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

  1. 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)


    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.

  2. [Artefacts of confocal microscopy]. (United States)

    Vekshin, N L; Frolov, M S


    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.

  3. 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)


    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.

  4. 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)


    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}

  5. Basic confocal microscopy

    Directory of Open Access Journals (Sweden)

    Manuela Monti


    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....

  6. 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...

  7. Biological confocal microscopy

    Directory of Open Access Journals (Sweden)

    Guy Cox


    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.

  8. Molecular confocal laser endomicroscopy

    DEFF Research Database (Denmark)

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


    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...

  9. Confocal laser endomicroscopy

    DEFF Research Database (Denmark)

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


    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...

  10. Confocal Raman Microscopy

    CERN Document Server

    Dieing, Thomas; Toporski, Jan


    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.

  11. 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


    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.

  12. Hyperspectral confocal microscope. (United States)

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


    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.

  13. 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)


    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)

  14. Confocal coded aperture imaging

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  15. Twin-Photon Confocal Microscopy

    CERN Document Server

    Simon, D S


    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.


    Directory of Open Access Journals (Sweden)

    Merete Krog Raarup


    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.

  17. Confocal endomicroscopy of the larynx (United States)

    Just, T.; Wiechmann, T.; Stachs, O.; Stave, J.; Guthoff, R.; Hüttmann, G.; Pau, H. W.


    Beside the good image quality with the confocal laser scanning microscope (HRTII) and the Rostock Cornea Module (RCM), this technology can not be used to investigate the human larynx in vivo. To accomplish this, a rigid custom-made endoscope (KARL STORZ GmbH & Co. KG; Tuttlingen Germany) was developed. A connector was developed to connect the scanner head of the HRTII to the rigid endoscope. With the connector, the starting plane can be set manually. To achieve optical sectioning of the laryngeal tissue (80 μm per volume scan), the scanning mechanism of the HRTII needs to be activated using a foot switch. The devices consisting of the endoscope, HRTII, and the connector supply images of 400 x 400 μm and reach average penetration depths of 100-300 μm (λ/4 plate of the scanner head of the HRTII was removed). The lateral and axial resolutions are about 1-2 μm and 2 μm, respectively. In vivo rigid confocal endoscopy is demonstrated with an acquisition time for a volume scan of 6 s. The aim of this study was to differentiate pre-malignant laryngeal lesions from micro-invasive carcinoma of the larynx. 22 patients with suspicious lesions of the true vocal cords were included. This pilot study clearly demonstrates the possibility to detect dysplastic cells close to the basal cell layer and within the subepithelial space in lesions with small leukoplakia (thin keratin layer). These findings may have an impact on microlaryngoscopy to improve the precision for biopsy and on microlaryngoscopic laser surgery of the larynx to identify the margins of the pre-malignant lesion.

  18. Optical tweezers for confocal microscopy (United States)

    Hoffmann, A.; Meyer zu Hörste, G.; Pilarczyk, G.; Monajembashi, S.; Uhl, V.; Greulich, K. O.


    In confocal laser scanning microscopes (CLSMs), lasers can be used for image formation as well as tools for the manipulation of microscopic objects. In the latter case, in addition to the imaging lasers, the light of an extra laser has to be focused into the object plane of the CLSM, for example as optical tweezers. Imaging as well as trapping by optical tweezers can be done using the same objective lens. In this case, z-sectioning for 3D imaging shifts the optical tweezers with the focal plane of the objective along the optical axis, so that a trapped object remains positioned in the focal plane. Consequently, 3D imaging of trapped objects is impossible without further measures. We present an experimental set-up keeping the axial trapping position of the optical tweezers at its intended position whilst the focal plane can be axially shifted over a distance of about 15 μm. It is based on fast-moving correctional optics synchronized with the objective movement. First examples of application are the 3D imaging of chloroplasts of Elodea densa (Canadian waterweed) in a vigorous cytoplasmic streaming and the displacement of zymogen granules in pancreatic cancer cells (AR42 J).

