocal microscopy

    Science.gov (United States)

    Pozzi, P.; Wilding, D.; Soloviev, O.; Vdovin, G.; Verhaegen, M.

    2016-03-01

    This proceeding reports early results in the development of a new technique for adaptive optics in confocal microscopy. The term adaptive optics refers to the branch of optics in which an active element in the optical system is used to correct inhomogeneities in the media through which light propagates. In its most classical form, mostly used in astronomical imaging, adaptive optics is achieved through a closed loop in which the actuators of a deformable mirror are driven by a wavefront sensor. This approach is severely limited in fluorescence microscopy, as the use of a wavefront sensor requires the presence of a bright, point like source in the field of view, a condition rarely satisfied in microscopy samples. Previously reported approaches to adaptive optics in fluorescence microscopy are therefore limited to the inclusion of fluorescent microspheres in the sample, to use as bright stars for wavefront sensors, or time consuming sensorless optimization procedures, requiring several seconds of optimization before the acquisition of a single image. We propose an alternative approach to the problem, implementing sensorless adaptive optics in a Programmable array microscope. A programmable array microscope is a microscope based on a digital micromirror device, in which the single elements of the micromirror act both as point sources and pinholes.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  4. Confocal laser scanning microscopy in study of bone calcification

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-12-01

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

  5. A confocal scanning laser ophthalmoscope for retinal vessel oximetry

    Science.gov (United States)

    Lompado, Arthur

    Measurement of a person's blood oxygen saturation has long been recognized as a useful metric for the characterizing ailments ranging from chronic respiratory disorders to acute, potentially life threatening, traumas. The ubiquity of oxygen saturation monitors in the medical field, including portable pulse oximeters and laboratory based CO-oximeters, is a testament to the importance of this technique. The work presented here documents the design, fabrication and development of a unique type of oxygen saturation monitor, a confocal scanning retinal vessel oximeter, with the potential to expand the usefulness of the present devices. A large part of the knowledge base required to construct the instrument comes from the consideration of light scattering by red blood cells in a blood vessel. Therefore, a substantial portion of this work is devoted to the process of light scattering by whole human blood and its effects on the development of a more accurate oximeter. This light scattering effect has been both measured and modeled stochastically to determine its contribution to the measured oximeter signal. It is shown that, although well accepted in the published literature, the model only correlates marginally to the measurements due to inherent limitations imposed by the model assumptions. Nonetheless, enough material has been learned about the scattering to allow development of a mathematical model for the interaction of light with blood in a vessel, and this knowledge has been applied to the data reduction of the present oximeter. This data reduction technique has been tested in a controlled experiment employing a model eye with a blood filled mock retinal vessel. It will be shown that the presently developed technique exhibited strong correlation between the known blood oxygen saturation and that calculated by the new system.

  6. Optimum Combined Lenses for Confocal Biochip Scanning System

    Institute of Scientific and Technical Information of China (English)

    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.

  7. Laser ablation of basal cell carcinomas guided by confocal microscopy

    Science.gov (United States)

    Sierra, Heidy; Cordova, Miguel; Nehal, Kishwer; Rossi, Anthony; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

    2016-02-01

    Laser ablation offers precise and fast removal of superficial and early nodular types of basal cell carcinomas (BCCs). Nevertheless, the lack of histological confirmation has been a limitation. Reflectance confocal microscopy (RCM) imaging combined with a contrast agent can offer cellular-level histology-like feedback to detect the presence (or absence) of residual BCC directly on the patient. We conducted an ex vivo bench-top study to provide a set of effective ablation parameters (fluence, number of passes) to remove superficial BCCs while also controlling thermal coagulation post-ablation to allow uptake of contrast agent. The results for an Er:YAG laser (2.9 um and pulse duration 250us) show that with 6 passes of 25 J/cm2, thermal coagulation can be effectively controlled, to allow both the uptake of acetic acid (contrast agent) and detection of residual (or absence) BCCs. Confirmation was provided with histological examination. An initial in vivo study on 35 patients shows that the uptake of contrast agent aluminum chloride) and imaging quality is similar to that observed in the ex vivo study. The detection of the presence of residual tumor or complete clearance was confirmed in 10 wounds with (additional) histology and in 25 lesions with follow-up imaging. Our results indicate that resolution is sufficient but further development and use of appropriate contrast agent are necessary to improve sensitivity and specificity. Advances in RCM technology for imaging of lateral and deep margins directly on the patient may provide less invasive, faster and less expensive image-guided approaches for treatment of BCCs.

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

    Science.gov (United States)

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

  9. In vivo confocal microscopy in different types of posterior polymorphous dystrophy

    Directory of Open Access Journals (Sweden)

    Babu Kalpana

    2007-01-01

    Full Text Available Posterior polymorphous dystrophy is a rare corneal dystrophy, usually detected by chance. This case series describes the morphologic features in the three different types of posterior polymorphous dystrophy using confocal microscopy.

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

    OpenAIRE

    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 ...

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

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

    Science.gov (United States)

    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.

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

    OpenAIRE

    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,...

  15. Optimizing the acquisition and analysis of confocal images for quantitative single-mobile-particle detection.

    Science.gov (United States)

    Friaa, Ouided; Furukawa, Melissa; Shamas-Din, Aisha; Leber, Brian; Andrews, David W; Fradin, Cécile

    2013-08-01

    Quantification of the fluorescence properties of diffusing particles in solution is an invaluable source of information for characterizing the interactions, stoichiometry, or conformation of molecules directly in their native environment. In the case of heterogeneous populations, single-particle detection should be the method of choice and it can, in principle, be achieved by using confocal imaging. However, the detection of single mobile particles in confocal images presents specific challenges. In particular, it requires an adapted set of imaging parameters for capturing the confocal images and an adapted event-detection scheme for analyzing the image. Herein, we report a theoretical framework that allows a prediction of the properties of a homogenous particle population. This model assumes that the particles have linear trajectories with reference to the confocal volume, which holds true for particles with moderate mobility. We compare the predictions of our model to the results as obtained by analyzing the confocal images of solutions of fluorescently labeled liposomes. Based on this comparison, we propose improvements to the simple line-by-line thresholding event-detection scheme, which is commonly used for single-mobile-particle detection. We show that an optimal combination of imaging and analysis parameters allows the reliable detection of fluorescent liposomes for concentrations between 1 and 100 pM. This result confirms the importance of confocal single-particle detection as a complementary technique to ensemble fluorescence-correlation techniques for the studies of mobile particle.

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

    Science.gov (United States)

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

    2008-02-01

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

  17. In vivo confocal microscopy of meibomian glands in primary blepharospasm

    Science.gov (United States)

    Lin, Tong; Gong, Lan

    2016-01-01

    Abstract The aim of the study was to evaluate the morphological changes of meibomian glands (MGs) in primary blepharospasm (PBS) by in vivo laser scanning confocal microscopy (LSCM) and to investigate the correlations between clinical data of PBS and LSCM parameters of MGs. This prospective and case–control study recruited 30 consecutive PBS patients and 30 age- and gender-matched healthy controls. After questionnaire assessments of ocular surface disease index (OSDI), Jankovic rating scale, and blepharospasm disability index, all subjects underwent blink rate evaluation, tear film break-up time (TBUT), corneal fluorescein staining (CFS), Schirmer test, MG expressibility, meibum quality, MG dropout, and LSCM examination of the MGs. The main LSCM outcomes included the mean MG acinar area and density, orifice diameter, meibum secretion reflectivity, acinar irregularity, and inhomogeneity of interstice and acinar wall. The PBS patients had significantly higher blink rate, higher OSDI and CFS scores, lower TBUT and Schirmer test value, and worse MG expressibility than the controls (All P  0.05). The PBS patients showed lower values of MG acinar area, orifice diameter and meibum secretion reflectivity, and higher scores of acinar irregularity and inhomogeneity of interstices than the controls (All P < 0.05). For the PBS patients, the severity of blepharospasm evaluated by JCR scale was strong correlated with MG acinar area (P < 0.001), orifice diameter (P = 0.002), meibum secretion reflectivity (P = 0.002), and MG acinar irregularity (P = 0.013). The MG expressibility was significantly correlated to MG acinar area (P = 0.039), orifice diameter (P < 0.001), and MG acinar irregularity (P = 0.014). The OSDI score was moderate correlated with MG acinar irregularity (P = 0.016), whereas the TBUT value was positively correlated with MG acinar area (P = 0.045) and negatively correlated to MG acinar irregularity (P = 0.016). The CFS score was negatively

  18. In vivo corneal confocal microscopic analysis in patients with keratoconus

    Institute of Scientific and Technical Information of China (English)

    Gulfidan; Bitirgen; Ahmet; Ozkagnici; Banu; Bozkurt; Rayaz; A; Malik

    2015-01-01

    AIM: To quantify corneal ultrastructure using laser scanning in vivo confocal microscopy(IVCM) in patients with keratoconus and control subjects. METHODS: Unscarred corneas of 78 keratoconic subjects without a history of contact lens use and 36age-matched control subjects were evaluated with slit-lamp examination(SLE), corneal topography and laser scanning IVCM. One eye was randomly chosen for analysis. Anterior and posterior stromal keratocyte,endothelial cell and basal epithelial cell densities and sub-basal nerve structure were evaluated.RESULTS: IVCM qualitatively demonstrated enlarged basal epithelial cells, structural changes in sub-basal and stromal nerve fibers, abnormal stromal keratocytes and keratocyte nuclei, and pleomorphism and enlargement of endothelial cells. Compared with control subjects, significant reductions in basal epithelial cell density( 5817 ± 306 cells / mm2 vs 4802 ±508 cells/mm2,P < 0. 001), anterior stromal keratocyte density(800 ±111 cells/mm2 vs 555 ±115 cells/mm2, P <0.001),posterior stromal keratocyte density(333±34 cells/mm2vs270 ±47 cells/mm2, P <0.001), endothelial cell density(2875 ±223 cells/mm2 vs 2686 ±265 cells/mm2, P <0.001),sub-basal nerve fiber density(31.2 ±8.4 nerves/mm2vs18.1 ±9.2 nerves/mm2, P <0.001), sub-basal nerve fiber length(21.4±3.4 mm/mm2 vs 16.1±5.1 mm/mm2, P <0.001),and sub-basal nerve branch density(median 50.0(first quartile 31.2- third quartile 68.7) nerve branches/mm2 vs median 25.0(first quartile 6.2- third quartile 45.3) nerve branches/mm2, P <0.001) were observed in patients with keratoconus.CONCLUSION: Significant microstructural abnormalities were identified in all corneal layers in the eyes of subjects with keratoconus using IVCM. This non-invasive in vivo technique provides an important means to define and follow progress of microstructural changes in patients with keratoconus.

  19. Impression cytology and in vivo confocal microscopy in corneas with total limbal stem cell deficiency

    Directory of Open Access Journals (Sweden)

    Aline Lütz de Araújo

    2013-10-01

    Full Text Available PURPOSES: To describe corneal changes seen on in vivo confocal microscopy in patients with total limbal stem cell deficiency and to correlate them with cytological findings. METHODS: A prospective case series including 13 eyes (8 patients with total limbal deficiency was carried out. Stem cell deficiency was diagnosed clinically and by corneal impression cytology. Confocal images of the central cornea were taken with the Heidelberg Retina Tomograph II, Rostock Corneal Module (Heidelberg Engineering, Heidelberg, Germany. RESULTS: Impression cytology of the cornea revealed conjunctival epithelial cells and goblet cells in all cases. In vivo confocal microscopy showed disruption of normal layers of the corneal epithelium in all eyes. Confocal images showed cells with characteristics of conjunctival epithelium at the cornea in 76.9% of the total. These findings on confocal microscopy were compatible to limbal stem cell deficiency. Additionally, goblet cells, squamous metaplasia, inflammatory cells and dendritic cells were observed. The sub-basal nerve plexus was not identified in any of the corneas. Corneal neovessels were observed at the epithelium and stroma. All cases showed diffuse hyper-reflective images of the stroma corresponding to opacity of the tissue. CONCLUSIONS: Limbal stem cell deficiency had been confirmed by impression cytology in all cases, and 76.9% of the cases could also be diagnosed by in vivo confocal microscopy through the conjunctival epithelial cell visualization on the corneal surface. Frequent confocal microscopy findings were abnormal cells at the cornea (conjunctival epithelial, goblet and inflammatory cells, corneal neovessels and diffuse hyper-reflection of the stroma.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Directory of Open Access Journals (Sweden)

    Saubaméa Bruno

    2010-03-01

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

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

    Science.gov (United States)

    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

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

    KAUST Repository

    Yong Wan,

    2009-11-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Directory of Open Access Journals (Sweden)

    Nohra E. Beltran

    2013-01-01

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2015-10-01

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

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

    International Nuclear Information System (INIS)

    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)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Strandh Christer

    2008-07-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA instrument for automated screening processes.

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

    Science.gov (United States)

    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.

  13. Improving contrast and sectioning power in confocal imaging by third harmonic generation in SiOx nanocrystallites

    Institute of Scientific and Technical Information of China (English)

    Gilbert Boyer; Karsten Plamann

    2007-01-01

    We present a new optical microscope in which the light transmitted by a sample-scanned transmission confocal microscope is frequency-tripled by SiOx nanocrystallites in lieu of being transmitted by a confocal pinhole. This imaging technique offers an increased contrast and a high scattered light rejection. It is demonstrated that the contrast close to the Sparrow resolution limit is enhanced and the sectioning power are increased with respect to the linear confocal detection mode. An experimental implementation is presented and compared with the conventional linear confocal mode.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-12-28

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

  18. Determination of Nanogram Microparticles from Explosives after Real Open-Air Explosions by Confocal Raman Microscopy.

    Science.gov (United States)

    Zapata, Félix; García-Ruiz, Carmen

    2016-07-01

    Explosives are increasingly being used for terrorist attacks to cause devastating explosions. The detection of their postblast residues after an explosion is a high challenge, which has been barely investigated, particularly using spectroscopic techniques. In this research, a novel methodology using confocal Raman microscopy has been developed for the analysis of postblast residues from 10 open-air explosions caused by 10 different explosives (TNT, RDX, PETN, TATP, HMTD, dynamite, black powder, ANFO, chloratite, and ammonal) commonly used in improvised explosive devices. The methodology for the determination of postblast particles from explosives consisted of examining the samples surfaces with both the naked eye, first, and microscopically (10× and 50×), immediately afterward; and finally, analyzing the selected residues by confocal Raman spectroscopy in order to identify the postblast particles from explosives. Interestingly, confocal Raman microscopy has demonstrated to be highly suitable to rapidly, selectively, and noninvasively analyze postblast microscopic particles from explosives up to the nanogram range.

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

    Institute of Scientific and Technical Information of China (English)

    唐志列; 杨初平; 裴红津; 梁瑞生; 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.

  20. Detection of functional groups and antibodies on microfabricated surfaces by confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Nashat, A.H.; Ferrari, M. [Univ. of California, Berkeley, CA (United States); Moronne, M. [Lawrence Berkeley National Lab., CA (United States)

    1998-10-20

    Fluorescence confocal microscopy was used to characterize micron-sized microfabricated silicon particles and planar oxides surfaces after silanization and immobilization of IgG antibody. Surfaces treated with amino- and mercaptosilanes were tested by the presence of amine and sulfhydryl groups by labeling with specific fluorescein probes. In addition, human antibody (IgG) was immobilized to the thiol-coated microparticles using the heterobifunctional crosslinker succinimidyl 4-(N-maleimidolmthyl)-cyclohexane-1-carboxylate. Estimates of the surface density of IgG were consistent with 8.3% of a monolayer of covalently-bound antibody. Confocal images confirmed uniform layers of both silanes and antibodies on the microparticles. The sensitivity limit for the confocal measurements was determined to be as low as 1.5 x 10{sup {minus}5} fluors per nm{sup 2}.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2016-01-01

    Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes. PMID:27515076

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

    Science.gov (United States)

    Lunter, Dominique; Daniels, Rolf

    2016-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-28

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

  5. Sub-micron imaging of buried integrated circuit structures using scanning confocal electron microscopy.

    Energy Technology Data Exchange (ETDEWEB)

    Frigo, S. P.; Levine, Z.; Zaluzec, N. J.; Materials Science Division; Northern Arizona Univ.; NIST

    2002-09-09

    Two-dimensional images of model integrated circuit components were collected using the technique of scanning confocal electron microscopy. For structures embedded about 5 {mu}m below the surface of a silicon oxide dielectric, a lateral resolution of 76{+-}9 nm was measured. Elemental mapping via x-ray emission spectrometry is demonstrated. A parallax analysis of images taken for various tilt angles to the electron beam allowed determination of the spacing between two wiring planes. The results show that scanning confocal electron microscopy is capable of probing buried structures at resolutions that will be necessary for the inspection of next-generation integrated circuit technology.

  6. Comparison between optical techniques and confocal microscopy for defect detection on thin wires

    Energy Technology Data Exchange (ETDEWEB)

    Siegmann, Philip; Sanchez-Brea, Luis Miguel; Martinez-Anton, Juan Carlos; Bernabeu, Eusebio

    2004-11-15

    Conventional microscopy techniques, such as atomic force microscopy (AFM), scanning electron microscopy (SEM), and confocal microscopy (CM) are not suitable for on-line surface inspection of fine metallic wires. In the recent years, some optical techniques have been developed to be used for those tasks. However, they need a rigorous validation. In this work, we have used confocal microscopy to obtain the topography z(x,y) of wires with longitudinal defects, such as dielines. The topography has been used to predict the light scattered by the wire. These simulations have been compared with experimental results, showing a good agreement.

  7. Microscopia confocal in vivo no diagnóstico de ceratite fúngica: relato de caso In vivo confocal microscopy in the diagnosis of fungal keratitis: case report

    Directory of Open Access Journals (Sweden)

    Gustavo Victor

    2006-06-01

    Full Text Available Os autores relatam um caso em que a microscopia confocal in vivo ajudou no diagnóstico e acompanhamento de ceratite fúngica. Realizou-se a microscopia confocal in vivo em paciente com úlcera corneana, que há 30 dias estava sendo tratada, sem obter melhora com uso de diversos medicamentos tópicos. O paciente também tinha se submetido à coleta de material corneano para análise laboratorial, com resultado negativo e inconclusivo. Foi observado à microscopia confocal, hifas e coleções infecciosas fúngicas. Dez dias após o diagnóstico confocal, o resultado de nova coleta de material corneano revelou crescimento de Fusarium sp.The authors describe a case of fungal keratitis that the in vivo confocal microscopy helped in the diagnosis and follow-up. Confocal microscopy was done in a patient's ulcer that did not improve with several topical medicines. Corneal scrapings were obtained and culture results were without conclusion. We observed hyphae and infectious collections on confocal microscopy. New corneal culture showed Fusarium sp ten days after confocal diagnosis.

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

    Science.gov (United States)

    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.

  9. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case.

    Science.gov (United States)

    Shahriari, Neda; Grant-Kels, Jane M; Rabinovitz, Harold S; Oliviero, Margaret; Scope, Alon

    2016-07-01

    Reflectance confocal microscopy (RCM) is an FDA approved noninvasive optical imaging technique that acquires cellular level-resolution skin images in vivo. Herein, we report a case of histopathologically proven large cell acanthoma (LCA) whose RCM features simulate those of squamous cell carcinoma in situ. PMID:27648388

  10. Handheld multispectral dual-axis confocal microscope for cervical cancer screening

    Science.gov (United States)

    Sarapukdee, Pongsak; Rattanavarin, Santi; Jarujareet, Ungkarn; Khemthongcharoen, Numfon; Jolivot, Romuald; Jung, Il Woong; López, Daniel; Mandella, Michael J.; Piyawattanametha, Wibool

    2013-03-01

    Our work demonstrates a MEMS based handheld dual-axis confocal microscope for cervical cancer screening. Imaging demonstration is performed with plant and animal tissue biopsies. The data is collected and displayed in real time with 2-5 Hz frame rates.

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

    NARCIS (Netherlands)

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

    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

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

    NARCIS (Netherlands)

    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

  13. Fundamental parameter based quantification algorithm for confocal nano-X-ray fluorescence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Schoonjans, Tom, E-mail: Tom.Schoonjans@UGent.be [X-ray Microspectroscopy and Imaging Research Group (XMI), Department of Analytical Chemistry, Ghent University, Krijgslaan 281 S12, B-9000 Ghent (Belgium); Silversmit, Geert; Vekemans, Bart [X-ray Microspectroscopy and Imaging Research Group (XMI), Department of Analytical Chemistry, Ghent University, Krijgslaan 281 S12, B-9000 Ghent (Belgium); Schmitz, Sylvia [Geosciences Institute/Mineralogy, Goethe University Frankfurt, Altenhoeferallee 1, D-60438 Frankfurt (Germany); Burghammer, Manfred; Riekel, Christian [ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble Cedex (France); Brenker, Frank E. [Geosciences Institute/Mineralogy, Goethe University Frankfurt, Altenhoeferallee 1, D-60438 Frankfurt (Germany); Vincze, Laszlo, E-mail: Laszlo.Vincze@UGent.be [X-ray Microspectroscopy and Imaging Research Group (XMI), Department of Analytical Chemistry, Ghent University, Krijgslaan 281 S12, B-9000 Ghent (Belgium)

    2012-01-15

    A new method for the quantification of X-ray fluorescence (XRF) was derived based on the fundamental parameter method (FPM). The FPM equations were adapted to accommodate the special case of confocal nano-XRF, i.e. X-ray nano-beam excitation coupled with confocal detection, taking into account the special characteristics of the detector channel polycapillary. A thorough error estimation algorithm based on the Monte Carlo method was applied, producing a detailed analysis of the uncertainties of the quantification results. The new FPM algorithm was applied on confocal nano-XRF data obtained from cometary dust returned by NASA's Stardust mission, recorded at beamline ID13 of the European Synchrotron Radiation Facility. - Highlights: Black-Right-Pointing-Pointer A new method for the quantification of confocal XRF is presented. Black-Right-Pointing-Pointer The quantification is based on the fundamental parameter method (FPM). Black-Right-Pointing-Pointer The new FPM algorithm was applied for the analysis of unique cometary dust particles. Black-Right-Pointing-Pointer The cometary particles were returned by NASA's Stardust mission in 2006. Black-Right-Pointing-Pointer Error estimation is based on the Monte Carlo method.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    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

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

    NARCIS (Netherlands)

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

    2001-01-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    NARCIS (Netherlands)

    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

  2. Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool

    Science.gov (United States)

    Yaroslavsky, Anna N.; Salomatina, Elena; Novak, John; Amat-Roldan, Ivan; Castano, Ana; Hamblin, Michael

    2006-02-01

    Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The developed technique may provide an efficient real-time optical tool for detecting skin pathology.

  3. Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos

    Science.gov (United States)

    Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mous...

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

    NARCIS (Netherlands)

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

    NARCIS (Netherlands)

    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

  6. Visualisation of biopolymer mixtures using confocal scanning laser microscopy (CSLM) and covalent labelling techniques

    NARCIS (Netherlands)

    Velde, van de F.; Weinbreck, F.; Edelman, M.W.; Linden, van der E.; Tromp, R.H.

    2003-01-01

    Confocal scanning laser microscopy (CSLM) has been used to study the behaviour of mixtures of proteins, gelatine, whey proteins and ß-lactoglobulin, and polysaccharides, dextran, gellan gum, carrageenan, gum Arabic, and starch. CSLM proved to be a suitable technique to visualise the microstructure o

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    NARCIS (Netherlands)

    Besseling, T. H.; Jose, J.; Blaaderen, A. Van

    2015-01-01

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

  9. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case.

    Science.gov (United States)

    Shahriari, Neda; Grant-Kels, Jane M; Rabinovitz, Harold S; Oliviero, Margaret; Scope, Alon

    2016-07-01

    Reflectance confocal microscopy (RCM) is an FDA approved noninvasive optical imaging technique that acquires cellular level-resolution skin images in vivo. Herein, we report a case of histopathologically proven large cell acanthoma (LCA) whose RCM features simulate those of squamous cell carcinoma in situ.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

    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

  12. Correlation of Biomicroscopic Findings with Confocal Microscopy in Eyes with Amiodarone-Induced Cornea Verticillata

    Directory of Open Access Journals (Sweden)

    Emine Kaya

    2014-01-01

    Full Text Available Objectives: To investigate the correlation between biomicroscopic and confocal microscopic findings in eyes with amiodarone-induced cornea verticillata. Materials and Methods: Sixteen eyes of 8 patients with amiodarone-induced cornea verticillata were evaluated. Eyes with keratopathy were staged according to Orlando slit-lamp microscopy classification. Confocal laser-scanning microscopy was performed by Rostock cornea modulated to HRT II (Heidelberg Engineering GmbH, Heidelberg, Germany, and staging was done according to Falke’s classification that is based on the degree of epithelial basal cell deposit accumulation. The relation between biomicroscopic staging and corneal involvement detected on confocal microscopy was assessed by Spearman correlation analysis. Results: The mean age of the 8 patients (5 male, 3 female was 63.1±7.2 (50 to 69 years. The mean duration of drug treatment was 12.1±11.8 (3 to 36 months, and the mean drug treatment dose was 312.5±223.2 (100 to 800 mg/day. At the time of examination, 50% of the patients had already given up the treatment at a mean of 29.5±15.8 (6 to 40 months ago, whereas the other 50% were still on amiodarone therapy. Hyper-reflecting deposits were observed in the basal epithelium, anterior-, mid-and deep-stroma, and in the endothelium on confocal microscopic examination. Correlation was detected between biomicroscopic and confocal microscopic stages (r=0.770, p<0.001. Frequency of detecting deposits in the stroma and endothelium was found to be increasing as the biomicroscopic stage increased (r=0.844; p<0.001 and r=0.551; p<0.01, respectively. Conclusion: In amiodarone-induced cornea verticillata, correlated results were detected between biomicroscopic and confocal microscopic staging. Therefore, in clinics where confocal microscopy is not available, biomicroscopic staging can be used as a guiding parameter in eyes with amiodarone-induced cornea verticillata. (Turk J Ophthalmol 2014; 44: 63-67

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

    Science.gov (United States)

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

    2016-03-01

    Confocal microscopy (CM) has been shown to correlate with oral mucosal histopathology in vivo. The purposes of this review are to summarize what we know so far about in vivo CM applications for oral mucosal pathologies, to highlight some current developments with CM devices relevant for oral applications, and to formulate where in vivo CM could hold further application for oral mucosal diagnosis and management. Ovid Medline® and/or Google® searches were performed using the terms 'microscopy, confocal', 'mouth neoplasms', 'mouth mucosa', 'leukoplakia, oral', 'oral lichen planus', 'gingiva', 'cheilitis', 'taste', 'inflammatory oral confocal', 'mucosal confocal' and 'confocal squamous cell oral'. In summary, inclusion criteria were in vivo use of any type of CM for the human oral mucosa and studies on normal or pathological oral mucosa. Experimental studies attempting to identify proteins of interest and microorganisms were excluded. In total 25 relevant articles were found, covering 8 main topics, including normal oral mucosal features (n=15), oral dysplasia or neoplasia (n=7), inflamed oral mucosa (n=3), taste impairment (n=3), oral autoimmune conditions (n=2), pigmented oral pathology/melanoma (n=1), delayed type hypersensitivity (n=1), and cheilitis glandularis (n=1). The evidence for using in vivo CM in these conditions is poor, as it is limited to mainly small descriptive studies. Current device developments for oral CM include improved probe design. The authors propose that future applications for in vivo oral CM may include burning mouth syndrome, intra-operative mapping for cancer surgery, and monitoring and targeted biopsies within field cancerization. PMID:26786962

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

    Science.gov (United States)

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

    2011-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Kobayashi A

    2014-02-01

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

  16. Learning reflectance confocal microscopy of melanocytic skin lesions through histopathologic transversal sections.

    Directory of Open Access Journals (Sweden)

    Juliana Casagrande Tavoloni Braga

    Full Text Available Histopathologic interpretation of dermoscopic and reflectance confocal microscopy (RCM features of cutaneous melanoma was timidly carried out using perpendicular histologic sections, which does not mimic the same plane of the image achieved at both techniques (horizontal plane. The aim of this study was to describe the transverse histologic sections research technique and correlate main dermoscopic features characteristic of cutaneous melanoma (atypical network, irregular globules and pseudopods with RCM and histopathology in perpendicular and transverse sections in order to offer a more precise interpretation of in vivo detectable features. Four melanomas and 2 nevi with different dermoscopic clues have been studied. Lesion areas that showed characteristic dermoscopic features were imaged by dermoscopy and confocal microscopy and directly correlated with histopathology in perpendicular and transverse sections. We presented the possibility to perform transverse sections as a new approach to understand RCM features. Atypical network showed different aspects in the 2 melanomas: in one case it was characterized by pleomorphic malignant melanocytes with tendency to form aggregates, whereas in the other elongated dendritic cells crowded around dermal papillae, some of them forming bridges that resembled the mitochondrial aspect at confocal and histopathology transversal sections. Pigment globules in melanomas and nevi differed for the presence of large atypical cells in the former, and pseudopods showed up as elongated nests protruded toward the periphery of the lesion. Transverse histologic research sections have a consistent dermoscopic and confocal correlate, and it may represent an help in confocal feature interpretation and an advance in improving melanoma diagnosis and knowledge of the biology of melanocytic lesions.

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

    Directory of Open Access Journals (Sweden)

    Zavislan James M

    2009-08-01

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

  18. Determination of sex by exfoliative cytology using acridine orange confocal microscopy: A short study

    Directory of Open Access Journals (Sweden)

    D Shyam Prasad Reddy

    2012-01-01

    Full Text Available Context: Establishing individuality is an imperative aspect in any investigation procedure. Sometimes, in identifying an individual, it becomes necessary to determine the sex of that particular individual. Combining rapidity with reliability, an innovative idea has been put forward using a confocal microscope in exfoliative cytology. In the present study, we have determined the sex of the individual from buccal mucosal scrapings. The exfoliative cells were observed for Barr bodies under a confocal microscope, and the percentage of Barr-body-positive cells was determined. Aims: The main objective of this study is to assess confocal microscopy for the determination of sex by observing Barr bodies in the exfoliative cells of both men and women. Settings and Design: Samples of buccal mucosa smears were made followed by acridine orange staining. The stained slides were observed under a confocal microscope and the data obtained was subjected for statistical analysis, especially for mean and standard deviation. Materials and Methods: Samples of buccal mucosa smears from 20 men and 20 women were obtained by scraping with flat wooden sticks (exfoliative cytology. The smears were fixed in 100% alcohol for 15 min, followed by acridine orange (AO staining as described by Von Bertalanffy et al. Smears stained with AO were examined under a confocal microscope and the percentage of Barr-body-positive cells was determined. Statistical Analysis Used: Data obtained was subjected for statistical analysis, especially for mean and standard deviation. Results: Two non-overlapping ranges for the percentage of Barr-body-positive cells have been obtained for men and women. It was observed that in the male samples, the percentage of Barr-body-positive cells ranged from 0-3%. In the female samples, the percentage of Barr-body-positive cells ranged from 18-72%, and all the females showed the presence of Barr bodies. Conclusion: The study showed that the presence of Barr

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

    International Nuclear Information System (INIS)

    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. Reflectance Confocal Microscopy as an Aid to Dermoscopy to Improve Diagnosis on Equivocal Lesions: Evaluation of Three Bluish Nodules

    Directory of Open Access Journals (Sweden)

    Sara Bassoli

    2010-01-01

    Full Text Available Nodular lesions can be difficult to diagnose under dermoscopy alone, since they often lack specific diagnostic features. Confocal microscopy can be used as an aid to dermoscopy, to increase the diagnostic accuracy on equivocal skin lesions. We report three cases of bluish nodular lesions, difficult to diagnose under dermoscopy alone. Confocal features were very useful in these cases to lead us to the correct diagnosis, recognizing benign versus malignant entities. Histopathology is also reported, with high correspondence compared to the confocal imaging.

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

    Directory of Open Access Journals (Sweden)

    Markus Piwko

    2015-08-01

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

  2. OCT and in vivo confocal microscopy of a pigmented corneal tumor-like lesion.

    Science.gov (United States)

    Szaflik, Jacek P; Oldak, Monika; Ulinska, Magdalena; Ulnska, Magdalena; Tesla, Piotr; Szaflik, Jerzy

    2009-01-01

    A 43-year-old woman presented with a pigmented flat tumor situated at the posterior surface of the cornea nasally in her left eye. Anterior-segment optical coherence tomography revealed that the lesion was similar to the iris leaf, was limited to the cornea, and did not communicate with the iridocorneal angle. In vivo scanning slit confocal microscopy imaged dense hyperreflective tissue behind the endothelium and bright spots dispersed on the adjacent endothelial surface. Multiple hyporeflective formations resembling cell nuclei were visualized within the hyperreflective mass and the cell borders were distinguished. The diagnosis of pigmented nevus or retrocorneal membrane was suspected. The authors conclude that anterior-segment optical coherence tomography and in vivo scanning slit confocal microscopy are useful in assessing the microstructure and penetration of pigmented corneal lesions.

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

    KAUST Repository

    Lin, Neil Y. C.

    2014-03-01

    We present a new design for a confocal rheoscope that enables uniform uniaxial or biaxial shear. The design consists of two precisely positioned parallel plates with a gap that can be adjusted down to 2 ±0.1 μm, allowing for the exploration of confinement effects. By using our shear cell in conjunction with a biaxial force measurement device and a high-speed confocal microscope, we are able to measure the real-time biaxial stress while simultaneously imaging the material three-dimensional structure. We illustrate the importance of the instrument capabilities by discussing the applications of this instrument in current and future research topics in colloidal suspensions. © 2014 AIP Publishing LLC.

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

    Science.gov (United States)

    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

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

    CERN Document Server

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

    2016-01-01

    We present a new design of a confocal rheoscope that enables us to precisely impose a uniform uniaxial or biaxial shear. The design consists of two precisely-positioned parallel plates. Our design allows us to adjust the gap between the plates to be as small as 2$\\pm$0.1 $\\mu$m, allowing for the exploration of confinement effects. By using our shear cell in conjunction with a biaxial force measurement device and a high-speed confocal microscope, we are able to measure the real-time biaxial stress while simultaneously imaging the material 3D structure. We illustrate the importance of the instrument capabilities by discussing the applications of this instrument in current and future research topics in colloidal suspensions.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Directory of Open Access Journals (Sweden)

    Francesca Salvatori

    2012-01-01

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

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

    Science.gov (United States)

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

    2014-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Truernit Elisabeth

    2008-06-01

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

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

    CERN Document Server

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

    2015-01-01

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

  11. 3-D Confocal microscopy track analysis: a promising tool for determining CR-39 response function

    Energy Technology Data Exchange (ETDEWEB)

    Vaginay, F. E-mail: francois.vaginay@univ-fcomte.fr; Fromm, M.; Pusset, D.; Meesen, G.; Chambaudet, A.; Poffijn, A

    2001-06-01

    A new method based on the use of the confocal microscope is described in order to evaluate the CR-39 response function for Li-7 ions with an incident energy of 10.77 MeV. This method uses the formulations developed by Fromm et al. and considers two etching velocities: V{sub B} represents the bulk etch rate and remains constant, and V{sub T} the track etch rate, which varies along the particle's path. The confocal microscope seems to bring big improvements for track analysis. The first results of V{sub T} versus the particle range are presented and compared with the curves obtained by the sequential etching method. The obtained V{sub T} are plotted and compared to LET, REL{sub 350} and the cumulative radial dose.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Ramla Gary

    2016-02-01

    Full Text Available 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.

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

    OpenAIRE

    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...

  15. Reflectance Confocal Mıcroscopy; Morphology of Normal Skin and The Use in Melanocytic Lesions

    OpenAIRE

    Aslı Vefa Turgut Erdemir; Ayşe Esra Koku Aksu

    2013-01-01

    In vivo reflectance confocal microscopy (RCM) is a noninvasive method that allows imaging of cells and structures in living skin with resolution close to the histological analysis. RCM allows the assessment of dynamic events in the epidermis, papillary dermis and superficial reticular dermis. From the stratum corneum to a depth of a maximum of 350 μm can be analysed. The noninvasive feature of this technique, allows repetitive imaging without any damage to the affected area and to evaluate th...

  16. The use of reflectance confocal microscopy for examination of benign and malignant skin tumors

    OpenAIRE

    Wielowieyska-Szybińska, Dorota; Białek-Galas, Kamila; Podolec, Katarzyna; Wojas-Pelc, Anna

    2014-01-01

    Reflectance confocal microscopy (RCM) is a modern, non-invasive diagnostic method that enables real-time imaging of epidermis and upper layers of the dermis with a nearly histological precision and high contrast. The application of this technology in skin imaging in the last few years has resulted in the progress of dermatological diagnosis, providing virtual access to the living skin erasing the need for conventional histopathology. The RCM has a potential of wide application in the dermatol...

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

    OpenAIRE

    Alexandrov, Theodore; Lasch, Peter

    2013-01-01

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

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

    OpenAIRE

    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...

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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....

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

    Science.gov (United States)

    Ulrich, Martina; Lange-Asschenfeldt, Susanne

    2013-06-01

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

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

    OpenAIRE

    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 ...

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

    OpenAIRE

    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...

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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.

  8. 3D elemental imaging of the crustacean Ceriodaphnia by means of SR confocal micro-XRF

    OpenAIRE

    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...

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

    Institute of Scientific and Technical Information of China (English)

    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.

  10. Confocal Ellipsoidal Reflector System for a Mechanically Scanned Active Terahertz Imager

    OpenAIRE

    Llombart, Nuria; Cooper, Ken B.; Dengler, Robert J.; Bryllert, Tomas; Siegel, Peter H.

    2010-01-01

    We present the design of a reflector system that can rapidly scan and refocus a terahertz beam for high-resolution standoff imaging applications. The proposed optical system utilizes a confocal Gregorian geometry with a small mechanical rotating mirror and an axial displacement of the feed. For operation at submillimeter wavelengths and standoff ranges of many meters, the imaging targets are electrically very close to the antenna aperture. Therefore the main reflector surface must be an ellip...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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 ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Jacquemin, P.B., E-mail: pbjacque@nps.edu [Mechanical Engineering, University of Victoria, EOW 548,800 Finnerty Road, Victoria, BC (Canada); Herring, R.A. [Mechanical Engineering, University of Victoria, EOW 548,800 Finnerty Road, Victoria, BC (Canada)

    2012-06-15

    A confocal holography microscope developed at the University of Victoria uniquely combines holography with a scanning confocal microscope to non-intrusively measure fluid temperatures in three-dimensions (Herring, 1997), (Abe and Iwasaki, 1999), (Jacquemin et al., 2005). The Confocal Scanning Laser Holography (CSLH) microscope was built and tested to verify the concept of 3D temperature reconstruction from scanned holograms. The CSLH microscope used a focused laser to non-intrusively probe a heated fluid specimen. The focused beam probed the specimen instead of a collimated beam in order to obtain different phase-shift data for each scan position. A collimated beam produced the same information for scanning along the optical propagation z-axis. No rotational scanning mechanisms were used in the CSLH microscope which restricted the scan angle to the cone angle of the probe beam. Limited viewing angle scanning from a single view point window produced a challenge for tomographic 3D reconstruction. The reconstruction matrices were either singular or ill-conditioned making reconstruction with significant error or impossible. Establishing boundary conditions with a particular scanning geometry resulted in a method of reconstruction with low error referred to as 'wily'. The wily reconstruction method can be applied to microscopy situations requiring 3D imaging where there is a single viewpoint window, a probe beam with high numerical aperture, and specified boundary conditions for the specimen. The issues and progress of the wily algorithm for the CSLH microscope are reported herein. -- Highlights: Black-Right-Pointing-Pointer Evaluation of an optical confocal holography device to measure 3D temperature of a heated fluid. Black-Right-Pointing-Pointer Processing of multiple holograms containing the cumulative refractive index through the fluid. Black-Right-Pointing-Pointer Reconstruction issues due to restricting angular scanning to the numerical aperture of the

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    OpenAIRE

    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.

  20. Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.