  19. Confocal Annular Josephson Tunnel Junctions (United States)

    Monaco, Roberto


    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.

  20. Confocal Endomicroscopy of Colorectal Polyps

    Directory of Open Access Journals (Sweden)

    Vivian M. Ussui


    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.

  1. 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: [Laboratoire d' Optique P. M. Duffieux, UMR-6603 CNR/Universite de Franche-Comte. 16 route de Gray, 25030 Besancon Cedex (France)


    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.

  2. Confocal Laser Endomicroscopy in Inflammatory Bowel Disease

    DEFF Research Database (Denmark)

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


    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...

  3. Confocal microlaparoscope for imaging the fallopian tube (United States)

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


    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.

  4. 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)


    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

  5. Confocal microlaparoscope for imaging the fallopian tube (United States)

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


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

  6. Spatial heterodyne scanning laser confocal holographic microscopy

    CERN Document Server

    Liu, Changgeng


    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...

  7. Confocal multiview light-sheet microscopy (United States)

    Medeiros, Gustavo de; Norlin, Nils; Gunther, Stefan; Albert, Marvin; Panavaite, Laura; Fiuza, Ulla-Maj; Peri, Francesca; Hiiragi, Takashi; Krzic, Uros; Hufnagel, Lars


    Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage. PMID:26602977

  8. Confocal Microscopy in Biopsy Proven Argyrosis

    Directory of Open Access Journals (Sweden)

    Melis Palamar


    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.

  9. Confocal microscopy via multimode fibers: fluorescence bandwidth (United States)

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe


    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.

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

    Directory of Open Access Journals (Sweden)

    Aqib H Zehri


    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.

  11. A Confocal Endoscope for Cellular Imaging

    Directory of Open Access Journals (Sweden)

    Jiafu Wang


    Full Text Available Since its inception, endoscopy has aimed to establish an immediate diagnosis that is virtually consistent with a histologic diagnosis. In the past decade, confocal laser scanning microscopy has been brought into endoscopy, thus enabling in vivo microscopic tissue visualization with a magnification and resolution comparable to that obtained with the ex vivo microscopy of histological specimens. The major challenge in the development of instrumentation lies in the miniaturization of a fiber-optic probe for microscopic imaging with micron-scale resolution. Here, we present the design and construction of a confocal endoscope based on a fiber bundle with 1.4-μm lateral resolution and 8-frames per second (fps imaging speed. The fiber-optic probe has a diameter of 2.6 mm that is compatible with the biopsy channel of a conventional endoscope. The prototype of a confocal endoscope has been used to observe epithelial cells of the gastrointestinal tracts of mice and will be further demonstrated in clinical trials. In addition, the confocal endoscope can be used for translational studies of epithelial function in order to monitor how molecules work and how cells interact in their natural environment.

  12. Confocal Microscopy Imaging of the Biofilm Matrix

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise


    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...

  13. Practical aspects of quantitative confocal microscopy. (United States)

    Murray, John M


    Confocal microscopes are in principle well suited for quantitative imaging. The 3D fluorophore distribution in a specimen is transformed by the microscope optics and detector into the 2D intensity distribution of a digital image by a linear operation, a convolution. If multiple 2D images of the specimen at different focal planes are obtained, then the original 3D distribution in the specimen can be reconstructed. This reconstruction is a low-pass spatially filtered representation of the original, but quantitatively preserves relative fluorophore concentrations, with of course some limitations on accuracy and precision due to aberrations and noise. Given appropriate calibration, absolute fluorophore concentrations are accessible. A few simple guidelines are given for setting up confocal microscopes and checking their performance. With a little care, the images collected should be suitable for most types of quantitative analysis.

  14. Digital confocal microscopy through a multimode fiber

    CERN Document Server

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


    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.

  15. A near-infrared confocal scanner (United States)

    Lee, Seungwoo; Yoo, Hongki


    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.