    Directory of Open Access Journals (Sweden)

    Franck P Martial

    Full Text Available Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded

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

    Energy Technology Data Exchange (ETDEWEB)

    Goetzinger, S [Institut fuer Physik, Humboldt-Universitaet zu Berlin, D-10117 Berlin (Germany); Menezes, L de S [Institut fuer Physik, Humboldt-Universitaet zu Berlin, D-10117 Berlin (Germany); Benson, O [Institut fuer Physik, Humboldt-Universitaet zu Berlin, D-10117 Berlin (Germany); Talapin, D V [Institut fuer Physikalische Chemie, Universitaet Hamburg, D-20146 Hamburg (Germany); Gaponik, N [Institut fuer Physikalische Chemie, Universitaet Hamburg, D-20146 Hamburg (Germany); Weller, H [Institut fuer Physikalische Chemie, Universitaet Hamburg, D-20146 Hamburg (Germany); Rogach, A L [Sektion Physik und CeNS, LMU Muenchen, D-80799 Munich (Germany); Sandoghdar, V [Laboratory of Physical Chemistry, Swiss Federal Institute of Technology (ETH), CH-8093 Zurich (Switzerland)

    2004-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    O. L. Pustovalova

    2009-01-01

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

  4. Histometric data obtained by in vivo confocal laser scanning microscopy in patients with systemic sclerosis

    Directory of Open Access Journals (Sweden)

    Altmeyer Peter

    2002-08-01

    Full Text Available Abstract Background It would be a benefit if time-saving, non-invasive methods could give hints for diagnosing systemic sclerosis. To investigate the skin of patients with systemic sclerosis using confocal laser scanning microscopy in vivo and to develop histometric parameters to describe characteristic cutaneous changes of systemic sclerosis observed by this new technique, we conducted an exploratory study. Materials and Methods Fifteen patients with systemic sclerosis treated with extracorporal photopheresis were compared with 15 healthy volunteers and 10 patients with other disorders also treated with extracorporal photopheresis. All subjects were investigated using confocal laser scanning microscopy in vivo. Results Micromorphologic characteristics of skin of patients with systemic sclerosis and measuring parameters for melanisation, epidermal hypotrophy, and fibrosis for dislocation of capillaries by collagen deposits in the papillary dermis were evaluated. An interesting finding was an increased thickness of the tissue in the dermal papillae superior to the first dermal papilla vessel. It was also possible to reproduce characteristic histologic features by confocal laser scanning microscopy in vivo. Histometric parameters for fibrosis and vascular features developed in this study showed significant differences in patients with systemic sclerosis compared to controls. Conclusions Although the predominant histopathological features in systemic sclerosis are findings of the reticular dermis and the subcutis, and in histopathological investigation the epidermis seems to remain unaffected by the disease, we have demonstrate some characteristic differences in the epidermis and papillary dermis by confocal laser scanning microscopy in vivo. Some of them have not been described so far. However, to use this technique as a tool for diagnosis and/or staging of systemic sclerosis, further studies are needed investigating the sensitivity and

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

  7. Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

    Science.gov (United States)

    Gareau, Dan; Bar, Anna; Snaveley, Nicholas; Lee, Ken; Chen, Nathaniel; Swanson, Neil; Simpson, Eric; Jacques, Steve

    2012-06-01

    For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4× histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kouichi Tsuji [Department of Applied Chemistry, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku Osaka 558-8585 (Japan); PRESTO-JST - Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan); Kazuhiko Nakano [Department of Applied Chemistry, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku Osaka 558-8585 (Japan)

    2007-05-15

    A new 3D micro-XRF instrument based on a confocal setup using two independent poly-capillary x-ray lenses and two x-ray sources (Cr and Mo targets) was developed. A full poly-capillary x-ray lens was attached to each x-ray tube. Another half poly-capillary lens was attached to a silicon drift x-ray detector (SDD). The focal spots of the three lenses were adjusted to a common position. The depth resolutions that were evaluated by use of a 10-{mu}m thick Au foil were approximately 90 {mu}m for the x-ray energy of Au L{alpha}. The effects of the dual Cr-Mo x-ray beam excitation were investigated. It was confirmed that the XRF intensity of light elements was increased by applying the Cr-target x-ray tube in a confocal configuration. In the proposed confocal configuration, 3D elemental mapping of the major elements of an amaranth seed was performed nondestructively at ambient air pressure. Each element of the seed showed different mapping images in the different depth layers. (authors)

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

    Science.gov (United States)

    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.

  10. Confocal reflectance quantitative phase microscopy system for cell biology studies (Conference Presentation)

    Science.gov (United States)

    Singh, Vijay Raj; So, Peter T. C.

    2016-03-01

    Quantitative phase microscopy (QPM), used to measure the refractive index, provides the optical path delay measurement at each point of the specimen under study and becomes an active field in biological science. In this work we present development of confocal reflection phase microscopy system to provide depth resolved quantitative phase information for investigation of intracellular structures and other biological specimen. The system hardware development is mainly divided into two major parts. First, creates a pinhole array for parallel confocal imaging of specimen at multiple locations simultaneously. Here a digital micro mirror device (DMD) is used to generate pinhole array by turning on a subset micro-mirrors arranged on a grid. Second is the detection of phase information of confocal imaging foci by using a common path interferometer. With this novel approach, it is possible to measure the nuclei membrane fluctuations and distinguish them from the plasma membrane fluctuations. Further, depth resolved quantitative phase can be correlated to the intracellular contents and 3D map of refractive index measurements.

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

    Science.gov (United States)

    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.

  12. Off-confocal Raman spectroscopy (OCRS) for subsurface measurements in layered turbid samples

    Science.gov (United States)

    Khan, Khan Mohammad; Ghosh, Nirmalya; Majumder, Shovan Kumar

    2016-09-01

    We report, for the first time, the development of a depth-sensitive Raman spectroscopy system for investigating subsurface depths in a layered turbid sample using the concept of varying Raman collection zones, while keeping the point of illumination fixed on the surface of the target sample. The system makes use of a conventional confocal Raman configuration and realizes the variation in Raman collection zones employing off-confocal detection. This is effected by moving the tip of the Raman detection fiber (acting as the pinhole aperture) from the focus of the Raman collection objective either by taking the point of detection away from the objective (along its axis) or bringing it closer to the objective (along the same axis), thereby essentially offering two ways of enabling subsurface interrogation at a given time. Another important attraction of the approach is that it can be used for analyzing layered turbid samples at depths beyond the reach of the conventional confocal Raman, though not at the cost of any further modifications in its instrumentation. Furthermore, the illumination point remains fixed on the sample surface and no adjustment is required in the sample arm, which indeed are significant advantages for depth-sensitive measurements in situ from layered turbid samples, particularly those having irregular surfaces (like biological tissues). The ability of the system to recover Raman spectra of the subsurface layer was demonstrated using a layered non-biological phantom and a biological tissue sample.

  13. The Enhancement of Confocal Images of Tissues at Bulk Optical Immersion

    CERN Document Server

    Meglinski, I V; Bashkatov, A N; Genina, E A; Tuchin, V V

    2003-01-01

    The purpose of the present work is a theoretical examination of how localized skin-tissue dehydration affects the depth of the confocal probing and what depth of effective detection can be reached with the chemical administration of skin tissues. A semi-infinite multilayer Monte Carlo model is used to estimate spatial localization of the output signal offered by a confocal probe. A solution of glycerol is taken in the capacity of innocuous osmotic agent. Diffusion of this bio-compatible chemical agent into the skin temporarily pushes water out of the tissues and results in the matching of the refractive indices of skin structural elements. This temporarily decreases scattering and increases transparency of topical skin layers, which allows for unrestricted light to permeate deeper into the skin. The results of simulation show that signal spatial localization offered by a confocal probe in the skin tissues during their clearing is usable for the monitoring of deep reticular dermis and improving the image contr...

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

    Science.gov (United States)

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

    2010-08-01

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

  15. 3-D reconstruction of neurons from multichannel confocal laser scanning image series.

    Science.gov (United States)

    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.

  16. Simultaneous confocal fluorescence microscopy and optical coherence tomography for drug distribution and tissue integrity assessment

    Science.gov (United States)

    Rinehart, Matthew T.; LaCroix, Jeffrey; Henderson, Marcus; Katz, David; Wax, Adam

    2011-03-01

    The effectiveness of microbicidal gels, topical products developed to prevent infection by sexually transmitted diseases including HIV/AIDS, is governed by extent of gel coverage, pharmacokinetics of active pharmaceutical ingredients (APIs), and integrity of vaginal epithelium. While biopsies provide localized information about drug delivery and tissue structure, in vivo measurements are preferable in providing objective data on API and gel coating distribution as well as tissue integrity. We are developing a system combining confocal fluorescence microscopy with optical coherence tomography (OCT) to simultaneously measure local concentrations and diffusion coefficients of APIs during transport from microbicidal gels into tissue, while assessing tissue integrity. The confocal module acquires 2-D images of fluorescent APIs multiple times per second allowing analysis of lateral diffusion kinetics. The custom Fourier domain OCT module has a maximum a-scan rate of 54 kHz and provides depth-resolved tissue integrity information coregistered with the confocal fluorescence measurements. The combined system is validated by imaging phantoms with a surrogate fluorophore. Time-resolved API concentration measured at fixed depths is analyzed for diffusion kinetics. This multimodal system will eventually be implemented in vivo for objective evaluation of microbicide product performance.

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    Safak Korkmaz

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    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

  1. Observation of posterior corneal vesicles with in vivo confocal microscopy and anterior segment OCT

    Directory of Open Access Journals (Sweden)

    Ryou Watanabe

    2010-10-01

    Full Text Available Ryou Watanabe, Toru Nakazawa, Nobuo FuseDepartment of Ophthalmology, Tohoku University Graduate School of Medicine, Sendai, JapanAbstract: The histopathology of posterior corneal vesicles (PCV has not yet been revealed. A 15-year-old girl, who was diagnosed by slit-lamp microscopy as PCV, was examined using specular microscopy, in vivo confocal microscopy, and anterior segment OCT (optical coherence tomography. Anterior segment OCT showed that the thickness of both corneas was within normal limits. At the same time, in vivo confocal microscopy revealed endothelial cells in the rounded dark areas, acellular hyporeflective layers on the Descemet’s membrane, and hyperreflective linear lesions. These findings were not reported previously by slit-lamp and specular microscopy. The abnormal findings only existed at the Descemet’s membrane and corneal endothelial layer. Previous reports dealing with posterior polymorphous dystrophy (PPMD examined using in vivo confocal microscopy reported almost the same findings, suggesting that PCV and PPMD may be the same at the microstructural level.Keywords: cornea, Descemet’s membrane, imaging

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

    Directory of Open Access Journals (Sweden)

    Elif Demirkilinc Biler

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Cotarla Ion

    2008-01-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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.

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

    Directory of Open Access Journals (Sweden)

    Hayyam Kiratli

    2016-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Cosimo Mazzotta

    2014-04-01

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

  6. Dual-detection confocal microscopy: high-speed surface profiling without depth scanning

    Science.gov (United States)

    Lee, Dong-Ryoung; Gweon, Dae-Gab; Yoo, Hongki

    2016-03-01

    We propose a new method for three-dimensional (3-D) imaging without depth scanning that we refer to as the dual-detection confocal microscopy (DDCM). Compared to conventional confocal microscopy, DDCM utilizes two pinholes of different sizes. DDCM generates two axial response curves which have different stiffness according to the pinhole diameters. The two axial response curves can draw the characteristics curve of the system which shows the relationship between the axial position of the sample and the intensity ratio. Utilizing the characteristic curve, the DDCM reconstructs a 3-D surface profile with a single 2-D scanning. The height of each pixel is calculated by the intensity ratio of the pixel and the intensity ratio curve. Since the height information can be obtained directly from the characteristic curve without depth scanning, a major advantage of DDCM over the conventional confocal microscopy is a speed. The 3-D surface profiling time is dramatically reduced. Furthermore, DDCM can measure 3-D images without the influence of the sample condition since the intensity ratio is independent of the quantum yield and reflectance. We present two types of DDCM, such as a fluorescence microscopy and a reflectance microscopy. In addition, we extend the measurement range axially by varying the pupil function. Here, we demonstrate the working principle of DDCM and the feasibility of the proposed methods.

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

    Energy Technology Data Exchange (ETDEWEB)

    Apedo, K.L., E-mail: apedo@unistra.fr [ICube, Université de Strasbourg, CNRS, 2 rue Boussingault, 67000 Strasbourg (France); Munzer, C.; He, H. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France); Montgomery, P. [ICube, Université de Strasbourg, CNRS, 23 rue du Loess, 67037 Strasbourg (France); Serres, N. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France); Fond, C. [ICube, Université de Strasbourg, CNRS, 2 rue Boussingault, 67000 Strasbourg (France); Feugeas, F. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France)

    2015-02-15

    Scanning electron microscopy and scanning probe microscopy have been used for several decades to better understand the microstructure of cementitious materials. Very limited work has been performed to date to study the roughness of cementitious materials by optical microscopy such as coherence scanning interferometry (CSI) and chromatic confocal sensing (CCS). The objective of this paper is to better understand how CSI can be used as a tool to analyze surface roughness and topography of cement pastes. Observations from a series of images acquired using this technique on both polished and unpolished samples are described. The results from CSI are compared with those from a STIL confocal microscopy technique (SCM). Comparison between both optical techniques demonstrates the ability of CSI to measure both polished and unpolished cement pastes. - Highlights: • Coherence scanning interferometry (CSI) was used to analyze cement paste surfaces. • The results from the CSI were compared with those from a confocal microscopy. • 3D roughness parameters were obtained using the window resizing method. • Polished and unpolished cement pastes were studied.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease.

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

    Directory of Open Access Journals (Sweden)

    Oranat Chuchuen

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

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

    Science.gov (United States)

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

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2±0.3% and 98.0±0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2±0.3% and 94.0±2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

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

    Directory of Open Access Journals (Sweden)

    Chao Huang

    Full Text Available Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000. We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81 and nodal tissue (n = 81. In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively. Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease.

  15. One shot confocal microscopy based on wavelength/space conversion by use of multichannel spectrometer

    Science.gov (United States)

    Miyamoto, Shuji; Hase, Eiji; Ichikawa, Ryuji; Mnamikawa, Takeo; Yasui, Takeshi; Yamamoto, Hirotugu

    2016-03-01

    Confocal laser microscope (CLM) has been widely used in the fields of the non-contact surface topography, biomedical imaging, and other applications, because of two-dimensional (2D) or three-dimensional (3D) imaging capability with the confocal effect and the stray light elimination. Although the conventional CLM has acquired the 2D image by mechanical scanning of the focused beam spot, further reduction of image acquisition time and the robustness to various disturbances are strongly required. To this end, it is essential to omit mechanical scanning for the image acquisition. In this article, we developed the scan-less, full-field CLM by combination of the line-focused CLM with the wavelength/1D-space conversion. This combination enables us to form the 2D focal array of a 2D rainbow beam on a sample and to encode the 2D image information of a sample on the 2D rainbow beam. The image-encoded 2D rainbow beam was decoded as a spectral line image by a multi-channel spectrometer equipped with a CMOS camera without the need for the mechanical scanning. The confocal full-field image was acquired during 0.23 ms with the lateral resolution of 26.3μm and 4.9μm for the horizontal and vertical directions, respectively, and the depth resolution of 34.9μm. We further applied this scan-less, full-field CLM for biomedical imaging of a sliced specimen and non-contact surface topography of an industry products. These demonstrations highlight a high potential of the proposed scan-less, full-field CLM.

  16. Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy

    Science.gov (United States)

    Rich, Thomas C.; Annamdevula, Naga; Britain, Andrea L.; Mayes, Samuel; Favreau, Peter F.; Leavesley, Silas J.

    2016-03-01

    Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors -- Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization -- whether epifluorescence or confocal microscopy -- may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

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

    OpenAIRE

    sprotocols

    2015-01-01

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

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

    International Nuclear Information System (INIS)

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

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

    Institute of Scientific and Technical Information of China (English)

    KechengLi; DouglasW.Reeve

    2004-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Kecheng Li; Douglas W. Reeve

    2004-01-01

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

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

    CERN Document Server

    Papageorge, Alexander T; Lev, Benjamin L

    2016-01-01

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

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

    OpenAIRE

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ugur Turk

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Sosa, Carlos M. [IFEG-CONICET, (X5016LAE) Ciudad Universitaria, Córdoba (Argentina); Sánchez, H. Jorge [IFEG-CONICET, (X5016LAE) Ciudad Universitaria, Córdoba (Argentina); FAMAF, Universidad Nacional de Córdoba, (X5016LAE) Ciudad Universitaria, Córdoba (Argentina); Pérez, Carlos A. [Laboratório Nacional de Luz Síncrotron – LNLS, POB 6192, 13084-971 Campinas, SP (Brazil); Perez, Roberto D., E-mail: danperez@famaf.unc.edu.ar [IFEG-CONICET, (X5016LAE) Ciudad Universitaria, Córdoba (Argentina); FAMAF, Universidad Nacional de Córdoba, (X5016LAE) Ciudad Universitaria, Córdoba (Argentina)

    2014-01-15

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

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

    OpenAIRE

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

    2010-01-01

    We present the design and operation of a fiber-based cryogenic confocal microscope. It is designed as a compact cold-finger that fits inside the bore of a superconducting magnet, and which is a modular unit that can be easily swapped between use in a dilution refrigerator and other cryostats. We aimed at application in quantum optical experiments with electron spins in semiconductors and the design has been optimized for driving with and detection of optical fields with well-defined polarizat...

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

    Science.gov (United States)

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

    2013-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  8. Two-photon fluorescence properties of curcumin as a biocompatible marker for confocal imaging

    Science.gov (United States)

    Kumar, Abhishek; Li, Lian; Chaturvedi, Akanksha; Brzostowski, Joseph; Chittigori, Joshna; Pierce, Susan; Samuelson, Lynne A.; Sandman, Daniel; Kumar, Jayant

    2012-05-01

    Two-photon (TP) fluorescence properties of an antioxidant and anti-tumor molecule, curcumin, were investigated. The two-photon absorption (TPA) action cross-section was measured in organic solvents and found to be 6 GM in tetrahydrofuran and 2 GM in dimethyl sulfoxide. The measured TPA cross-section is comparable to that of rhodamine 6G. One-photon and TP confocal microscopy has demonstrated that curcumin is internalized in cells and can be used for imaging applications. Our investigation indicates that curcumin is a viable biocompatible TP fluorescent marker.

  9. Insights into esophagus tissue architecture using two-photon confocal microscopy

    Science.gov (United States)

    Liu, Nenrong; Wang, Yue; Feng, Shangyuan; Chen, Rong

    2013-08-01

    In this paper, microstructures of human esophageal mucosa were evaluated using the two-photon laser scanning confocal microscopy (TPLSCM), based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). The distribution of epithelial cells, muscle fibers of muscularis mucosae has been distinctly obtained. Furthermore, esophageal submucosa characteristics with cancer cells invading into were detected. The variation of collagen, elastin and cancer cells is very relevant to the pathology in esophagus, especially early esophageal cancer. Our experimental results indicate that the MPM technique has the much more advantages for label-free imaging, and has the potential application in vivo in the clinical diagnosis and monitoring of early esophageal cancer.

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

    Directory of Open Access Journals (Sweden)

    Attila Bokros

    2013-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Gauthier, E., E-mail: eric.gauthier@cea.fr [CEA/DSM/IRFM, CEA Cadarache, Saint-Paul-lez-Durance (France); Brosset, C.; Roche, H.; Tsitrone, E.; Pégourié, B.; Martinez, A. [CEA/DSM/IRFM, CEA Cadarache, Saint-Paul-lez-Durance (France); Languille, P. [PIIM, CNRS-Université de Provence, Centre de St Jérôme, 13397 Marseille, Cedex 20 (France); Courtois, X.; Lallier, Y. [CEA/DSM/IRFM, CEA Cadarache, Saint-Paul-lez-Durance (France); Salami, M. [AVANTIS CONCEPT, 75 Rue Marcelin Berthelot, 13858 Aix en Provence (France)

    2013-07-15

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2000-03-09

    Our original proposal was presented to the LDRD committee on February 18, 1999. The revised proposal that followed incorporated changes that addressed the issues, concerns, and suggestions put forth by the committee members both during the presentation and in subsequent discussions we've had with individual committee members. The goal of the proposal was to establish an SMD confocal microscopy capability and technology base at LLNL. Here we report on our progress during the 6-month period for which funding was available.

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

    Directory of Open Access Journals (Sweden)

    Pang A

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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

  15. Network formation in colloid-liquid crystal mixtures studied by confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J; Poon, W C K [School of Physics and the Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), JCMB, University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh EH9 3JZ (United Kingdom)

    2004-05-19

    We studied the formation of particle networks in colloid + liquid crystal mixtures cooled below the isotropic-nematic transition temperature by time-resolved laser scanning confocal microscopy. Our observations confirm a recent suggestion that alkane impurities play a crucial role in slowing down the speed of the isotropic-nematic interface. This enables the growing nematic droplets to 'push' particles into increasingly concentrated regions, ultimately resulting in a cellular network solid. We also found that faster cooling rates resulted in increasingly hierarchical cellular structures.

  16. Imaging Single ZnO Vertical Nanowire Laser Cavities using UV-Laser Scanning Confocal Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gargas, D.J.; Toimil-Molares, M.E.; Yang, P.

    2008-11-17

    We report the fabrication and optical characterization of individual ZnO vertical nanowire laser cavities. Dilute nanowire arrays with interwire spacing>10 ?m were produced by a modified chemical vapor transport (CVT) method yielding an ideal platform for single nanowire imaging and spectroscopy. Lasing characteristics of a single vertical nanowire are presented, as well as high-resolution photoluminescence imaging by UV-laser scanning confocal microscopy. In addition, three-dimensional (3D) mapping of the photoluminescence emission performed in both planar and vertical dimensions demonstrates height-selective imaging useful for vertical nanowires and heteronanostructures emerging in the field of optoelectronics and nanophotonics.

  17. Microscopia confocal reflectante aplicada ao diagnóstico do melanoma cutâneo Reflectance confocal microscopy in the diagnosis of cutaneous melanoma

    Directory of Open Access Journals (Sweden)

    Cintia Rito

    2009-12-01

    Full Text Available O melanoma cutâneo é um problema de saúde pública a nível mundial. Sua incidência tem aumentado, de forma marcante, nos últimos anos, e o diagnóstico e excisão precoces são essenciais para o bom prognóstico dos pacientes. Neste contexto, a dermatoscopia ganhou grande importância, nas últimas duas décadas, melhorando, de forma significativa, a acurácia do diagnóstico do melanoma, em estágios iniciais. Porém, existem algumas lesões benignas que apresentam dermatoscopia duvidosa, levando à realização de cirurgias desnecessárias. Mais recentemente, a microscopia confocal reflectante vem sendo introduzida como método diagnóstico auxiliar promissor, por ser um exame não-invasivo, realizado in vivo, de forma simples, indolor e de rápida execução. É a única técnica capaz de identificar estruturas celulares e examinar a epiderme e a derme papilar, com resolução semelhante à da histopatologia, com uma sensibilidade de 97,3%, e especificidade de 72,3% para o diagnóstico do melanoma cutâneo. É uma importante ferramenta diagnóstica, visto que não substitui o exame histopatológico realizado no pós-operatório, mas permite a abordagem racional das lesões com dermatoscopia duvidosa, evitando procedimentos cirúrgicos desnecessários.Skin melanoma is an international public health issue, with a considerable increase in frequency over the past few years. Early diagnosis and excision are essential for good patient prognosis. Over the past two decades dermoscopy has gained significance due to a major improvement in the accuracy of skin melanoma diagnosis in its early stage. However, there are some benign lesions of questionable dermoscopy, which may lead to the performance of unnecessary surgery. Recently, reflectance confocal microscopy has been introduced as a promising supplementary diagnostic method. It is a noninvasive, in vivo, simple, painless and quick exam. It is the only technique capable of identifying cellular

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-05-12

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

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

    Directory of Open Access Journals (Sweden)

    Karsten Rodenacker

    2011-05-01

    Full Text Available Populations of bacteria in sludge flocs and biofilm marked by fluorescence marked with fluorescent probes are digitised with a confocal laser scanning microscope. These data are used to analyse the microbial community structure, to obtain information on the localisation of specific bacterial groups and to examine gene expression. This information is urgently required for an in-depth understanding of the function and, more generally, the microbial ecology of biofilms. Methods derived from quantitative image analysis are applied to digitised data from confocal laser scanning microscopes to obtain quantitative descriptions of volumetric, topological (and topographical properties of different compartments of the components under research. In addition to free-moving flocs, also biofilms attached to a substratum in an experimental environment are analysed. Growth form as well as interaction of components are quantitatively described. Classical measurements of volume and intensity (shape, distribution and distance dependent interaction measurements using methods from mathematical morphology are performed. Mainly image (volume processing methods are outlined. Segmented volumes are globally and individually (in terms of 3Dconnected components measured and used for distance mapping transform as well as for estimation of geodesic distances from the substrate. All transformations are applied on the 3D data set. Resulting distance distributions are quantified and related to information on the identity and activity of the probe-identified bacteria.

  1. Spatial correlation of confocal Raman scattering and secondary ion mass spectrometric molecular images of lignocellulosic materials.

    Science.gov (United States)

    Li, Zhen; Chu, Li-Qiang; Sweedler, Jonathan V; Bohn, Paul W

    2010-04-01

    A detailed chemical and structural understanding of pre-enzymatic processing of lignocellulosic materials (LCMs) is a key objective in the development of renewable energy. Efficient rendering of biomass components into fermentable substrates for conversion into biofuel feedstocks would benefit greatly from the development of new technologies to provide high-quality, spatially resolved chemical information about LCMs during the various processing states. In an effort to realize this important goal, spatially correlated confocal Raman and mass spectrometric images allow the extraction of three-dimensional information from the perennial grass, Miscanthus x giganteus. An optical microscopy-based landmark registry scheme was developed that allows samples to be transferred between laboratories at different institutions, while retaining the capability to access the same physical regions of the samples. Subsequent to higher resolution imaging via confocal Raman microscopy and secondary ion mass spectrometry (SIMS), laser desorption-ionization mass spectrometry was used to place these regions within the overall sample architecture. Excellent sample registry was evident in the highly correlated Raman and SIMS images. In addition, the correlation of vibrational Raman scattering with mass spectra from specific spatial locations allowed confirmation of the assignment of intracellular globular structures to hemicellulose-rich lignin complexes, an assignment which could only be made tentatively from either image alone. PMID:20205411

  2. In vivo fluorescence confocal microscopy: indocyanine green enhances the contrast of epidermal and dermal structures

    Science.gov (United States)

    Skvara, Hans; Kittler, Harald; Schmid, Johannes A.; Plut, Ulrike; Jonak, Constanze

    2011-09-01

    In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

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

    Science.gov (United States)

    Hitt, Darren L.; Lowe, Mary L.

    1997-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Massimiliano eCardinale

    2014-03-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Confocal time lapse imaging as an efficient method for the cytocompatibility evaluation of dental composites.

    Science.gov (United States)

    Attik, Ghania Nina; Gritsch, Kerstin; Colon, Pierre; Grosgogeat, Brigitte

    2014-01-01

    It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

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

    Science.gov (United States)

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

    2012-07-01

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

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

    Science.gov (United States)

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

    1998-04-01

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

  9. In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract

    Science.gov (United States)

    Piyawattanametha, Wibool; Ra, Hyejun; Qiu, Zhen; Friedland, Shai; Liu, Jonathan T. C.; Loewke, Kevin; Kino, Gordon S.; Solgaard, Olav; Wang, Thomas D.; Mandella, Michael J.; Contag, Christopher H.

    2012-02-01

    Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 μm2 and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.

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

    Science.gov (United States)

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

    2016-10-01

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

  11. Surgical imaging catheter for confocal microendoscopy with advanced contrast delivery and focus systems

    Science.gov (United States)

    Tanbakuchi, Anthony A.; Rouse, Andrew R.; Udovich, Josh A.; Gmitro, Arthur F.

    2006-02-01

    We present a laparoscope for fluorescence confocal microendoscopy specifically designed for microscopic imaging during diagnostic laparoscopic surgery. The catheter consists of a disposable rigid distal tip which houses a flexible microendoscope and dye channel. The laparoscopic tip is a small disposable polycarbonate sheath containing two inner lumens with a glass window on the distal end. The sheath outer diameter suitable for use in a 5mm trocar. The smaller inner lumen provides a channel for delivering fluorescent contrast agents to the tissue through a 200um hole in the glass window. On the proximal end, the smaller lumen is coupled to a computer controlled fluid delivery system that controls the amount of contrast agent dispensed onto the tissue down to a fraction of a micro liter. The main lumen houses the microendoscope. The microendoscope incorporates a computer-controlled focus mechanism that can quickly and accurately focus while correcting for hysteresis. This fluorescence confocal micro-laparoscope will be tested in a small-scale clinical trial on women undergoing oophorectomy in the near future.

  12. Epiphany sealer penetration into dentinal tubules: Confocal laser scanning microscopic study

    Directory of Open Access Journals (Sweden)

    S V Ravi

    2014-01-01

    Full Text Available Aims: The aim of the following study was to evaluate the percentage and average depth of epiphany sealer penetration into dentinal tubules among the coronal, middle and apical thirds of the root using the confocal laser scanning microscopy (CLSM. Materials and Methods: A total of 10 maxillary central incisors were prepared and obturated with Resilon-Epiphany system. Sealer was mixed with fluorescent rhodamine B isothiyocyanate dye for visibility under confocal microscope. Teeth were cross-sectioned into coronal, middle and apical sections-2 mm thick. Sections were observed under CLSM. Images were analyzed for percentage and average depth of sealer penetration into dentinal tubules using the lasso tool in Adobe Photoshop CS3 (Adobe systems incorporated, San jose, CA and laser scanning microscopy (LSM 5 image analyzer. Statistical Analysis Used: One-way analysis of variance with Student Neuman Keuls post hoc tests, Kruskal-Wallis test and Wilcoxon signed-rank post hoc tests. Results: The results showed that a higher percentage of sealer penetration in coronal section-89.23%, followed by middle section-84.19% and the apical section-64.9%. Average depth of sealer penetration for coronal section was 526.02 μm, middle-385.26 μm and apical-193.49 μm. Conclusions: Study concluded that there was higher epiphany sealer penetration seen in coronal followed by middle and least at apical third of the roots.

  13. A fully automatic framework for cell segmentation on non-confocal adaptive optics images

    Science.gov (United States)

    Liu, Jianfei; Dubra, Alfredo; Tam, Johnny

    2016-03-01

    By the time most retinal diseases are diagnosed, macroscopic irreversible cellular loss has already occurred. Earlier detection of subtle structural changes at the single photoreceptor level is now possible, using the adaptive optics scanning light ophthalmoscope (AOSLO). This work aims to develop a fully automatic segmentation framework to extract cell boundaries from non-confocal split-detection AOSLO images of the cone photoreceptor mosaic in the living human eye. Significant challenges include anisotropy, heterogeneous cell regions arising from shading effects, and low contrast between cells and background. To overcome these challenges, we propose the use of: 1) multi-scale Hessian response to detect heterogeneous cell regions, 2) convex hulls to create boundary templates, and 3) circularlyconstrained geodesic active contours to refine cell boundaries. We acquired images from three healthy subjects at eccentric retinal regions and manually contoured cells to generate ground-truth for evaluating segmentation accuracy. Dice coefficient, relative absolute area difference, and average contour distance were 82±2%, 11±6%, and 2.0±0.2 pixels (Mean±SD), respectively. We find that strong shading effects from vessels are a main factor that causes cell oversegmentation and false segmentation of non-cell regions. Our segmentation algorithm can automatically and accurately segment photoreceptor cells on non-confocal AOSLO images, which is the first step in longitudinal tracking of cellular changes in the individual eye over the time course of disease progression.

  14. Dye-enhanced multimodal confocal microscopy for noninvasive detection of skin cancers in mouse models

    Science.gov (United States)

    Park, Jesung; Mroz, Pawel; Hamblin, Michael R.; Yaroslavsky, Anna N.

    2010-03-01

    Skin cancer is the most common form of human cancer. Its early diagnosis and timely treatment is of paramount importance for dermatology and surgical oncology. In this study, we evaluate the use of reflectance and fluorescence confocal microscopy for detecting skin cancers in an in-vivo trial with B16F10 melanoma and SCCVII squamous cell carcinoma in mice. For the experiments, the mice are anesthetized, then the tumors are infiltrated with aqueous solution of methylene blue and imaged. Reflectance images are acquired at 658 nm. Fluorescence is excited at 658 nm and registered in the range between 690 and 710 nm. After imaging, the mice are sacrificed. The tumors are excised and processed for hematoxylin and eosin histopathology, which is compared to the optical images. The results of the study indicate that in-vivo reflectance images provide valuable information on vascularization of the tumor, whereas the fluorescence images mimic the structural features seen in histopathology. Simultaneous dye-enhanced reflectance and fluorescence confocal microscopy shows promise for the detection, demarcation, and noninvasive monitoring of skin cancer development.

  15. Determination of nitric oxide mediating intracellular Ca2+ release on neurons based on confocal microscopy imaging

    Science.gov (United States)

    Zheng, Liqin; Wang, Yuhua; He, Yipeng; Zeng, Yixiu; Zhang, Yanding; Xie, Shusen

    2014-09-01

    The gas NO is a ubiquitous intercellular messenger that modulates a wide range of physiological and pathophysiological functions. But few studies were made to study the role of NO in the Ca2+ release in dorsal root ganglion (DRG) neurons by confocal microscopy. Thus the objective of this study was to assess if NO has a role in Ca2+ signaling in DRG neurons using confocal microscopy combined with special fluorescence probe Fluo-3/AM. A 100 μM concentration of the NO donors (Sodium Nitroprusside, Dihydrate, SNP) and NO synthase inhibitor (NG-Monomethyl-L-arginine, Monoacetate salt, L-NMMA) was used in the study. Results showed that the fluorescence intensity increased rapidly after injecting SNP, which indicated that SNP could enhance intracellular Ca2+ release. And the fluorescence intensity shrank gradually with time and kept at a low level for quite a long period after loading with L-NMMA which indicated that L-NMMA could block intracellular Ca2+ release. All these results demonstrated that NO was involved in the regulation of intracellular Ca2+ release in the DRG neurons.

  16. Impression Cytology in Eyes with Clinical and Confocal Scan Features of Acanthamoeba Keratitis

    Directory of Open Access Journals (Sweden)

    Mozhgan Rezaei Kanavi

    2013-01-01

    Full Text Available Purpose: To report impression cytology findings in specimens obtained from eyes with clinical and confocal microscopic features of Acanthamoeba keratitis (AK. Methods: In this interventional case series, impression cytology was obtained from corneas of patients with clinical and confocal microscopic features indicative of AK. Specimens were stained with Periodic acid-Schiff/Papanicolaou (PAS/PAP and examined for the presence of PAS-reactive Acanthamoeba cysts and/or hyperchromatic pear-shaped trophozoites. All specimens were then decolorized and re-stained with calcofluor white (CFW for the presence of chemofluorescent cysts. Results: Fifty-six eyes of 50 patients with mean age of 25.5±9.3 (range, 17 to 78 years were evaluated. Forty-one (82% cases were female and 51 (91.1% eyes had history of contact lens wear. PAS-reactive Acanthamoeba cysts and/or hyperchromatic pear-shaped trophozoites were identified in 53 eyes (94.6%, 2 of which demonstrated only trophozoitelike structures. CFW staining was able to reveal the presence of chemofluorescent cysts in all 51 specimens (91.1% in which cysts had been demonstrated with PAS/PAP staining. Trophozoites were not detected with CFW due to background staining of the cellulose acetate strip used for impression cytology. Conclusion: Corneal impression cytology, stained with PAS/PAP or with CFW, successfully detects Acanthamoeba and can be employed for early noninvasive diagnosis of AK.

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

    Science.gov (United States)

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

    2015-08-01

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

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2014-06-01

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

  20. Laser Scanning In Vivo Confocal Microscopy of Clear Grafts after Penetrating Keratoplasty

    Science.gov (United States)

    Wang, Dai; Song, Peng; Wang, Shuting; Sun, Dapeng; Wang, Yuexin; Zhang, Yangyang

    2016-01-01

    Purpose. To evaluate the changes of keratocytes and dendritic cells in the central clear graft by laser scanning in vivo confocal microscopy after penetrating keratoplasty (PK). Methods. Thirty adult subjects receiving PK at Shandong Eye Institute and with clear grafts and no sign of immune rejection after surgery were recruited into this study, and 10 healthy adults were controls. The keratocytes and dendritic cells in the central graft were evaluated by laser scanning confocal microscopy, as well as epithelium cells, keratocytes, corneal endothelium cells, and corneal nerves (especially subepithelial plexus nerves). Results. Median density of subepithelial plexus nerves, keratocyte density in each layer of the stroma, and density of corneal endothelium cells were all lower in clear grafts than in controls. The dendritic cells of five (16.7%) patients were active in Bowman's membrane and stromal membrane of the graft after PK. Conclusions. Activated dendritic cells and Langerhans cells could be detected in some of the clear grafts, which indicated that the subclinical stress of immune reaction took part in the chronic injury of the clear graft after PK, even when there was no clinical rejection episode. PMID:27034940

  1. Corneal confocal microscopy reveals trigeminal small sensory fiber neuropathy in Amyotrophic Lateral Sclerosis

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    Giulio eFerrari

    2014-10-01

    Full Text Available Although subclinical involvement of sensory neurons in amyotrophic lateral sclerosis (ALS has been previously demonstrated, corneal small fiber sensory neuropathy has not been reported to-date. We examined a group of sporadic ALS patients with corneal confocal microscopy, a recently developed imaging technique allowing in vivo observation of corneal small sensory fibers. Corneal confocal microscopy examination revealed a reduction of corneal small fiber sensory nerve number and branching in ALS patients. Quantitative analysis demonstrated an increase in tortuosity and reduction in length and fractal dimension of ALS patients’ corneal nerve fibers compared to age-matched controls. Moreover, bulbar function disability scores were significantly related to measures of corneal nerve fibers anatomical damage.Our study demonstrates for the first time a corneal small fiber sensory neuropathy in ALS patients. This finding further suggests a link between sporadic ALS and facial-onset sensory and motor neuronopathy (FOSMN syndrome, a rare condition characterized by early sensory symptoms (with trigeminal nerve distribution, followed by wasting and weakness of bulbar and upper limb muscles. In addition, the finding supports a model of neurodegeneration in ALS as a focally advancing process.

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

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    Elizabeth Kolos

    2015-06-01

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

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

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    Inceu Georgeta

    2014-12-01

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

  4. Corneal confocal microscopy reveals trigeminal small sensory fiber neuropathy in amyotrophic lateral sclerosis

    Science.gov (United States)

    Ferrari, Giulio; Grisan, Enrico; Scarpa, Fabio; Fazio, Raffaella; Comola, Mauro; Quattrini, Angelo; Comi, Giancarlo; Rama, Paolo; Riva, Nilo

    2014-01-01

    Although subclinical involvement of sensory neurons in amyotrophic lateral sclerosis (ALS) has been previously demonstrated, corneal small fiber sensory neuropathy has not been reported to-date. We examined a group of sporadic ALS patients with corneal confocal microscopy, a recently developed imaging technique allowing in vivo observation of corneal small sensory fibers. Corneal confocal microscopy (CCM) examination revealed a reduction of corneal small fiber sensory nerve number and branching in ALS patients. Quantitative analysis demonstrated an increase in tortuosity and reduction in length and fractal dimension of ALS patients’ corneal nerve fibers compared to age-matched controls. Moreover, bulbar function disability scores were significantly related to measures of corneal nerve fibers anatomical damage. Our study demonstrates for the first time a corneal small fiber sensory neuropathy in ALS patients. This finding further suggests a link between sporadic ALS and facial-onset sensory and motor neuronopathy (FOSMN) syndrome, a rare condition characterized by early sensory symptoms (with trigeminal nerve distribution), followed by wasting and weakness of bulbar and upper limb muscles. In addition, the finding supports a model of neurodegeneration in ALS as a focally advancing process. PMID:25360111

  5. In vivo corneal confocal microscopy in diabetes: Where we are and where we can get.

    Science.gov (United States)

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-09-15

    In vivo corneal confocal microscopy (IVCCM) is a novel, reproducible, easy and noninvasive technique that allows the study of the different layers of the cornea at a cellular level. As cornea is the most innervated organ of human body, several studies investigated the use of corneal confocal microscopy to detect diabetic neuropathies, which are invalidating and deadly complications of diabetes mellitus. Corneal nerve innervation has been shown impaired in subjects with diabetes and a close association between damages of peripheral nerves due to the diabetes and alterations in corneal sub-basal nerve plexus detected by IVCCM has been widely demonstrated. Interestingly, these alterations seem to precede the clinical onset of diabetic neuropathies, paving the path for prevention studies. However, some concerns still prevent the full implementation of this technique in clinical practice. In this review we summarize the most recent and relevant evidences about the use of IVCCM for the diagnosis of peripheral sensorimotor polyneuropathy and of autonomic neuropathy in diabetes. New perspectives and current limitations are also discussed.

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

    Science.gov (United States)

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

    2016-03-01

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

  7. Laser Scanning In Vivo Confocal Microscopy of Clear Grafts after Penetrating Keratoplasty

    Directory of Open Access Journals (Sweden)

    Dai Wang

    2016-01-01

    Full Text Available Purpose. To evaluate the changes of keratocytes and dendritic cells in the central clear graft by laser scanning in vivo confocal microscopy after penetrating keratoplasty (PK. Methods. Thirty adult subjects receiving PK at Shandong Eye Institute and with clear grafts and no sign of immune rejection after surgery were recruited into this study, and 10 healthy adults were controls. The keratocytes and dendritic cells in the central graft were evaluated by laser scanning confocal microscopy, as well as epithelium cells, keratocytes, corneal endothelium cells, and corneal nerves (especially subepithelial plexus nerves. Results. Median density of subepithelial plexus nerves, keratocyte density in each layer of the stroma, and density of corneal endothelium cells were all lower in clear grafts than in controls. The dendritic cells of five (16.7% patients were active in Bowman’s membrane and stromal membrane of the graft after PK. Conclusions. Activated dendritic cells and Langerhans cells could be detected in some of the clear grafts, which indicated that the subclinical stress of immune reaction took part in the chronic injury of the clear graft after PK, even when there was no clinical rejection episode.

  8. Confocal Microscopy–Guided Laser Ablation for Superficial and Early Nodular Basal Cell Carcinoma

    Science.gov (United States)

    Chen, Chih-Shan Jason; Sierra, Heidy; Cordova, Miguel; Rajadhyaksha, Milind

    2014-01-01

    Importance Laser ablation is a rapid and minimally invasive approach for the treatment of superficial skin cancers, but efficacy and reliability vary owing to lack of histologic margin control. High-resolution reflectance confocal microscopy (RCM) may offer a means for examining margins directly on the patient. Observations We report successful elimination of superficial and early nodular basal cell carcinoma (BCC) in 2 cases-, using RCM imaging to guide Er-:YAG laser ablation. Three-dimensional (3-D) mapping is feasible with RCM-, to delineate the lateral border and thickness of the tumor. Thus, the surgeon may deliver laser fluence and passes with localized control—ie, by varying the ablation parameters in sub-lesional areas with specificity that is governed by the 3-D topography of the BCC. We further demonstrate intra-operative detection of residual BCC after initial laser ablation and complete removal of remaining tumor by additional passes. Both RCM imaging and histologic sections confirm the final clearance of BCC. Conclusions and Relevance Confocal microscopy may enhance the efficacy and reliability of laser tumor ablation. This report represents a new translational application for RCM imaging, which, when combined with an ablative laser, may one day provide an efficient and cost-effective treatment for BCC. PMID:24827701

  9. Characterisation of the varnish-object interface using white light confocal profilometry

    Science.gov (United States)

    Wei, W.; Frohn, J.; Weber, M.