  16. Clinical applications of corneal confocal microscopy

    Directory of Open Access Journals (Sweden)

    Mitra Tavakoli


    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

  17. Digital differential confocal microscopy based on spatial shift transformation. (United States)

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


    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.

  18. 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)


    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.

  19. 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.

  20. Reflectance confocal microscopy features of facial angiofibromas (United States)

    Millán-Cayetano, José-Francisco; Yélamos, Oriol; Rossi, Anthony M.; Marchetti, Michael A.; Jain, Manu


    Facial angiofibromas are benign tumors presenting as firm, dome-shaped, flesh-colored to pink papules, typically on the nose and adjoining central face. Clinically and dermoscopically they can mimic melanocytic nevi or basal cell carcinomas (BCC). Reflectance confocal microscopy (RCM) is a noninvasive imaging tool that is useful in diagnosing melanocytic and non-melanocytic facial lesions. To date no studies have described the RCM features of facial angiofibromas. Herein, we present two cases of facial angiofibromas that were imaged with RCM and revealed tumor island-like structures that mimicked BCC, leading to skin biopsy.

  1. Confocal laser endomicroscopy in ulcerative colitis

    DEFF Research Database (Denmark)

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


    was to correlate colonic confocal laser endomicroscopy (CLE) in ulcerative colitis with histopathology and macroscopic appearance before and after intensification of medical treatment. METHODS: Twenty-two patients with ulcerative colitis in clinical relapse and 7 control subjects referred for colonoscopy were...... colitis compared with inactive ulcerative colitis...... is an emerging endoscopic technique that reproducibly identifies mucosal changes in ulcerative colitis. With the exception of crypt changes, endomicroscopic features appear to improve slowly with time after medical treatment. ( CLINICAL TRIAL REGISTRATION NUMBER: NCT01684514.)....

  2. Fungal Keratitis - Improving Diagnostics by Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Esben Nielsen


    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.

  3. Confocal Raman microspectroscopy of the skin. (United States)

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


    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.

  4. Reflectance Confocal Microscopy for Inflammatory Skin Diseases. (United States)

    Agozzino, M; Gonzalez, S; Ardigò, M


    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.

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


    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.

  6. Biological applications of confocal fluorescence polarization microscopy (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

  7. Fluorescence confocal endomicroscopy in biological imaging (United States)

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


    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 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 rodent disease models, in vivo endomicroscopy with appropriate fluorescent agents allowed examination of thrombosis formation, tumour microvasculature and liver metastases, diagnosis and staging of ulcerative colitis, liver necrosis and glomerulonephritis. Miniaturised confocal endomicroscopy allows rapid in vivo molecular and subsurface microscopy of normal and pathologic tissue at high resolution in small and large whole animal models

  8. Confocal Terahertz Imaging of Ancient Manuscripts (United States)

    Flammini, Mariano; Bonsi, Claudia; Ciano, Chiara; Giliberti, Valeria; Pontecorvo, Emanuele; Italia, Paola; DelRe, Eugenio; Ortolani, Michele


    Terahertz imaging has the potential to identify and decipher portions of ancient manuscripts, which may be unreadable at infrared and visible wavelengths. We use a scanning confocal terahertz microscope to scan a medieval parchment with music notes and pentagrams written with different inks. The microscope is based on a continuous-wave solid-state source at 0.3 THz, emitting in the free space with a horn antenna, and a high numerical-aperture ellipsoidal reflector. We present terahertz images with diffraction-limited lateral resolution of approximately 0.5 mm, where the different inks all give similar high contrast. Symbols written on the "verso" side of the parchment, barely glimpsed in the near-infrared photograph, leave a clear imprint in the terahertz images. Artifacts due to imperfect flatness of the parchment are also briefly discussed.