    2007-07-01

    An ongoing concern of conservators when restoring paintings and polychrome objects is the possibility of irreversible damage to the original paint layers. This is the case when removal and replacement of aged varnishes is being considered. Although much work has been conducted to determine the effect of the removal of a varnish on the condition of the (painted) surface, there is still some uncertainty in the conclusions because it has, until recently, been impossible to characterize the original surface under the varnish in situ. A promising solution to this problem is the use of white light confocal profilometry. An initial study has been conducted using this technique to measure the roughness of pencil lines and oil paint under a varnish. Measurements were made on areas up to several mm2 in size, at lateral resolutions down to 1 μm, and vertical (roughness) resolutions of 0.1-0.3 μm. It was found that the surface roughness of surfaces under relatively thick varnishes can be measured at relatively low magnification (20x). For thin varnish layers typical of paintings, higher magnifications are required. White light confocal profilometry has thus been shown to be a useful tool for in situ studies of the (sub)surface properties of (painted) objects.

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

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    Vineeta Nikhil

    2013-01-01

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

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

    Science.gov (United States)

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

    2014-08-15

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

  12. In-vivo characterization of DALM in ulcerative colitis with high-resolution probe-based confocal laser endomicroscopy

    Institute of Scientific and Technical Information of China (English)

    Giovanni D De Palma; Stefania Staibano; Saverio Siciliano; Francesco Maione; Maria Siano; Dario Esposito; Giovanni Persico; Yang Yi

    2011-01-01

    Recently, the use of confocal laser endomicroscopy (CLE)in the diagnosis of chronic ulcerative colitis (CUC) was reported. In this brief report we aimed to assess the application of probe-based CLE to characterize colonic mucosa and dysplasia in CUC. The study involved a patient presenting long-standing CUC. Confocal imaging of both the inflamed mucosa, a circumscribed lesion (dysplasiaassociated lesional mass), and adjacent colonic mucosa are demonstrated and the correlation between the CLE and histological images. Inflamed mucosa and dysplasia showed specific alteration of crypt architecture, cellular infiltration, and vessel architecture with an excellent correlation between CLE and standard histological examination.

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

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    Babu Kalpana

    2010-01-01

    Full Text Available We report an interesting ocular finding of bilateral multiple coin-shaped epithelial lesions along with the confocal microscopy findings in a patient following an acute attack of erythema multiforme (EM minor. A 30-year-old male presented with a history of watering and irritation in both eyes of three days duration. He was diagnosed to have EM minor and was on oral acyclovir. Slit-lamp examination revealed multiple coin-shaped epithelial lesions. Confocal microscopy showed a corresponding conglomerate of hyper-reflective epithelial lesions. The corneal lesions resolved over six weeks with oral steroids and acyclovir. An immunological mechanism is suspected.

  14. [Studies on Effect of Alkali Pretreatment on Anaerobic Digestion of Rice Straw with Confocal Raman Microscopy].

    Science.gov (United States)

    Xia, Yi-hua; Luo, Liu-bin; Li, Xiao-li; He, Yong; Sheng, Kui-chuan

    2015-03-01

    NaOH pretreatment is a convenient and effective method which is widely used in rice straw anaerobic digestion. But the mechanism of the alkaline (NaOH) hydrolysis of biopolymers compositions and polymeric cross-linked network structures of rice straw cell wall need further study. This paper firstly studied the effect and mechanism of alkali pretreatment on anaerobic digestion and biogas production of rice straw by using a combination of confocal Raman microscopy and transmission electron microscope. First, the original rice straw and the rice straw pretreated by NaOH were taken for mapping scanning by confocal Raman microscopy with micron-scale spatial resolution. Then principal component analysis was adopted to extract main information of Raman spectra, it could be found that the two types of samples were respectively presented with ray-like distribution in the first two principal component space, which were with cumulative contribution of 99%. And there was a clear boundary between the two types of samples without any overlapping, indicating that there was a significant difference of Raman spectral characteristic between original rice leaf and rice leaf pretreated by NaOH. Further analysis of the loading weights of the first two principal components showed that the Raman peaks at 1 739, 1 508 and 1 094 cm(-1) were the important bands, and these three Raman peaks were attributed to the scattering of hemicellulose, cellulose and lignin respectively. Following, chemical imaging analysis of hemicellulose, cellulose and lignin were achieved by combining these Raman peaks and microscopic image information. It could be found that the NaOH pretreatment resulted in a loss of dense spatial uniformity structure of tissue and great decreases of the contents of these three ingredients, particularly lignin. It can be concluded that it is feasible to non-destructively measure hemicellulose, lignin and cellulose in rice straw tissue by confocal Raman microscopy, and to achieve

  15. Roughness of biopores and cracks in Bt-horizons by confocal laser scanning microscopy

    Science.gov (United States)

    Leue, Martin; Gerke, Horst H.

    2016-04-01

    During preferential flow events in structured soils, the movement of water and reactive solutes is mostly restricted to larger inter-aggregate pores, cracks, and biopores. The micro-topography of such macropores in terms of pore shapes, geometry, and roughness is crucial for describing the exchange of water and solutes between macropores and the soil matrix. The objective of this study was to determine the surface roughness of intact structural surfaces from the Bt-horizon of Luvisols by confocal laser scanning microscopy. For this purpose, samples with the structural surface types including cracks with and without clay-organic coatings from Bt-horizons developed on loess and glacial till were compared. The surface roughness of these structures was calculated in terms of three parameters from selected surface regions of 0.36 mm² determined with a confocal laser scanning microscope of the type Keyence VK-X100K. These data were evaluated in terms of the root-mean-squared roughness, Rq, the curvature, Rku, and the ratio between surface area and base area, RA. Values of Rq and RA were smaller for coated as compared to uncoated cracks and earthworm burrows of the Bt-horizons from both parent materials. The results indicated that the illuviation of clayey material led to a "smoothing" of the crack surfaces, which was similar for the coarser textured till-Bt and the finer-textured loess-Bt surfaces. The roughness indicated by Rq and RA values was only slightly smaller and that indicated by Rku slightly higher for the structural surfaces from the loess as compared to those from the glacial till. These results suggest a minor importance of the parent material on the roughness of structural surfaces in the Bt-horizon. The similarity of Rq, RA, and Rku values between surfaces of earthworm burrows and uncoated cracks did not confirm an expected smoothing effect of the burrow walls by the earthworm. In contrast to burrow walls, root channels from the loess-Bt were smoother

  16. In-Vivo Slit Scanning Confocal Microscopy of Normal Corneas in Indian Eyes

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    Vanathi Murugesan

    2003-01-01

    Full Text Available Objective: To study the cellular populations of healthy corneas of Indian eyes using confocal microscopy and to evaluate the correlation with age, gender and laterality. Methods: The central corneas of 100 eyes of 50 healthy subjects were examined using an i n-vivo slit scanning confocal microscope (Confoscan 2. Images were analysed for cell densities of the epithelium, stroma and endothelium. Results: Good quality images enabling analysis of all cell layer populations were obtained in 74 eyes of 43 healthy subjects (22 males and 21 females with a mean age of 31.89 ± 13.47 (range 19-71 years. The basal epithelial cell density was 3601.38 ± 408.19 cells/mm2 (range 3017.3 -4231.1cells/mm2. The mean keratocyte nuclei density in the anterior stroma was 1005.02 ± 396.86 cells/mm2 (range 571.6 - 1249.6 cells/mm2 and in the posterior stroma was 654.32 ± 147.09 cells/mm2 (range 402.6 - 1049.1 cells/mm2. Posterior keratocyte nuclei density was 30.76% less than the anterior stromal keratocyte nuclei density. The difference in keratocyte nuclei density was statistically significant (P=0.001. The mean endothelial cell density was 2818.1 ± 361.03 cells/mm2 (range 2118.9 - 4434 cells/mm2 and the mean endothelial cell area was found to be 385.44 ± 42.66 mm2 (range 268.9 - 489.2 mm2. Hexagonal cells formed 22.5 - 69.4% of the endothelial cell populations (mean 42.04 ± 11.81%. Mean coefficient of cell size variation was 32.29 ± 3.06 (range 27.2 - 39.2. No statistically significant differences were found in cell densities of any corneal layer either between female and male patients or between right and left eyes. Basal epithelial cell density, anterior stromal keratocyte nuclei and posterior stromal keratocyte nuclei density were unaffected by age (r= 0.12, 0.07, - 0.12 respectively (P= 0.001. There was a statistically significant negative correlation between mean endothelial cell density and increase in age (r= - 0.42, P=0.001. Coefficient of cell size

  17. Changes in corneal parameters at confocal microscopy in treated glaucoma patients

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    Ranno S

    2011-07-01

    Full Text Available Stefano Ranno1, Paolo Fogagnolo2, Luca Rossetti3, Nicola Orzalesi3, Paolo Nucci11Eye Clinic, San Giuseppe Hospital, University of Milan, Milan, Italy; 2GB Bietti Foundation for Study and Research in Ophthalmology, Rome, Italy; 3Eye Clinic, Department of Medicine, San Paolo Hospital, University of Milan, Milan, ItalyBackground: The purpose of this study was to evaluate corneal parameters in treated glaucoma patients, nontreated glaucoma patients, and normal subjects using confocal microscopy.Methods: Forty patients with primary open-angle glaucoma and 22 untreated controls underwent confocal microscopy of the cornea using the Heidelberg retinal tomograph cornea module. The glaucoma group was divided into two subgroups, ie, patients on medical treatment for at least two years before inclusion (with beta-blockers or prostaglandin analogs and nontreated glaucoma patients. The following corneal parameters were evaluated: endothelial cell density and number, reflectivity, and tortuosity of sub-basal nerves. For reflectivity and tortuosity, a dedicated grading scale ranging from 0 to 4 was used. Differences between treatments were also evaluated in the treated glaucoma group.Results: Number of fibers and reflectivity of the sub-basal plexus were significantly lower in glaucoma patients as compared with controls (2.5 ± 0.7 versus 2.9 ± 0.9, P = 0.006, and 2.3 ± 0.8 versus 2.7 ± 0.9, P = 0.04, respectively, whereas tortuosity was significantly higher (2.6 ± 1 versus 2.0 ± 0.8, P = 0.007. Endothelial cell density (measured as cells per mm2 was lower in the glaucoma group comparing treated patients with nontreated patients (2826 ± 285 versus 3124 ± 272, P = 0.0003. Comparing treated patients with nontreated patients, relevant differences were found in number (2.3 ± 0.7 versus 2.8 ± 0.8, P = 0.004, tortuosity (2.8 ± 1 versus 2.2 ± 0.8, P = 0.004, and reflectivity (2.2 ± 0.8 versus 2.6 ± 0.8, P = 0.04. No differences in corneal parameters were

  18. 1KW Power Transmission Using Wireless Acoustic-Electric Feed-Through (WAEF)

    Science.gov (United States)

    Sherrit, S.; Bao, X.; Badescu, M.; Aldrich, J.; Bar-Cohen, Y.; Biederman, W.

    2008-01-01

    A variety of space applications require the delivery of power into sealed structures. Since the structural integrity can be degraded by holes for cabling we present an alternative method of delivering power and information using stress waves to the internal space of a sealed structure. One particular application of this technology is in sample return missions where it is critical to preserve the sample integrity and to prevent earth contamination. Therefore, the container has to be hermetically sealed and the integrity of the seal must be monitored in order to insure to a high degree of reliability the integrity of the sample return vessel. In this study we investigated the use of piezoelectric acoustic-electric power feed-through devices to transfer electric power wirelessly through a solid wall by using elastic or acoustic waves. The technology is applicable to a range of space and terrestrial applications where power is required by electronic equipment inside sealed containers, vacuum or pressure vessels, etc., where holes in the wall are prohibitive or may result in significant structural performance degradation or unnecessarily complex designs. To meet requirements of higher power applications, the feasibility to transfer kilowatts level power was investigated. Pre-stressed longitudinal piezoelectric feed-through devices were analyzed by finite element models and an equivalent circuit model was developed to predict the power transfer characteristics to different electric loads. Based on the results of the analysis a prototype device was designed, fabricated and a demonstration of the transmission of electric power up to 1.068-kW was successfully conducted. Efficiencies in the 80-90% range were also demonstrated and methods to increase the efficiency further are currently being considered.

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2010-03-01

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

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

    Science.gov (United States)

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

    2008-08-01

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

  2. Reflectance Confocal Mıcroscopy; Morphology of Normal Skin and The Use in Melanocytic Lesions

    Directory of Open Access Journals (Sweden)

    Aslı Vefa Turgut Erdemir

    2013-09-01

    Full Text Available In vivo reflectance confocal microscopy (RCM is a noninvasive method that allows imaging of cells and structures in living skin with resolution close to the histological analysis. RCM allows the assessment of dynamic events in the epidermis, papillary dermis and superficial reticular dermis. From the stratum corneum to a depth of a maximum of 350 μm can be analysed. The noninvasive feature of this technique, allows repetitive imaging without any damage to the affected area and to evaluate the response to the treatment. RCM, provides great convenience in assesment of normal skin and particularly in the evaluation of melanocytic lesions due to high contrast features of melanin and melanosomes. In this article morphology of normal skin and features of melanocytic lesions is described with RCM.

  3. A two dimensional optical input to one dimensional serial pulse transformation using confocal reflectors.

    Science.gov (United States)

    Hulse, George

    2014-01-01

    An optical approach using confocal parabolic reflectors is used to transform 2D input data based on spatial position to a 1D sequenced serial string. The optical input data are set up as a 2D array. Individual channels are established between the input array and the final output detector, which reads the data as a time based serial data. The transformation is achieved by changing the optical path length associated with each pixel and its channel to the output detector. The 2D data can be images or individual sources but the light must be parallel. This paper defines how to establish the channels and the calculations required to achieve the desired transformation.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Patrick Krauss

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

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

    Science.gov (United States)

    Cianconi, Luigi; Conte, Gabriele; Mancini, Manuele

    2011-01-01

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

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

    Science.gov (United States)

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

    2013-09-01

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

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

    Science.gov (United States)

    de Jonge, Niels [Oak Ridge, TN

    2010-08-17

    A confocal scanning transmission electron microscope which includes an electron illumination device providing an incident electron beam propagating in a direction defining a propagation axis, and a precision specimen scanning stage positioned along the propagation axis and movable in at least one direction transverse to the propagation axis. The precision specimen scanning stage is configured for positioning a specimen relative to the incident electron beam. A projector lens receives a transmitted electron beam transmitted through at least part of the specimen and focuses this transmitted beam onto an image plane, where the transmitted beam results from the specimen being illuminated by the incident electron beam. A detection system is placed approximately in the image plane.

  9. Quantitative Assessment of Birefringent Skin Structures in Scattered Light Confocal Imaging Using Radially Polarized Light

    Directory of Open Access Journals (Sweden)

    Natallia Eduarda Uzunbajakava

    2013-09-01

    Full Text Available The polarization characteristics of birefringent tissues could be only partially obtained using linearly polarized light in polarization sensitive optical imaging. Here we analyze the change in polarization of backscattered light from birefringent structures versus the orientations of the incident polarizations using linearly, circularly and radially polarized light in a cross-polarized confocal microscope. A spatially variable retardation plate composed of eight sectors of λ/2 wave plates was used to transform linearly polarized light into a radially polarized light. Based on the experimental data obtained from ex-vivo measurements on human scalp hairs and in-vivo measurements on hair and skin, we exemplify that the underestimation of the birefringence content resulting from the orientation related effects associated with the use of linearly polarized light for imaging tissues containing wavy birefringent structures could be minimized by using radially polarized light.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    1991-05-01

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

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

    Science.gov (United States)

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

    2000-01-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

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

    Science.gov (United States)

    Mathey, A; Lukins, P B

    2001-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-15

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

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

    Science.gov (United States)

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

    2010-11-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yin Liu

    2016-01-01

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

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2013-05-01

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

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

    Science.gov (United States)

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

    2007-01-01

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

  2. Three-dimensional imaging of DNA fragments during electrophoresis using a confocal detector

    Science.gov (United States)

    Brewer, Laurence R.; Davidson, Courtney; Balch, Joseph W.; Carrano, Anthony

    1995-04-01

    We have measured the 3D distribution of DNA fragments within an electrophoretic band. The measurements were made using a confocal microscope and a photon counting photomultiplier detector. A DNA sequencing standard was loaded into glass microchannel plates containing polyacrylamide gel. The measurements were made by scanning the plates in three dimensions using a mechanical stage under computer control, while electrophoresis was taking place. We found that the distribution of DNA was the same for all the bands measured in the sequencing ladder with an approximate Gaussian distribution along all three axis. These measurements are important to understand what physical forces shape electrophoretic bands confined by a channel and also as an aid in the design of high throughput DNA sequencers.

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

    International Nuclear Information System (INIS)

    The signal-to-noise ratio (SNR)-resolution trade-off is of great importance to bio-imaging applications where the aim is to image the sample using as little light as possible without significantly sacrificing image quality. In this paper the inherent SNR-resolution tradeoff in Confocal Fluorescence Microscopy (CFM) systems is presented by means of an effective tradeoff curve. A CFM system that employs a differential pinhole detection scheme has recently been shown to offer increased resolution, but at the expense of SNR. An optimum profile for the differential pinhole is identified in this paper that offers improved performance over a conventional (circular pinhole) system. The performance enhancement is illustrated through computer simulation. (paper)

  4. Pharmaceutical applications of confocal laser scanning microscopy: the physical characterisation of pharmaceutical systems.

    Science.gov (United States)

    Pygall, Samuel R; Whetstone, Joanne; Timmins, Peter; Melia, Colin D

    2007-12-10

    The application of confocal laser scanning microscopy (CLSM) to the physicochemical characterisation of pharmaceutical systems is not as widespread as its application within the field of cell biology. However, methods have been developed to exploit the imaging capabilities of CLSM to study a wide range of pharmaceutical systems, including phase-separated polymers, colloidal systems, microspheres, pellets, tablets, film coatings, hydrophilic matrices, and chromatographic stationary phases. Additionally, methods to measure diffusion in gels, bioadhesives, and for monitoring microenvironmental pH change within dosage forms have been utilised. CLSM has also been used in the study of the physical interaction of dosage forms with biological barriers such as the eye, skin and intestinal epithelia, and in particular, to determine the effectiveness of a plethora of pharmaceutical systems to deliver drugs through these barriers. In the future, there is continuing scope for wider exploitation of existing techniques, and continuing advancements in instrumentation.

  5. In vivo amyloid aggregation kinetics tracked by time-lapse confocal microscopy in real-time.

    Science.gov (United States)

    Villar-Piqué, Anna; Espargaró, Alba; Ventura, Salvador; Sabate, Raimon

    2016-01-01

    Amyloid polymerization underlies an increasing number of human diseases. Despite this process having been studied extensively in vitro, aggregation is a difficult process to track in vivo due to methodological limitations and the slow kinetics of aggregation reactions in cells and tissues. Herein we exploit the amyloid properties of the inclusions bodies (IBs) formed by amyloidogenic proteins in bacteria to address the kinetics of in vivo amyloid aggregation. To this aim we used time-lapse confocal microscopy and a fusion of the amyloid-beta peptide (A β42) with a fluorescent reporter. This strategy allowed us to follow the intracellular kinetics of amyloid-like aggregation in real-time and to discriminate between variants exhibiting different in vivo aggregation propensity. Overall, the approach opens the possibility to assess the impact of point mutations as well as potential anti-aggregation drugs in the process of amyloid formation in living cells.

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

    Science.gov (United States)

    Kakade, Rohan; Walker, John G.; Phillips, Andrew J.

    2016-01-01

    The signal-to-noise ratio (SNR)-resolution trade-off is of great importance to bio-imaging applications where the aim is to image the sample using as little light as possible without significantly sacrificing image quality. In this paper the inherent SNR-resolution tradeoff in Confocal Fluorescence Microscopy (CFM) systems is presented by means of an effective tradeoff curve. A CFM system that employs a differential pinhole detection scheme has recently been shown to offer increased resolution, but at the expense of SNR. An optimum profile for the differential pinhole is identified in this paper that offers improved performance over a conventional (circular pinhole) system. The performance enhancement is illustrated through computer simulation.

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

    KAUST Repository

    Nappini, Silvia

    2011-04-07

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

  8. Local order in a supercooled colloidal fluid observed by confocal microscopy

    CERN Document Server

    Gasser, U; Weitz, D A

    2003-01-01

    The local order in a supercooled monodisperse colloidal fluid is studied by direct imaging of the particles with a laser scanning confocal microscope. The local structure is analysed with a bond order parameter method, which allows one to discern simple structures that are relevant in this system. As expected for samples that crystallize eventually, a large fraction of the particles are found to sit in surroundings with dominant face-centred cubic or hexagonally close-packed character. Evidence for local structures that contain fragments of icosahedra is found, and, moreover, the icosahedral character increases with volume fraction phi, which indicates that it might play an important role at volume fractions near the glass transition.