  9. Near-infrared hyperspectral reflective confocal microscopy (United States)

    Huang, Wei; Zhang, Yunhai; Miao, Xin; Xue, Xiaojun; Xiao, Yun


    A Near-Infrared HyperSpectral Reflective Confocal Microscopy (NIHS-RCM) is proposed in order to get high resolution images of deep biological tissues such as skin. The microscopy system uses a super-continuum laser for illumination, an acousto-optic tunable filter (AOTF) for rapid selection of near-infrared spectrum, a resonant galvanometer scanner for high speed imaging (15f/s) and near-infrared avalanche diode as detector. Porcine skin and other experiments show that the microscopy system could get deep tissue images (180 μm), and show the different ingredients of tissue with different wavelength of illumination. The system has the ability of selectively imaging of multiple ingredients at deep tissue which can be used in skin diseases diagnosis and other fields.

  10. Imaging white adipose tissue with confocal microscopy. (United States)

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


    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.

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

    NARCIS (Netherlands)

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


    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

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

    NARCIS (Netherlands)

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


    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

  13. 4D confocal microscopy for visualisation of bone remodelling

    NARCIS (Netherlands)

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


    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

  14. Evaluation and purchase of confocal microscopes: Numerous factors to consider (United States)

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

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

    Institute of Scientific and Technical Information of China (English)


    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

  16. In Vivo Confocal Microscopy expanding horizons in corneal imaging

    NARCIS (Netherlands)

    T. Hillenaar (Toine)


    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

  17. Confocal Raman Microspectroscopy of Oral Streptococci (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

  18. Optimization of confocal scanning laser ophthalmoscope design. (United States)

    LaRocca, Francesco; Dhalla, Al-Hafeez; Kelly, Michael P; Farsiu, Sina; Izatt, Joseph A


    Confocal scanning laser ophthalmoscopy (cSLO) enables high-resolution and high-contrast imaging of the retina by employing spatial filtering for scattered light rejection. However, to obtain optimized image quality, one must design the cSLO around scanner technology limitations and minimize the effects of ocular aberrations and imaging artifacts. We describe a cSLO design methodology resulting in a simple, relatively inexpensive, and compact lens-based cSLO design optimized to balance resolution and throughput for a 20-deg field of view (FOV) with minimal imaging artifacts. We tested the imaging capabilities of our cSLO design with an experimental setup from which we obtained fast and high signal-to-noise ratio (SNR) retinal images. At lower FOVs, we were able to visualize parafoveal cone photoreceptors and nerve fiber bundles even without the use of adaptive optics. Through an experiment comparing our optimized cSLO design to a commercial cSLO system, we show that our design demonstrates a significant improvement in both image quality and resolution.

  19. Calculation of confocal microscope images of cholesteric blue phases (United States)

    Fukuda, Jun-ichi; Okumura, Yasushi; Kikuchi, Hirotsugu


    Real-space images of bulk cholesteric blue phases (BPs) have been successfully obtained by confocal microscopy observations using structural color without doping fluorescent dye. However, theoretical interpretation of these images (for example, the understanding of the relation between intensity distribution and the ordering of BPs) remains challenging because typical lattice spacing of BPs is of the order of the wavelength of visible light, and therefore geometrical optics is entirely useless. In this work, we present a numerical approach to calculate the confocal images of BPs by solving the Maxwell equations. Calculated confocal images are consistent with experimental observations in terms of in-plane symmetry.

  20. Confocal supercritical angle fluorescence microscopy for cell membrane imaging

    CERN Document Server

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


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

  1. Microelectrophoresis of Silica Rods Using Confocal Microscopy. (United States)

    Bakker, Henriëtte E; Besseling, Thijs H; Wijnhoven, Judith E G J; Helfferich, Peter H; van Blaaderen, Alfons; Imhof, Arnout