  9. Examination of indentation geometry-constitutive behaviour relations with confocal microscopy and finite element modeling

    Energy Technology Data Exchange (ETDEWEB)

    Santos, C. [California Univ., Santa Barbara, CA (United States). Dept. of Materials; Odette, G.R.; Lucas, G.E. [California Univ., Santa Barbara, CA (United States). Dept. of Mechanical and Environmental Engineering]|[California Univ., Santa Barbara, CA (United States). Dept. of Chemical and Nuclear Engineering; Yamamoto, T. [Tohoku Univ., Sendai (Japan). Inst. for Materials Research

    1998-10-01

    Microhardness measurements have been of general interest in irradiated materials testing as a monitor of strength changes, and the geometry of the pile-up of material around the indentation has been found to be related to the work-hardening behavior. This relationship has been further examined here. Vickers microhardness tests were performed on a variety of metal alloys including low alloy, high Cr, and austenitic stainless steels and a Nb-Ti alloy. The pile-ups around the identations were quantified using confocal microscopy techniques. In addition, the indentation process and pile-up geometry was simulated using finite element techniques and the corresponding constitutive equations for each of the test materials. The results from these methods have been used to develop an improved understanding and quantification between the pile-up geometry and the constitutive behavior of the test material. (orig.) 10 refs.

  10. Local order in a supercooled colloidal fluid observed by confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gasser, U [Department of Physics and Division of Engineering and Applied Sciences, Harvard University, Cambridge, MA (United States); Schofield, Andrew [Department of Physics and Astronomy, University of Edinburgh, Edinburgh (United Kingdom); Weitz, D A [Department of Physics and Division of Engineering and Applied Sciences, Harvard University, Cambridge, MA (United States)

    2003-01-15

    The local order in a supercooled monodisperse colloidal fluid is studied by direct imaging of the particles with a laser scanning confocal microscope. The local structure is analysed with a bond order parameter method, which allows one to discern simple structures that are relevant in this system. As expected for samples that crystallize eventually, a large fraction of the particles are found to sit in surroundings with dominant face-centred cubic or hexagonally close-packed character. Evidence for local structures that contain fragments of icosahedra is found, and, moreover, the icosahedral character increases with volume fraction {phi}, which indicates that it might play an important role at volume fractions near the glass transition.

  11. Measurement of buried undercut structures in microfluidic devices by laser fluorescent confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Li Shiguang; Liu Jing; Nguyen, Nam-Trung; Fang Zhongping; Yoon, Soon Fatt

    2009-11-20

    Measuring buried, undercut microstructures is a challenging task in metrology. These structures are usually characterized by measuring their cross sections after physically cutting the samples. This method is destructive and the obtained information is incomplete. The distortion due to cutting also affects the measurement accuracy. In this paper, we first apply the laser fluorescent confocal microscopy and intensity differentiation algorithm to obtain the complete three-dimensional profile of the buried, undercut structures in microfluidic devices, which are made by the soft lithography technique and bonded by the oxygen plasma method. The impact of material wettability and the refractive index (n) mismatch among the liquid, samples, cover layer, and objective on the measurement accuracy are experimentally investigated.

  12. Evaluation of the Cytotoxic Behavior of Fungal Extracellular Synthesized Ag Nanoparticles Using Confocal Laser Scanning Microscope

    Directory of Open Access Journals (Sweden)

    Taher A. Salaheldin

    2016-03-01

    Full Text Available Silver nanoparticles have been synthesized by subjecting a reaction medium to a Fusarium oxysporum biomass at 28 °C for 96 h. The biosynthesized Ag nanoparticles were characterized on the basis of their anticipated peak at 405 nm using UV-Vis-NIR spectroscopy. Structural confirmation was evident from the characteristic X-ray diffraction (XRD pattern, high-resolution transmission electron Microscopy (HRTEM and the particle size analyzer. The Ag nanoparticles were of dimension 40 ± 5 nm and spherical in shape. The study mainly focused on using the confocal laser scanning microscope (CLSM to examine the cytotoxic activities of fungal synthesized Ag nanoparticles on a human breast carcinoma cell line MCF7 cell, which featured remarkable vacuolation, thus indicating a potent cytotoxic activity.

  13. Confocal Fluorescence Imaging Enables Noninvasive Quantitative Assessment of Host Cell Populations In Vivo Following Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Soumya Mitra, Oleg Mironov, Thomas H. Foster

    2012-01-01

    Full Text Available We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH-mediated photodynamic therapy (PDT of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV administration of 1 μmol kg-1 HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1+/CD11b+ leukocytes and major histocompatibility complex class II (MHC-II+ cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1+ cells in response to therapy. The maximum accumulation of Gr1+ cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1+ cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1+ cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2013-03-01

    Fibrin gels are biological networks that play a fundamental role in blood coagulation and other patho/physiological processes, such as thrombosis and cancer. Electron and confocal microscopies show a collection of fibers that are relatively monodisperse in diameter, not uniformly distributed, and connected at nodal points with a branching order of ∼3-4. Although in the confocal images the hydrated fibers appear to be quite straight (mass fractal dimension D(m) = 1), for the overall system 1, joined at randomly distributed nodal points. The resulting 3D network strikingly resembles real fibrin gels and can be sketched as an assembly of densely packed fractal blobs, i.e., regions of size ξ, where the fiber concentration is higher than average. The blobs are placed at a distance ξ0 between their centers of mass so that they are overlapped by a factor η =ξ/ξ0 and have D(m) ∼1.2-1.6. The in silico gels' structure is quantitatively analyzed by its 3D spatial correlation function g(3D)(r) and corresponding power spectrum I(q) = FFT(3D[g3D(r)]), from which ρ, d, D(m), η, and ξ0 can be extracted. In particular, ξ0 provides an excellent estimate of the gel mesh size. The in silico gels' I(q) compares quite well with real gels' elastic light-scattering measurements. We then derived an analytical form factor for accurately fitting the scattering data, which allowed us to directly recover the gels' structural parameters.

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

    Steinbach, Gábor; Kaňa, Radek

    2016-04-01

    Photosynthesis research employs several biophysical methods, including the detection of fluorescence. Even though fluorescence is a key method to detect photosynthetic efficiency, it has not been applied/adapted to single-cell confocal microscopy measurements to examine photosynthetic microorganisms. Experiments with photosynthetic cells may require automation to perform a large number of measurements with different parameters, especially concerning light conditions. However, commercial microscopes support custom protocols (through Time Controller offered by Olympus or Experiment Designer offered by Zeiss) that are often unable to provide special set-ups and connection to external devices (e.g., for irradiation). Our new system combining an Arduino microcontroller with the Cell⊕Finder software was developed for controlling Olympus FV1000 and FV1200 confocal microscopes and the attached hardware modules. Our software/hardware solution offers (1) a text file-based macro language to control the imaging functions of the microscope; (2) programmable control of several external hardware devices (light sources, thermal controllers, actuators) during imaging via the Arduino microcontroller; (3) the Cell⊕Finder software with ergonomic user environment, a fast selection method for the biologically important cells and precise positioning feature that reduces unwanted bleaching of the cells by the scanning laser. Cell⊕Finder can be downloaded from http://www.alga.cz/cellfinder. The system was applied to study changes in fluorescence intensity in Synechocystis sp. PCC6803 cells under long-term illumination. Thus, we were able to describe the kinetics of phycobilisome decoupling. Microscopy data showed that phycobilisome decoupling appears slowly after long-term (>1 h) exposure to high light. PMID:27050040

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Schlachter, Simon C.; Kang, DongKyun; Gora, Michalina J.; Vacas-Jacques, Paulino; Wu, Tao; Carruth, Robert W.; Wilsterman, Eric J.; 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 resolution. SECM imaging of the esophagus has been previously demonstrated at relatively low line rates (5 kHz). In this paper, we demonstrate SECM imaging of large regions of esophageal tissues at a high line imaging rate of 100 kHz. The SECM system comprises a wavelength-swept source with a fast sweep rate (100 kHz), high output power (80 mW), and a detector unit with a large bandwidth (100 MHz). The sensitivity of the 100-kHz SECM system was measured to be 60 dB and the transverse resolution was 1.6 µm. Excised swine and human esophageal tissues were imaged with the 100-kHz SECM system at a rate of 6.6 mm2/sec. Architectural and cellular features of esophageal tissues could be clearly visualized in the SECM images, including papillae, glands, and nuclei. These results demonstrate that large-area SECM imaging of esophageal tissues can be successfully conducted at a high line imaging rate of 100 kHz, which will enable whole-organ SECM imaging in vivo. PMID:24049684

  2. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2011-03-01

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

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

    Science.gov (United States)

    Steinbach, Gábor; Kaňa, Radek

    2016-04-01

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

  5. Confocal laser scanning microscopy, a new in vivo diagnostic tool for schistosomiasis.

    Directory of Open Access Journals (Sweden)

    Carlos Fritzsche

    Full Text Available BACKGROUND: The gold standard for the diagnosis of schistosomiasis is the detection of the parasite's characteristic eggs in urine, stool, or rectal and bladder biopsy specimens. Direct detection of eggs is difficult and not always possible in patients with low egg-shedding rates. Confocal laser scanning microscopy (CLSM permits non-invasive cell imaging in vivo and is an established way of obtaining high-resolution images and 3-dimensional reconstructions. Recently, CLSM was shown to be a suitable method to visualize Schistosoma mansoni eggs within the mucosa of dissected mouse gut. In this case, we evaluated the suitability of CLSM to detect eggs of Schistosoma haematobium in a patient with urinary schistosomiasis and low egg-shedding rates. METHODOLOGY/PRINCIPAL FINDINGS: The confocal laser scanning microscope used in this study was based on a scanning laser system for imaging the retina of a living eye, the Heidelberg Retina Tomograph II, in combination with a lens system (image modality. Standard light cystoscopy was performed using a rigid cystoscope under general anaesthesia. The CLSM endoscope was then passed through the working channel of the rigid cystoscope. The mucosal tissue of the bladder was scanned using CLSM. Schistoma haematobium eggs appeared as bright structures, with the characteristic egg shape and typical terminal spine. CONCLUSION/SIGNIFICANCE: We were able to detect schistosomal eggs in the urothelium of a patient with urinary schistosomiasis. Thus, CLSM may be a suitable tool for the diagnosis of schistosomiasis in humans, especially in cases where standard diagnostic tools are not suitable.

  6. In vivo evaluation of DSAEK interface with scanning-laser confocal microscopy

    Directory of Open Access Journals (Sweden)

    Ferrari Giulio

    2012-08-01

    Full Text Available Abstract Background Descemet Stripping Automated Endothelial Keratoplasty (DSAEK allows selective replacement of the endothelium. Post-operative haze and particles can affect the interface quality and, ultimately, visual outcome. In this study, we evaluated DSAEK interface with in vivo laser confocal microscopy (LCM in order to: (i correlate interface status with best corrected visual acuity, and (ii with time from surgery; (iii correlate interface particle number with best corrected visual acuity. Host-donor interface was imaged and graded using a published reflectivity scale. Particles at the interface were counted. Methods 18 eyes of 16 patients (6 males and 10 females; mean age: 74 ± 8.3 years which underwent DSAEK were examined by means of in vivo laser confocal microscopy between 1 and 24 months after surgery. Host-donor interface was imaged and graded using a published reflectivity scale. Particles present at the interface were counted. Results Interface reflectivity was 2.17 ± 1.2 and significantly correlated with visual acuity (Spearman correlation coefficient −0.83; P  Conclusion DSAEK interface imaged with LCM is helpful in diagnosing poor host-donor interface quality in DSAEK surgery. A good quality interface is related to a better visual acuity. Moreover, the quality of the interface appears to improve as time passes from the surgery. Interface quality is related with visual acuity and improves with time from surgery. LCM should be considered as an added tool in post-DSAEK follow-up of patients. Finally, our study shows that the presence of particles does not influence visual outcome.

  7. Spectrally encoded confocal microscopy (SECM) for rapid assessment of breast excision specimens (Conference Presentation)

    Science.gov (United States)

    Brachtel, Elena F.; Johnson, Nicole B.; Huck, Amelia E.; Rice-Stitt, Travis L.; Vangel, Mark G.; Smith, Barbara L.; Tearney, Guillermo J.; Kang, DongKyun

    2016-03-01

    Unacceptably large percentage (20-40%) of breast cancer lumpectomy patients are required to undergo multiple surgeries when positive margins are found upon post-operative histologic assessment. If the margin status can be determined during surgery, surgeon can resect additional tissues to achieve tumor-free margin, which will reduce the need for additional surgeries. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to image the entire surgical margin within a short procedural time. Previously, SECM was shown to rapidly image a large area (10 mm by 10 mm) of human esophageal tissue within a short procedural time (15 seconds). When used in lumpectomy, SECM will be able to image the entire margin surface of ~30 cm2 in around 7.5 minutes. SECM images will then be used to determine margin status intra-operatively. In this paper, we present results from a study of testing accuracy of SECM for diagnosing malignant breast tissues. We have imaged freshly-excised breast specimens (N=46) with SECM. SECM images clearly visualized histomorphologic features associated with normal/benign and malignant breast tissues in a similar manner to histologic images. Diagnostic accuracy was tested by comparing SECM diagnoses made by three junior pathologists with corresponding histologic diagnoses made by a senior pathologist. SECM sensitivity and specificity were high, 0.91 and 0.93, respectively. Intra-observer agreement and inter-observer agreement were also high, 0.87 and 0.84, respectively. Results from this study showed that SECM has a potential to accurately determine margin status during breast cancer lumpectomy.

  8. An in vitro Comparative Evaluation of Three Remineralizing Agents using Confocal Microscopy

    Science.gov (United States)

    Chokshi, Achala; Konde, Sapna; Shetty, Sunil Raj; Chandra, Kumar Narayan; Jana, Sinjana; Mhambrey, Sanjana; Thakur, Sneha

    2016-01-01

    Introduction The caries process has been thought to be irreversible, resulting in the permanent loss of tooth substance and eventually the development of a cavity. Recent approaches focused on application of remineralizing agents to incipient carious lesions, aim at controlling demineralization and promoting remineralization. Remineralizing agents create a supersaturated environment around the lesion; thus, preventing mineral loss and forces calcium and phosphate ions in the vacant areas. Aim To compare and evaluate the remineralization potential of Fluoride Varnish, CPP-ACP Paste (Casein Phosphopeptide-Amorphous Calcium Phosphate) and fTCP Paste (functionalized Tricalcium Phosphate) using confocal microscope. Materials and Methods Two windows of 3X3mm were created on the labial cervical and incisal thirds in 60 permanent maxillary central incisors. The teeth were demineralized to create artificial caries and divided into three groups of 20 each. Group I specimens were coated with Fluoride Varnish once whereas those in CPP-ACP paste group and fTCP group were brushed for 2 minutes, twice daily for 20 and 40 days. The specimens were stored in artificial saliva during the study period and were later sectioned and observed under confocal microscope. Data obtained was statistically analyzed using Fischer’s exact test, ANOVA and post-hoc Bonferroni’s test. Results Fluoride Varnish, CPP-ACP Paste and fTCP Paste showed remineralization of artificial carious lesions at both the time intervals. Fluoride varnish showed the highest remineralization followed by CPP-ACP Paste and fTCP Paste. A statistically significant increase in remineralization potential of CPP-ACP Paste and fTCP Paste was observed at the end of 40 days as compared to 20 days. Conclusion Fluoride varnish showed the greatest remineralization potential of artificial carious lesions followed by CPP-ACP Paste and fTCP Paste respectively. PMID:27504408

  9. Confocal microscopy for simultaneous imaging of Cu electrodeposit morphology and adsorbate fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Chung, D.S.; Alkire, R.C. [Univ. of Illinois, Urbana, IL (United States)

    1997-05-01

    Confocal laser scanning microscopy was used in situ during electrochemical experiments to track localized fluorescence patterns of adsorbed organic agents and to correlate such adsorption with changes in surface morphology accompanying electrolysis. In solutions of 5 {micro}M DiOC{sub 6}(3)/0.01 M H{sub 2}SO{sub 4}, with and without 0.05 M CuSo{sub 4}, confocal imaging revealed that DiOC{sub 6}(3) adsorbed to polycrystalline Au and inhibited cathodic processes occurring there. In the absence of dissolved Cu, DiOC{sub 6}(3) adsorption on Au remained unaltered by changes in cathodic potential up to {minus}750 mV (SSE). During Cu electrodeposition at {minus}550 and at {minus}650 mV (SSE), adsorbed DiOC{sub 6}(3) restricted nucleation of Cu to a small number of active sites where Cu grew hemispherically; and DiOC{sub 6}(3) adsorption was maintained across regions where nucleation had not occurred. Instantaneous nucleation was approached under such conditions. When DiOC{sub 6}(3) was present, copper growth proceeded according to the Volmer-Weber mechanism at {minus}650 mV (SSE). Results from secondary ion mass spectrometry indicated that DiOC{sub 6}(3), or a derivative of it, was incorporated into the deposit during Cu electrodeposition. During Electrodissolution of Cu on Au at 0 mV (SSE), adsorption of DiOC{sub 6}(3) occurred predominantly at surface sites of Cu rather than Au.

  10. Spectrally Encoded Confocal Microscopy (SECM) for Diagnosing of Breast Cancer in Excision and Margin Specimens

    Science.gov (United States)

    Brachtel, Elena F.; Johnson, Nicole B.; Huck, Amelia E.; Rice-Stitt, Travis L.; Vangel, Mark G.; Smith, Barbara L.; Tearney, Guillermo J.; Kang, Dongkyun

    2016-01-01

    A large percentage of breast cancer patients treated with breast conserving surgery need to undergo multiple surgeries due to positive margins found during post-operative margin assessment. Carcinomas could be removed completely during the initial surgery and additional surgery avoided if positive margins can be determined intra-operatively. Spectrally-encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to rapidly image the entire surgical margin at sub-cellular resolution and accurately determine margin status intra-operatively. In this paper, in order to test feasibility of using SECM for intra-operative margin assessment, we have evaluated the diagnostic accuracy of SECM for detecting various types of breast cancers. Forty-six surgically-removed breast specimens were imaged with a SECM system. Side-by-side comparison between SECM and histologic images showed that SECM images can visualize key histomorphologic patterns of normal/benign and malignant breast tissues. Small (500 µm × 500 µm) spatially-registered SECM and histologic images (n=124 for each) were diagnosed independently by three pathologists with expertise in breast pathology. Diagnostic accuracy of SECM for determining malignant tissues was high, average sensitivity of 0.91, specificity of 0.93, positive predictive value of 0.95, and negative predictive value of 0.87. Intra-observer agreement and inter-observer agreement for SECM were also high, 0.87 and 0.84, respectively. Results from this study suggest that SECM may be developed into an intra-operative margin assessment tool for guiding breast cancer excisions. PMID:26779830

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

    Directory of Open Access Journals (Sweden)

    Chao eHuang

    2014-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Coralie Fouquet

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

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

    Science.gov (United States)

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

    2011-12-01

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

  14. Microrheology: Structural evolution under static and dynamic conditions by simultaneously analysis of confocal microscopy and diffusing wave spectroscopy

    NARCIS (Netherlands)

    Nicolas, Y.; Paques, M.; Knaebel, A.; Steyer, A.; Munch, J.P.; Blijdenstein, T.B.J.; Aken, van G.A.

    2003-01-01

    An oscillatory shear configuration was developed to improve understanding of structural evolution during deformation. It combines an inverted confocal scanning laser microscope (CSLM) and a special sample holder that can apply to the sample specific deformation: oscillatory shear or steady strain. I

  15. Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy

    DEFF Research Database (Denmark)

    Novak, Ivana; Nitschke, Roland; Amstrup, Jan

    2002-01-01

    Pancreatic ducts have several types of purinergic P2 receptors, however, nothing is known about P2 receptors in acini. The aim was to establish whether acini express functional P2 receptors coupled to intracellular Ca2+ signals and to measure the signals ratiometrically in a confocal laser scanni...

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

    NARCIS (Netherlands)

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

    2000-01-01

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

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  18. Spatial inhomogeneity in spectra and exciton dynamics in porphyrin micro-rods and micro-brushes: Confocal microscopy

    Indian Academy of Sciences (India)

    SHYAMTANU CHATTORAJ; KANKAN BHATTACHARYYA

    2016-11-01

    In an aqueous acidic solution, the porphyrin meso-tetra(4-sulfonatophenyl) porphyrin tetrasodium salt (TPPS) forms different kinds of assembly (micro-rods and micro-brush) depending on condition of evaporation. The exciton dynamics and emission spectra of the micro-rods and micro-brushes depend on spatialinhomogeneity. This is elucidated by time-resolved confocal microscopy.

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

    NARCIS (Netherlands)

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

    2006-01-01

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

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

    NARCIS (Netherlands)

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

    2000-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-02-17

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

  2. Improvement of Axial Resolution in Confocal Microscopy by an Optical Pupil Filter with Two Zones Phase-Shifted by π

    Institute of Scientific and Technical Information of China (English)

    林列; 王湘晖; 王肇圻; 母国光

    2003-01-01

    A filter with two zones phase-shifted by π is proposed to improve the axial resolution of confocal microscopes with a finite-sized detector. The optimum axial resolution for a given size of the detector can be achieved by adjusting the zone boundary of the filter. The experimental results are well in agreement with the theoretical predictions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fredrich, J.T.

    1999-02-10

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

  4. Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy

    DEFF Research Database (Denmark)

    Sørensen, Christiane Elisabeth; Novak, Ivana

    2001-01-01

    of this reaction in confocal microscopy, we monitored luciferin fluorescence as a sign of ATP release by single acini. In addition we used quinacrine to mark ATP stores, which were similar to those marked with fluorescent ATP, 2'-(or-3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate, but only partially...

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Science.gov (United States)

    De Simoni, Maria-Grazia

    2013-01-01

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

  8. Confocal Raman Microscopy of Hybrid-Supported Phospholipid Bilayers within Individual C18-Functionalized Chromatographic Particles.

    Science.gov (United States)

    Kitt, Jay P; Harris, Joel M

    2016-09-01

    Measuring lipid-membrane partitioning of small molecules is critical to predicting bioavailability and investigating molecule-membrane interactions. A stable model membrane for such studies has been developed through assembly of a phospholipid monolayer on n-alkane-modified surfaces. These hybrid bilayers have recently been generated within n-alkyl-chain (C18)-modified porous silica and used in chromatographic retention studies of small molecules. Despite their successful application, determining the structure of hybrid bilayers within chromatographic silica is challenging because they reside at buried interfaces within the porous structure. In this work, we employ confocal Raman microscopy to investigate the formation and temperature-dependent structure of hybrid-phospholipid bilayers in C18-modified, porous-silica chromatographic particles. Porous silica provides sufficient surface area within a confocal probe volume centered in an individual particle to readily measure, with Raman microscopy, the formation of an ordered hybrid bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the surface C18 chains. The DMPC surface density was quantified from the relative Raman scattering intensities of C18 and phospholipid acyl chains and found to be ∼40% of a DMPC vesicle membrane. By monitoring Raman spectra acquired versus temperature, the bilayer main phase transition was observed to be broadened and shifted to higher temperature compared to a DMPC vesicle, in agreement with differential scanning calorimetry (DSC) results. Raman scattering of deuterated phospholipid was resolved from protonated C18 chain scattering, showing that the lipid acyl and C18 chains melt simultaneously in a single phase transition. The surface density of lipid in the hybrid bilayer, the ordering of both C18 and lipid acyl chains upon bilayer formation, and decoupling of C18 methylene C-H vibrations by deuterated lipid acyl chains all suggest an interdigitated acyl chain

  9. Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles

    Science.gov (United States)

    Guggenheim, Emily J.; Khan, Abdullah; Pike, Jeremy; Chang, Lynne; Lynch, Iseult; Rappoport, Joshua Z.

    2016-01-01

    The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP

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

    Science.gov (United States)

    Hefti, Ryan Alf

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

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

    Directory of Open Access Journals (Sweden)

    Lennert S. Ploeger

    2005-01-01

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

  12. Influences of edges and steep slopes in 3D interference and confocal microscopy

    Science.gov (United States)

    Xie, Weichang; Hagemeier, Sebastian; Woidt, Carsten; Hillmer, Harmut; Lehmann, Peter

    2016-04-01

    Optical measurement techniques are widely applied in high-resolution contour, topography and roughness measurement. In this context vertical scanning white-light interferometers and confocal microscopes have become mature instruments over the last decades. The accuracy of measurement results is highly related not only to the type and physical properties of the measuring instruments, but also to the measurement object itself. This contribution focuses on measurement effects occurring at edges and height steps using white-light interferometers of different numerical apertures. If the edge is perfectly perpendicular, batwing effects appear at height steps. These batwings show maximum height if the height-to-wavelength-ratio (HWR) is about one forth or three forth, and they disappear if the HWR value is about an integer multiple of one half. The wavelength that is relevant in this context is the effective wavelength, i.e. the center wavelength of the illuminating light multiplied by a correction factor known as the numerical aperture correction. However, in practice the edges are usually not perfectly perpendicular. In this case, the measurement results depend also on the derivative of the surface height function and they may differ from theory and the prediction according to the HWR value. Measurements of such steps show systematical effects depending on the lateral resolution of the instrument. In this context, a Linnik interferometer with a magnification of 100x and NA = 0.9 is used to characterize the three dimensional topography of more or less rectangular calibration specimens and quasi-perpendicular structures produced by the nanoimprint technology. The Linnik interferometer is equipped with LED light sources emitting at different wavelengths, so that the HWR value can be changed. This is possible since the high NA objective lenses show a rather limited depth of focus such that the temporal coherence gating may be replaced by focal gating in this particular

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

    Directory of Open Access Journals (Sweden)

    Vyacheslav M Shkryl

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

  14. A brief discussion on confocal microscopy techniques%浅谈共聚焦显微技术

    Institute of Scientific and Technical Information of China (English)

    陈木旺

    2013-01-01

    Confocal microscopy boasts such advantages as high contrast, high resolution and easy to 3D reconstruction. Therefore, the device has been widely applied in the manufacture and detection of biology and medical research, micro-machining, semiconductor and macromolecule. There are several kinds of confocal microscopy, including laser scanning confocal microscopy ( LSCM), spinning-disk confocal microscopy (SDCM) and structured illumination microscopy (SIM). LSCM has such advantages as high contrast and resolution, but it is complicated, bulky, expensive and slow in imaging. SDCM can get the confocal image fast based on the lower resolution and more complicated technique. Though SIM is the simplest and cheapest confocal microscopy, it can offer high quality image with fast speed.%共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用.常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高.

  15. Differentiating gastrointestinal stromal tumors from gastric adenocarcinomas and normal mucosae using confocal Raman microspectroscopy

    Science.gov (United States)

    Hsu, Chih-Wei; Huang, Chia-Chi; Sheu, Jeng-Horng; Lin, Chia-Wen; Lin, Lien-Fu; Jin, Jong-Shiaw; Chen, Wenlung

    2016-07-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, and gastric adenocarcinomas are a common cancer worldwide. To differentiate GISTs from adenocarcinomas is important because the surgical processes for both are different; the former excises the tumor with negative margins, while the latter requires radical gastrectomy with lymph node dissection. Endoscopy with biopsy is used to distinguish GISTs from adenocarcinomas; however, it may cause tumor bleeding in GISTs. We reported here the confocal Raman microspectroscopy as an effective tool to differentiate GISTs, adenocarcinomas, and normal mucosae. Of 119 patients enrolled in this study, 102 patients underwent gastrectomy (40 GISTs and 62 adenocarcinomas), and 17 patients with benign lesions were obtained as normal mucosae. Raman signals were integrated for 100 s for each spot on the specimen, and 5 to 10 spots, depending on the sample size, were chosen for each specimen. There were significant differences among those tissues as evidenced by different Raman signal responding to phospholipids and protein structures. The spectral data were further processed and analyzed by using principal component analysis. A two-dimensional plot demonstrated that GISTs, adenocarcinomas, and normal gastric mucosae could be effectively differentiated from each other.

  16. Demeclocycline as a contrast agent for detecting brain neoplasms using confocal microscopy

    Science.gov (United States)

    Wirth, Dennis; Smith, Thomas W.; Moser, Richard; Yaroslavsky, Anna N.

    2015-04-01

    Complete resection of brain tumors improves life expectancy and quality. Thus, there is a strong need for high-resolution detection and microscopically controlled removal of brain neoplasms. The goal of this study was to test demeclocycline as a contrast enhancer for the intraoperative detection of brain tumors. We have imaged benign and cancerous brain tumors using multimodal confocal microscopy. The tumors investigated included pituitary adenoma, meningiomas, glioblastomas, and metastatic brain cancers. Freshly excised brain tissues were stained in 0.75 mg ml-1 aqueous solution of demeclocyline. Reflectance images were acquired at 402 nm. Fluorescence signals were excited at 402 nm and registered between 500 and 540 nm. After imaging, histological sections were processed from the imaged specimens and compared to the optical images. Fluorescence images highlighted normal and cancerous brain cells, while reflectance images emphasized the morphology of connective tissue. The optical and histological images were in accordance with each other for all types of tumors investigated. Demeclocyline shows promise as a contrast agent for intraoperative detection of brain tumors.

  17. Confocal ultrafast pump-probe spectroscopy: a new technique to explore nanoscale composites

    Science.gov (United States)

    Virgili, Tersilla; Grancini, Giulia; Molotokaite, Egle; Suarez-Lopez, Inma; Rajendran, Sai Kiran; Liscio, Andrea; Palermo, Vincenzo; Lanzani, Guglielmo; Polli, Dario; Cerullo, Giulio

    2012-03-01

    This article is devoted to the exploration of the benefits of a new ultrafast confocal pump-probe technique, able to study the photophysics of different structured materials with nanoscale resolution. This tool offers many advantages over standard stationary microscopy techniques because it directly interrogates excited state dynamics in molecules, providing access to both radiative and non-radiative deactivation processes at a local scale. In this paper we present a few different examples of its application to organic semiconductor systems. The first two are focussed on the study of the photophysics of phase-separated polymer blends: (i) a blue-emitting polyfluorene (PFO) in an inert matrix of PMMA and (ii) an electron donor polythiophene (P3HT) mixed with an electron acceptor fullerene derivative (PCBM). The experimental results on these samples demonstrate the capability of the technique to unveil peculiar interfacial dynamics at the border region between phase-segregated domains, which would be otherwise averaged out using conventional pump-probe spectroscopy. The third example is the study of the photophysics of isolated mesoscopic crystals of the PCBM molecule. Our ultrafast microscope could evidence the presence of two distinctive regions within the crystals. In particular, we could pinpoint for the first time areas within the crystals showing photobleaching/stimulated emission signals from a charge-transfer state.

  18. Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

    Science.gov (United States)

    Lange-Asschenfeldt, Susanne; Bob, Adrienne; Terhorst, Dorothea; Ulrich, Martina; Fluhr, Joachim; Mendez, Gil; Roewert-Huber, Hans-Joachim; Stockfleth, Eggert; Lange-Asschenfeldt, Bernhard

    2012-07-01

    There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

  19. Evaluation of confocal laser scanning microscopy for enumeration of virus-like particles in aquatic systems

    Science.gov (United States)

    Agis, Martin; Luef, Birgit

    2016-01-01

    Abstract Abundances of virus-like particles (VLPs, mostly bacteriophages) are high in aquatic environments; therefore, techniques for precise enumeration are essential in ecological monitoring. VLPs were determined after staining with SYBR Gold by conventional epifluorescence microscopy and compared to enumerations performed by confocal laser scanning microscopy (CLSM). In order to assess the potential of CLSM for viral direct counts (VDCs), we processed samples from different freshwater and marine systems. Optical sectioning by CLSM and production of an overlay picture of multiple scans enables the often uneven whole investigated filter area to be brought to the plane of focus. This allows for subsequent image analysis of digitally created high-quality images. Another advantage using the CLSM was that the short spot excitation of the stain via laser beam minimized fading of the stain. The VDC results show that there is no significant difference between the two methods. Regarding the known difficulties of viral abundance estimates on particulate material, CLSM was further applied to enumerate VLPs on a small set of marine transparent exopolymeric particles sampled from the Atlantic Ocean. Our data suggest that CLSM is a useful tool to count viruses in water samples as well as attached to certain types of aquatic aggregates. PMID:23108709

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

    International Nuclear Information System (INIS)

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

  1. Pseudoxanthoma elasticum and reflectance confocal microscopy: report of two affected young sisters

    Directory of Open Access Journals (Sweden)

    Victor Desmond Mandel

    2015-04-01

    Full Text Available Pseudoxanthoma elasticum (PXE is a rare inherited multisystem disorder that mainly affects skin, eyes and cardiovascular system. The associated clinical signs are due to progressive calcification of elastic fibres and blood vessels, despite normal levels of calcium and phosphorus in blood and urine. The first clinical description of the disease was done in 1881 by Rigal, and in 1896 it was named PXE by Darier. Transmission of the disease is autosomal recessive. PXE is caused by homozygous or compound heterozygous mutations in the ATP-binding cassette subfamily C member 6 (ABCC6 gene, which encodes a transmembrane transport ADP-dependent protein (MRP6. The gene is expressed predominantly in the liver and kidney, and found in low level in the tissue involved by PXE. The clinical expression of PXE is heterogeneous with considerable variation in age of onset, progression and severity of the disease, even in individuals of the same family with identical mutations.We present the case of two young sisters affected by PXE and the correlation between the histopathology and the reflectance confocal microscopy (RCM. Parents and brother carry one copy of the mutated gene, without showing signs and symptoms of the disorder. We report the main clinical aspects of PXE and we highlight the importance of early diagnosis of the disease for adequate therapeutical management of associated complications.

  2. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks.

    Science.gov (United States)

    Hames, Samuel C; Ardigò, Marco; Soyer, H Peter; Bradley, Andrew P; Prow, Tarl W

    2016-01-01

    Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20-30 and 50-70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin. PMID:27088865

  3. In vivo confocal microscopy of basal cell carcinoma: a systematic review of diagnostic accuracy.

    Science.gov (United States)

    Kadouch, D J; Schram, M E; Leeflang, M M; Limpens, J; Spuls, P I; de Rie, M A

    2015-10-01

    Basal cell carcinoma (BCC) is the most prevalent type of skin cancer. Histologic analysis of punch biopsy or direct excision specimen is used to confirm clinical diagnosis. In vivo reflectance confocal microscopy (RCM) is a non-invasive imaging modality that could facilitate early diagnosis and minimize unnecessary invasive procedures. We systematically reviewed diagnostic accuracy (sensitivity and specificity) of RCM in diagnosing primary BCCs to judge its usefulness. Eligible studies were reviewed for methodological quality using the QUADAS-2 tool. We used the bivariate random-effects model to calculate summary estimates of sensitivity and specificity. Six studies met the selection criteria and were included for analysis. The meta-analysis showed a summary estimate of sensitivity 0.97 (95% CI, 0.90-0.99) and specificity 0.93 (95% CI, 0.88-0.96). All but one of the QUADAS-2 items showed a high or unclear risk of bias with regards to patient selection. RCM may be a promising diagnostic tool, but the limited number of available studies and potential risk of bias of included studies do not allow us to draw firm conclusions. Future accuracy studies should take these limitations into account. PMID:26290493

  4. Quantification of Mesenchymal Stem Cell (MSC delivery to a target site using in vivo confocal microscopy.

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    Luke J Mortensen

    Full Text Available The ability to deliver cells to appropriate target tissues is a prerequisite for successful cell-based therapy. To optimize cell therapy it is therefore necessary to develop a robust method of in vivo cell delivery quantification. Here we examine Mesenchymal Stem Cells (MSCs labeled with a series of 4 membrane dyes from which we select the optimal dye combination for pair-wise comparisons of delivery to inflamed tissue in the mouse ear using confocal fluorescence imaging. The use of an optimized dye pair for simultaneous tracking of two cell populations in the same animal enables quantification of a test population that is referenced to an internal control population, thereby eliminating intra-subject variations and variations in injected cell numbers. Consistent results were obtained even when the administered cell number varied by more than an order of magnitude, demonstrating an ability to neutralize one of the largest sources of in vivo experimental error and to greatly reduce the number of cells required to evaluate cell delivery. With this method, we are able to show a small but significant increase in the delivery of cytokine pre-treated MSCs (TNF-α & IFN-γ compared to control MSCs. Our results suggest future directions for screening cell strategies using our in vivo cell delivery assay, which may be useful to develop methods to maximize cell therapeutic potential.

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

    Science.gov (United States)

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

    2013-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Federico eLuzzati

    2011-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Rajesh Kumar

    2015-04-01

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

  8. Estimating pH at the Air/Water Interface with a Confocal Fluorescence Microscope.

    Science.gov (United States)

    Yang, Haiya; Imanishi, Yasushi; Harata, Akira

    2015-01-01

    One way to determine the pH at the air/water interface with a confocal fluorescence microscope has been proposed. The relation between the pH at the air/water interface and that in a bulk solution has been formulated in connection with the adsorption equilibrium and the dissociation equilibrium of the dye adsorbed. Rhodamine B (RhB) is used as a surface-active fluorescent pH probe. The corrected fluorescence spectrum of RhB molecules at the air/water interface with the surface density of 1.0 nmol m(-2) level shows pH-dependent shifts representing an acid-base equilibrium. Two ways to determine the unknown acid-base equilibrium constant of RhB molecules at the air/water interface have been discussed. With surface-tension measurements, the adsorption properties, maximum surface density, and adsorption equilibrium constants were estimated for both cationic and zwitterionic forms of RhB molecules at the air/water interface.

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

    Directory of Open Access Journals (Sweden)

    Fabiana Fabbretti

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

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

    Galizia, C G; Kimmerle, B

    2004-01-01

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

  12. Assessing strain mapping by electron backscatter diffraction and confocal Raman microscopy using wedge-indented Si.

    Science.gov (United States)

    Friedman, Lawrence H; Vaudin, Mark D; Stranick, Stephan J; Stan, Gheorghe; Gerbig, Yvonne B; Osborn, William; Cook, Robert F

    2016-04-01

    The accuracy of electron backscatter diffraction (EBSD) and confocal Raman microscopy (CRM) for small-scale strain mapping are assessed using the multi-axial strain field surrounding a wedge indentation in Si as a test vehicle. The strain field is modeled using finite element analysis (FEA) that is adapted to the near-indentation surface profile measured by atomic force microscopy (AFM). The assessment consists of (1) direct experimental comparisons of strain and deformation and (2) comparisons in which the modeled strain field is used as an intermediate step. Direct experimental methods (1) consist of comparisons of surface elevation and gradient measured by AFM and EBSD and of Raman shifts measured and predicted by CRM and EBSD, respectively. Comparisons that utilize the combined FEA-AFM model (2) consist of predictions of distortion, strain, and rotation for comparison with EBSD measurements and predictions of Raman shift for comparison with CRM measurements. For both EBSD and CRM, convolution of measurements in depth-varying strain fields is considered. The interconnected comparisons suggest that EBSD was able to provide an accurate assessment of the wedge indentation deformation field to within the precision of the measurements, approximately 2×10(-4) in strain. CRM was similarly precise, but was limited in accuracy to several times this value. PMID:26939030

  13. Real time confocal laser scanning microscopy: Potential applications in space medicine and cell biology

    Science.gov (United States)

    Rollan, Ana; Ward, Thelma; McHale, Anthony P.

    Photodynamic therapy (PDT), in which tissues may be rendered fatally light-sensitive represents a relatively novel treatment for cancer and other disorders such as cardiovascular disease. It offers significant application to disease control in an isolated environment such as space flight. In studying PDT in the laboratory, low energy lasers such as HeNe lasers are used to activate the photosensitized cellular target. A major problem associated with these studies is that events occurring during actual exposure of the target cells to the system cannot be examined in real time. In this study HeLa cells were photosensitized and photodynamic activation was accomplished using the scanning microbeam from a confocal laser scanning microscope. This form of activation allowed for simultaneous photoactivation and observation and facilitated the recording of events at a microscopic level during photoactivation. Effects of photodynamic activation on the target cells were monitored using the fluorophores rhodamine 123 and ethidium homodimer-1. Potential applications of these forms of analyses to space medicine and cell biology are discussed.

  14. A light sheet confocal microscope for image cytometry with a variable linear slit detector

    Science.gov (United States)

    Hutcheson, Joshua A.; Khan, Foysal Z.; Powless, Amy J.; Benson, Devin; Hunter, Courtney; Fritsch, Ingrid; Muldoon, Timothy J.

    2016-03-01

    We present a light sheet confocal microscope (LSCM) capable of high-resolution imaging of cell suspensions in a microfluidic environment. In lieu of conventional pressure-driven flow or mechanical translation of the samples, we have employed a novel method of fluid transport, redox-magnetohydrodynamics (redox-MHD). This method achieves fluid motion by inducing a small current into the suspension in the presence of a magnetic field via electrodes patterned onto a silicon chip. This on-chip transportation requires no moving parts, and is coupled to the remainder of the imaging system. The microscopy system comprises a 450 nm diode 20 mW laser coupled to a single mode fiber and a cylindrical lens that converges the light sheet into the back aperture of a 10x, 0.3 NA objective lens in an epi-illumination configuration. The emission pathway contains a 150 mm tube lens that focuses the light onto the linear sensor at the conjugate image plane. The linear sensor (ELiiXA+ 8k/4k) has three lateral binning modes which enables variable detection aperture widths between 5, 10, or 20 μm, which can be used to vary axial resolution. We have demonstrated redox-MHD-enabled light sheet microscopy in suspension of fluorescent polystyrene beads. This approach has potential as a high-throughput image cytometer with myriad cellular diagnostic applications.

  15. Modeling enzymatic hydrolysis of lignocellulosic substrates using fluorescent confocal microscopy II: pretreated biomass.

    Science.gov (United States)

    Luterbacher, Jeremy S; Moran-Mirabal, Jose M; Burkholder, Eric W; Walker, Larry P

    2015-01-01

    In this study, we extend imaging and modeling work that was done in Part I of this report for a pure cellulose substrate (filter paper) to more industrially relevant substrates (untreated and pretreated hardwood and switchgrass). Using confocal fluorescence microscopy, we are able to track both the structure of the biomass particle via its autofluorescence, and bound enzyme from a commercial cellulase cocktail supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A. Imaging was performed throughout hydrolysis at temperatures relevant to industrial processing (50°C). Enzyme bound predominantly to areas with low autofluorescence, where structure loss and lignin removal had occurred during pretreatment; this confirms the importance of these processes for successful hydrolysis. The overall shape of both untreated and pretreated hardwood and switchgrass particles showed little change during enzymatic hydrolysis beyond a drop in autofluorescence intensity. The permanence of shape along with a relatively constant bound enzyme signal throughout hydrolysis was similar to observations previously made for filter paper, and was consistent with a modeling geometry of a hollowing out cylinder with widening pores represented as infinite slits. Modeling estimates of available surface areas for pretreated biomass were consistent with previously reported experimental results. PMID:25042048

  16. Quantitative analyses of Streptococcus mutans biofilms with quartz crystal microbalance, microjet impingement and confocal microscopy.