    The electrophoretic mobility and the zeta potential (ζ) of fluorescently labeled colloidal silica rods, with an aspect ratio of 3.8 and 6.1, were determined with microelectrophoresis measurements using confocal microscopy. In the case where the colloidal particles all move at the same speed parallel to the direction of the electric field, we record a xyz-stack over the whole depth of the capillary. This method is faster and more robust compared to taking xyt-series at different depths inside the capillary to obtain the parabolic flow profile, as was done in previous work from our group. In some cases, rodlike particles do not move all at the same speed in the electric field, but exhibit a velocity that depends on the angle between the long axis of the rod and the electric field. We measured the orientation-dependent velocity of individual silica rods during electrophoresis as a function of κa, where κ(-1) is the double layer thickness and a is the radius of the rod associated with the diameter. Thus, we determined the anisotropic electrophoretic mobility of the silica rods with different sized double layers. The size of the double layer was tuned by suspending silica rods in different solvents at different electrolyte concentrations. We compared these results with theoretical predictions. We show that even at already relatively high κa when the Smoluchowski limiting law is assumed to be valid (κa > 10), an orientation dependent velocity was measured. Furthermore, we observed that at decreasing values of κa the anisotropy in the electrophoretic mobility of the rods increases. However, in low polar solvents with κa < 1, this trend was reversed: the anisotropy in the electrophoretic mobility of the rods decreased. We argue that this decrease is due to end effects, which was already predicted theoretically. When end effects are not taken into account, this will lead to strong underestimation of the experimentally determined zeta potential.

  2. Interference Confocal Microscope Integrated with Spatial Phase Shifter. (United States)

    Wang, Weibo; Gu, Kang; You, Xiaoyu; Tan, Jiubin; Liu, Jian


    We present an interference confocal microscope (ICM) with a new single-body four-step simultaneous phase-shifter device designed to obtain high immunity to vibration. The proposed ICM combines the respective advantages of simultaneous phase shifting interferometry and bipolar differential confocal microscopy to obtain high axis resolution, large dynamic range, and reduce the sensitivity to vibration and reflectance disturbance seamlessly. A compact single body spatial phase shifter is added to capture four phase-shifted interference signals simultaneously without time delay and construct a stable and space-saving simplified interference confocal microscope system. The test result can be obtained by combining the interference phase response and the bipolar property of differential confocal microscopy without phase unwrapping. Experiments prove that the proposed microscope is capable of providing stable measurements with 1 nm of axial depth resolution for either low- or high-numerical aperture objective lenses.

  3. Divided-aperture differential confocal fast-imaging microscopy (United States)

    Wang, Yun; Qiu, Lirong; Zhao, Xiangye; Zhao, Weiqian


    A new method, laser divided-aperture differential confocal microscopy (DDCM), is proposed to achieve high-resolution 3D imaging of microstructures of large-scale sample surfaces. This method uses a divided-aperture confocal structure to significantly improve the axial resolution of confocal microscopy and keep a long working distance simultaneously; uses two radically offset point detectors to achieve differential detection to further improve the axial response sensitivity and realize fast imaging of a large-scale sample surface with a big axial scan-step interval. Theoretical analyses and experimental results show that the DDCM can reach an axial resolution of 5 nm with a 3.1 mm working distance with a 3 times imaging speed of a confocal system with the same resolution.

  4. Automated spherical aberration correction in scanning confocal microscopy

    NARCIS (Netherlands)

    Yoo, H.W.; Royen, M.E.; van Cappellen, W.A.; Houtsmuller, A.B.; Verhaegen, M.H.G.; Schitter, G.


    Mismatch between the refractive indexes of immersion media and glass coverslips introduces spherical aberrations in microscopes especially for high numerical aperture objectives. This contribution demonstrates an automated adjustment of the coverslip correction collar in scanning confocal microscopy


    Institute of Scientific and Technical Information of China (English)


    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.

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

    Directory of Open Access Journals (Sweden)

    K. Gawdzińska


    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.

  7. In vivo confocal microscopy in chloroquine-induced keratopathy

    Directory of Open Access Journals (Sweden)

    Iacopo Paladini


    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.