    Science.gov (United States)

    Kreth, J; Hagerman, E; Tam, K; Merritt, J; Wong, D T W; Wu, B M; Myung, N V; Shi, W; Qi, F

    2004-10-01

    Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm. PMID:16429589

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

    Science.gov (United States)

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

    2014-01-01

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

  18. Probe-Based Confocal Laser Endomicroscopy for Indeterminate Biliary Strictures: Refinement of the Image Interpretation Classification

    Directory of Open Access Journals (Sweden)

    Michel Kahaleh

    2015-01-01

    Full Text Available Background. Accurate diagnosis and clinical management of indeterminate biliary strictures are often a challenge. Tissue confirmation modalities during Endoscopic Retrograde Cholangiopancreatography (ERCP suffer from low sensitivity and poor diagnostic accuracy. Probe-based confocal laser endomicroscopy (pCLE has been shown to be sensitive for malignant strictures characterization (98% but lacks specificity (67% due to inflammatory conditions inducing false positives. Methods. Six pCLE experts validated the Paris Classification, designed for diagnosing inflammatory biliary strictures, using a set of 40 pCLE sequences obtained during the prospective registry (19 inflammatory, 6 benign, and 15 malignant. The 4 criteria used included (1 multiple thin white bands, (2 dark granular pattern with scales, (3 increased space between scales, and (4 thickened reticular structures. Interobserver agreement was further calculated on a separate set of 18 pCLE sequences. Results. Overall accuracy was 82.5% (n=40 retrospectively diagnosed versus 81% (n=89 prospectively collected for the registry, resulting in a sensitivity of 81.2% (versus 98% for the prospective study and a specificity of 83.3% (versus 67% for the prospective study. The corresponding interobserver agreement for 18 pCLE clips was fair (k=0.37. Conclusion. Specificity of pCLE using the Paris Classification for the characterization of indeterminate bile duct stricture was increased, without impacting the overall accuracy.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

  1. Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

    Science.gov (United States)

    Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2015-03-01

    High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas.

  2. Penetration of resin-based materials into initial erosion lesion: A confocal microscopic study.

    Science.gov (United States)

    Ionta, Franciny Querobim; Boteon, Ana Paula; Moretto, Marcelo Juliano; Júnior, Odair Bim; Honório, Heitor Marques; Silva, Thiago Cruvinel; Wang, Linda; Rios, Daniela

    2016-02-01

    The application of resin-based materials is an alternative of treatment for eroded lesions. Nevertheless, there are no studies about the penetration of these materials into eroded lesion, which might affect its adhesion. Therefore, this study evaluated the penetration of four resin-based materials, with and without enamel etching. By using an in vitro protocol, types of treatment were studied at five levels (AdheSE(®) , Tetric N-Bond(®) , Single Bond 2(®) , Helioseal Clear(®) , Icon(®) ) and types of enamel etching in two levels (with and without). Materials were stained with 0.02 mg/mL ethanolic solution of tetramethylrhodamine isothiocyanate. Bovine enamel samples (4 × 4 mm) were immersed in 0.01 M HCl, pH 2.3, for 30 seconds to produce initial eroded lesions. Afterward, the materials were applied on half of sample enamel surface following the manufacturer's instructions. On the other half of sample, the materials were applied without etching the enamel. Materials penetration into the enamel was assessed by Confocal Laser Scanning Microscopy on reflection and fluorescence modes. The penetration depth (PD) was measured using ImageJ software. Data were analyzed by two-way ANOVA and Tukey test (P material, etched enamel resulted in higher PD than non-etched (P  0.05). It can be concluded that prior enamel etching increased the materials penetration into eroded enamel and the Icon(®) -infiltrant presented highest penetration.

  3. Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging.

    Science.gov (United States)

    Kuznetsov, Andrey V; Hermann, Martin; Troppmair, Jakob; Margreiter, Raimund; Hengster, Paul

    2010-01-01

    Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission-fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1-0.3 mum/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 mum/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems. PMID:19382913

  4. Measurement of diffusion of fluorescent compounds and autofluorescence in skin in vivo using a confocal instrument

    Science.gov (United States)

    Buttenschoen, K. K.; Sutton, E. E.; Daly, D.; Girkin, J. M.

    2016-02-01

    Using compact and affordable instrumentation based upon fluorescent confocal imaging we have tracked the movement of autofluorescent compounds through skin in near real time with high temporal and spatial resolution and sensitivity. The ability to measure the diffusion of compounds through skin with such resolution plays an important role for applications such as monitoring the penetration of pharmaceuticals applied to skin and assessing the integrity of the skin barrier. Several measurement methods exist, but they suffer from a number of problems such as being slow, expensive, non-portable and lacking sensitivity. To address these issues, we adapted a technique that we previously developed for tracking fluorescent compounds in the eye to measure the autofluorescence and the diffusion of externally applied fluorescent compounds in skin in vivo. Results are presented that show the change in autofluorescence of the volar forearm over the course of a week. We furthermore demonstrate the ability of the instrument to measure the diffusion speed and depth of externally applied fluorescent compounds both in healthy skin and after the skin barrier function has been perturbed. The instrument is currently being developed further for increased sensitivity and multi-wavelength excitation. We believe that the presented instrument is suitable for a large number of applications in fields such as assessment of damage to the skin barrier, development of topical and systemic medication and tracking the diffusion of fluorescent compounds through skin constructs as well as monitoring effects of skin products and general consumer products which may come into contact with the skin.

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

    Science.gov (United States)

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

    2016-08-15

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

  6. Morphological confocal microscopy in arthropods and the enhancement of autofluorescence after proteinase K extraction.

    Science.gov (United States)

    Valdecasas, Antonio G; Abad, Angela

    2011-02-01

    Procedures to study the molecular and morphological characteristics of microscopic organisms are often incompatible with each other. Therein, the realization of alternatives that make the characterization of these features compatible and simultaneously permit the deposition of the original material as a voucher sample into a reference collection is one of the foremost goals of biodiversity studies. In this study, we show that genomic extraction does not necessarily compromise the detailed study of the external morphology of microscopic organisms, and to do so, we used a group of aquatic mites (Acari, Hydrachnidia) as a test group. Hydrachnidia morphology is difficult to study when specimens have been stored in pure ethanol; however, proteinase K extraction leaves them flexible and easy to dissect, while, at the same time, maintaining all of their diagnostic features intact. Furthermore, autofluorescence is significantly enhanced after proteinase extraction. Our study was conducted with aquatic mites that were stored in absolute ethanol in the field and processed for DNA extraction using a Qiagen QIAamp minikit. Before and after molecular extraction, a laser scanning confocal microscopy morphological examination was carried out.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Marco Antonio Botelho

    2014-02-01

    Full Text Available OBJECTIVE: To determine the safety and efficacy of a transdermal nanostructured formulation of progesterone (10% combined with estriol (0.1% + estradiol (0.25% for relieving postmenopausal symptoms. METHODS: A total of 66 postmenopausal Brazilian women with climacteric symptoms of natural menopause received transdermal nanostructured formulations of progesterone and estrogens in the forearm daily for 60 months to mimic the normal ovarian secretory pattern. Confocal Raman spectroscopy of hormones in skin layers was performed. Clinical parameters, serum concentrations of estradiol and follicle-stimulating hormone, blood pressure, BI-RADS classification from bilateral mammography, and symptomatic relief were compared between baseline and 60 months post-treatment. Clinicaltrials.gov: NCT02033512. RESULTS: An improvement in climacteric symptoms was reported in 92.5% of women evaluated before and after 60 months of treatment. The serum concentrations of estradiol and follicle-stimulating hormone changed significantly (p<0.05 after treatment; the values of serum follicle-stimulating hormone decreased after 60 months from 82.04±4.9 to 57.12±4.1 IU/mL. A bilateral mammography assessment of the breasts revealed normal results in all women. No adverse health-related events were attributed to this hormone replacement therapy protocol. CONCLUSION: The nanostructured formulation is safe and effective in re-establishing optimal serum levels of estradiol and follicle-stimulating hormone and relieving the symptoms of menopause. This transdermal hormone replacement therapy may alleviate climacteric symptoms in postmenopausal women.

  9. Evaluation of human serum of severe rheumatoid arthritis by confocal Raman spectroscopy

    Science.gov (United States)

    Carvalho, C. S.; Raniero, L.; Santo, A. M. E.; Pinheiro, M. M.; Andrade, L. E. C.; Cardoso, M. A. G.; Junior, J. S.; Martin, A. A.

    2010-02-01

    Rheumatoid Arthritis is a systemic chronic inflammatory disease, recurrent and systemic, initiated by autoantibodies and maintained by inflammatory mechanisms cellular applicants. The evaluation of this disease to promote early diagnosis, need an associations of many tools, such as clinical, physical examination and thorough medical history. However, there is no satisfactory consensus due to its complexity. In the present work, confocal Raman spectroscopy was used to evaluate the biochemical composition of human serum of 40 volunteers, 24 patients with rheumatoid arthritis presenting clinical signs and symptoms, and 16 healthy donors. The technique of latex agglutination for the polystyrene covered with human immunoglobulin G and PCR (protein c-reactive) was performed for confirmation of possible false-negative results within the groups, facilitating the statistical interpretation and validation of the technique. This study aimed to verify the changes for the characteristics Raman peaks of biomolecules such as immunoglobulins amides and protein. The results were highly significant with a good separation between groups mentioned. The discriminant analysis was performed through the principal components and correctly identified 92% of the donors. Based on these results, we observed the behavior of arthritis autoimmune, evident in certain spectral regions that characterize the serological differences between the groups.

  10. Implementation of fluorescence confocal mosaicing microscopy by "early adopter" Mohs surgeons: a review of recent progress

    Science.gov (United States)

    Jain, Manu; Rajadhyaksha, Milind; Nehal, Kishwer

    2016-03-01

    Confocal mosaicing microscopy (CMM) enables rapid imaging of large areas of fresh tissue ex vivo without the processing that is necessary for conventional histology. When performed with fluorescence mode using acridine orange (nuclear specific dye) it enhances nuclei-to-dermis contrast that enables detection of all types of BCCs including thin strands of infiltrative basal cell carcinomas (BCCs). Thus far, this technique has been mostly validated in research setting for the analysis of BCC tumor margins. Recently, CMM has been adopted and implemented in real clinical settings by some surgeons as an alternative tool to frozen section (FS) during Mohs surgery. In this review article we summarize the development of CMM guided imaging of ex vivo tissues from bench to bedside. We also present its current state of application in routine clinical workflow not only for the assessment of BCC margin but also for other skin cancers such as melanoma, SCC, and some infectious diseases where FS is not routinely performed. Lastly, we also discuss the potential limitations of this technology as well as future developments. As this technology advances further, it may serve as an adjunct to standard histology and enable rapid surgical pathology of skin cancers at the bedside.

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

    Energy Technology Data Exchange (ETDEWEB)

    Botelho, Marco Antonio; Queiroz, Dinalva Brito; Barros, Gisele; Guerreiro, Stela; Umbelino, Sonia; Lyra, Arao; Borges, Boniek; Freitas, Allan, E-mail: marcobotelho@pq.cnpq.br [Universidade Potiguar, Natal, RN (Brazil). Lab. de Nanotecnologia; Fechine, Pierre [Universidade Federal do Ceara (GQMAT/UFCE), Fortaleza, CE (Brazil). Dept. de Quimica Analitica. Grupo Avancado de Biomateriais em Quimica; Queiroz, Danilo Caldas de [Instituto Federal de Ciencia e Tecnologia (IFCT), Fortaleza, CE (Brazil). Lab. de Biotecnologia; Ruela, Ronaldo [Instituto de Biotecnologia Aplicada (INBIOS), Fortaleza, CE (Brazil); Almeida, Jackson Guedes [Universidade Federal do Vale de Sao Francisco (UNIVALE), Petrolina, PE (Brazil). Fac. de Ciencias Farmaceuticas; Quintans Junior, Lucindo [Universidade Federal de Sergipe (UFSE), Sao Cristovao, SE (Brazil). Dept. de Fisiologia

    2014-06-01

    Objective:to determine the safety and efficacy of a transdermal nanostructured formulation of progesterone (10%) combined with estriol (0.1%) + estradiol (0.25%) for relieving postmenopausal symptoms. Methods: a total of 66 postmenopausal Brazilian women with climacteric symptoms of natural menopause received transdermal nanostructured formulations of progesterone and estrogens in the forearm daily for 60 months to mimic the normal ovarian secretory pattern. Confocal Raman spectroscopy of hormones in skin layers was performed. Clinical parameters, serum concentrations of estradiol and follicle-stimulating hormone, blood pressure, BI-RADS classification from bilateral mammography, and symptomatic relief were compared between baseline and 60 months post-treatment. Clinicaltrials.gov: NCT02033512. Results: an improvement in climacteric symptoms was reported in 92.5% of women evaluated before and after 60 months of treatment. The serum concentrations of estradiol and follicle-stimulating hormone changed significantly (p<0.05) after treatment; the values of serum follicle-stimulating hormone decreased after 60 months from 82.04 ± 4.9 to 57.12 ± 4.1 IU/mL. A bilateral mammography assessment of the breasts revealed normal results in all women. No adverse health-related events were attributed to this hormone replacement therapy protocol. Conclusion: the nanostructured formulation is safe and effective in re-establishing optimal serum levels of estradiol and follicle-stimulating hormone and relieving the symptoms of menopause. This transdermal hormone replacement therapy may alleviate climacteric symptoms in postmenopausal women. (author)

  12. Confocal microscopic evaluation of mixing performance for three-dimensional microfluidic mixer.

    Science.gov (United States)

    Yasui, Takao; Omoto, Yusuke; Osato, Keiko; Kaji, Noritada; Suzuki, Norikazu; Naito, Toyohiro; Okamoto, Yukihiro; Tokeshi, Manabu; Shamoto, Eiji; Baba, Yoshinobu

    2012-01-01

    We developed a confocal microscopic method for a quantitative evaluation of the mixing performance of a three-dimensional microfluidic mixer. We fabricated a microfluidic baker's transformation (MBT) mixer as a three-dimensional passive-type mixer for the efficient mixing of solutions. Although the MBT mixer is one type of ideal mixers, it is hard to evaluate its mixing performance, since the MBT mixer is based on several cycles of complicated three-dimensional microchannel structures. We applied the method developed here to evaluate the mixing of water and a fluorescein isothiocyanate (FITC; diffusion coefficient, 4.9 × 10(-10) m(2) s(-1)) solution by the MBT mixer. This method enables us to capture vertical section images for the fluid distributions of FITC and water at different three-dimensional microchannel structures of the MBT device. These images are in good agreement with those of mixing images based on numerical simulations. The mixing ratio could be calculated by the fluorescence intensity at each pixel of the vertical section image; complete mixing is recognized by a mixing ratio of more than 90%. The mixing ratios are measured at different cycles of the MBT mixer by changing the flow rate; the mixing performance is evaluated by comparisons with the mixing ratio of the straight microchannel without the MBT mixer.

  13. Effect of Hypericin on Confocal Imaging of Ca2+ Signaling in Cultured Human Retinal Pigment Epithelium

    Institute of Scientific and Technical Information of China (English)

    Qianying Gao; Yannian Hui; Yusheng Wang; Lin Wang

    2001-01-01

    Purpose: To investigate the mechanism of the Ca2 + signaling in cultured human retinal pigment epithelial(RPE) cells with the protein kinase C(PKC) specific inhibitor-hypericin stimulation.Methods: Cultured human RPE cells were analyzed using the fluorescence Ca2+ dye fluo-3 AM and laser scanning confocal microscope(LSCM) after stimulation with 100nM phorbol 12-myristate 13-acetate(PMA) and (or)5 concentrations of hypericin(1, 2, 3, 4 and 5 μM).Results: The normal fluorescence in RPE cells was strong and distributed throughout the cells. The nucleus appeared to be more fluorescent than the cytoplasm. After stimulation with PMA alone or 5 concentrations of hypericin, a rapid decrease in flurescence intensity was observed. There was no obvious difference in decreased curve among 5concentrations. However, after stimulation with a 24 hr preincubation of PMA and 5 concentrations of hypericin, a further decrease was not observed.Conclusion: Fluo-3 AM appears to be a good indicator of the change in Ca2+ occurring in RPE cells and hypericin is a strong inhibitor of Ca2 + influx channel. Hypericin has potential as a therapeutic drug for proliferative vitreoretinopathy(PVR), the inhibitory effect on PVR might be caused by blocking the PKC activity and inhibiting Ca2+ influxpathway.

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

    Science.gov (United States)

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

    2014-03-01

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

  15. Confocal microscopy with cylindrical vector beams and spectroscopy of single silicon nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Chizhik, Anna; Chizhik, Alexey; Baer, Sebastian; Meixner, Alfred [Inst. of Physical and Theor. Chem., Univ. of Tuebingen (Germany); Schmidt, Torsten; Huisken, Friedrich [Lab. Astrophys., Group of the MPI for Astronomy at the Inst. of Solid State Phys., Univ. of Jena (Germany)

    2010-07-01

    Being the paramount material silicon revealed new magnificent outlooks with the development of nanotechnology. During last years the research on silicon nanoparticles has been one of the hottest topics. However, many of their photoluminescence (PL) properties are still unclear. Combining the confocal microscopy, spectroscopy, and cylindrical vector beams (also known as higher order laser modes) we reveal new details of fundamental PL properties of Si/SiO{sub 2} core-shell systems and hollow SiO{sub 2} shells. We show that the emission from both systems may originate from defects of the SiO{sub 2} structure or at the Si-SiO{sub 2} interface. This result demonstrates the effect of ''break-down'' of the quantum confinement in small Si/SiO{sub 2} nanoparticles, which limits the PL tunability and thus, applications in Si optical nanostructures, especially in the short wavelength range. Using the technique of cylindrical vector beams we demonstrate that SiO{sub 2} nanoparticles and Si/SiO{sub 2} nanocrystals, where the PL originates from defects, possess linear transition dipole moment (TDM). Moreover, we precisely determine the 3-dimensional orientation of single nanoparticle TDM and show such dynamical effects as TDM sudden flipping.

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

    Directory of Open Access Journals (Sweden)

    Qiaoling Wei

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Wei LI

    2013-01-01

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

  18. Excitonic emission of CuInS{sub 2} crystals using confocal microscopy system

    Energy Technology Data Exchange (ETDEWEB)

    Horikawa, Yusuke; Matsuo, Shingo; Wakita, Kazuki [Department of Electrical, Electronics and Computer Engineering, Chiba Institute of Technology, 2-17-1, Tsudanuma, Narashino, Chiba 275-0016 (Japan); Shim, YongGu [Department of Physics and Electronics, Osaka Prefecture University, 1-1 Gakuencho, Naka-ku, Sakai, Osaka 599-8531 (Japan)

    2013-08-15

    Photoluminescence (PL) spectra in the band-edge region on bulk single-crystals of CuInS{sub 2} grown by the traveling heater method have been investigated using a confocal microscopy system. The observed PL spectra are separated into two Lorentzian peaks which are assigned to be A and B free excitons, by the analysis of the excitation intensity dependence of the emissions. Consequently, we present the behaviour of B free exciton within a wide range of temperatures. The time-resolved emissions of A free exciton have also been examined. The decay of the emissions is analyzed using a double exponential curve. Fast and slow components are attributed to nonradiative relaxation and radiative recombination, respectively. The decay-time constant of the slow component corresponds to the radiative lifetime of A free exciton and is obtained over the wide temperature region until 300 K. (copyright 2013 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  19. Quantitative characterization of the fracture surface of Si single crystals by confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Xin, Y.B.; Hsia, K.J.; Lange, D.A. [Univ. of Illinois, Urbana, IL (United States)

    1995-12-01

    Experiments are conducted to study the dislocation nucleation conditions at the crack tip in {l_brace}110{r_brace}<110> oriented Si single crystals. Specimens with surface cracks are first statically loaded at elevated temperatures for a prolonged period of time to initiate and move dislocations away from the crack tip, then cooled down to room temperature and loaded to fracture to measure the fracture toughness. Fractographic analysis of the fracture surfaces is performed. Distinct wavy patterns on the fracture surface at the initial cleavage crack front are observed, which is attributed to the existence of local mixed mode 1/mode 3 stresses resulting from the inhomogeneous dislocation activity. Confocal microscopy is employed to quantify the fracture surface roughness. The results show that the increase of fracture toughness is directly associated with the increased area of the rough surface, which is characterized by the roughness number or the fractal dimension increment. The results also demonstrate that dislocation nucleation can occur only at discrete sites. The spacing between these dislocation nucleation sources is of the order of 1 {micro}m. A simple model is developed for the relationship between the fracture toughness and the surface roughness parameters, which is in good agreement with the experimental results.

  20. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks

    Science.gov (United States)

    Hames, Samuel C.; Ardigò, Marco; Soyer, H. Peter; Bradley, Andrew P.; Prow, Tarl W.

    2016-01-01

    Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20–30 and 50–70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin. PMID:27088865