  8. A New Multichannel Spectral Imaging Laser Scanning Confocal Microscope

    Directory of Open Access Journals (Sweden)

    Yunhai Zhang


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

  9. Measuring Corneal Haze by Using Scheimpflug Photography and Confocal Microscopy (United States)

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


    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

  10. Fused oblique incidence reflectometry and confocal fluorescence microscopy (United States)

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


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

  11. Confocal and Two-Photon Microscopy: Foundations, Applications and Advances (United States)

    Diaspro, Alberto


    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.

  12. 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


    @@ 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.

  13. Simple high-speed confocal line-scanning microscope. (United States)

    Im, Kang-Bin; Han, Sumin; Park, Hwajoon; Kim, Dongsun; Kim, Beop-Min


    Using a line scan camera and an acousto-optic deflector (AOD), we constructed a high-speed confocal laser line-scanning microscope that can generate confocal images (512 x 512 pixels) with up to 191 frames/s without any mechanically moving parts. The line scanner consists of an AOD and a cylindrical lens, which creates a line focus sweeping over the sample. The measured resolutions in z (depth), x (perpendicular to line focus), and y (direction of line focus) directions are 3.3 mum, 0.7 mum and 0.9 mum, respectively, with a 50x objective lens. This confocal microscope may be useful for analyzing fast phenomena during biological and chemical interactions and for fast 3D image reconstruction.

  14. 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


    @@ 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.

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

    Directory of Open Access Journals (Sweden)

    Yamazaki N


    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

  16. Confocal Raman microscopy for identification of bacterial species in biofilms (United States)

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


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

  17. A Pico Projector Source for Confocal Fluorescence and Ophthalmic Imaging. (United States)

    Muller, Matthew S


    A Pico digital light projector has been implemented as an integrated illumination source and spatial light modulator for confocal imaging. The target is illuminated with a series of rapidly projected lines or points to simulate scanning. Light returning from the target is imaged onto a 2D rolling shutter CMOS sensor. By controlling the spatio-temporal relationship between the rolling shutter and illumination pattern, light returning from the target is spatially filtered. Confocal retinal, fluorescence, and Fourier-domain optical coherence tomography implementations of this novel imaging technique are presented.

  18. Microscopia confocal in vivo na cistinose: relato de caso

    Directory of Open Access Journals (Sweden)

    Victor Gustavo


    Full Text Available A cistinose é doença autossômica recessiva rara caracterizada pelo acúmulo do aminoácido cistina livre dentro dos lisossomos e geralmente é fatal na primeira década de vida na ausência de transplante renal. O presente estudo tem por objetivo relatar os achados da microscopia confocal in vivo em paciente adulto com cistinose infantil. O exame de microscopia confocal in vivo revelou que há diferenças quanto à intensidade de acometimento, tamanho e forma dos depósitos nas diversas camadas corneanas.

  19. EUS-Guided Needle-Based Confocal Laser Endomicroscopy

    DEFF Research Database (Denmark)

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


    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...

  20. Optomechatronics Design and Control for Confocal Laser Scanning Microscopy

    NARCIS (Netherlands)

    Yoo, H.W.


    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

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

    NARCIS (Netherlands)

    K. Maquelin (Kees)


    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

  2. Laser differential fitting confocal microscopy with high imaging efficiency. (United States)

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


    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.

  3. 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))


    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.

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

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.


    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.

  5. 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)


    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).

  6. Comprehensive confocal endomicroscopy of the esophagus in vivo (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.


    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

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

    Institute of Scientific and Technical Information of China (English)


    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.

  8. 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


    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.

  9. Three-Dimensional Visualization of Interfacial Phenomena Using Confocal Microscopy (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


    Institute of Scientific and Technical Information of China (English)

    Wang Yonghong; Yu Xiaofen


    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.

  11. Laser differential confocal ultra-long focal length measurement. (United States)

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


    A new laser differential confocal focal-length measurement method is proposed for the measurement of an ultra-long focal-length. The approach proposed uses the property of an axial intensity curve that the absolute zero precisely corresponds to the focus of the objective in a differential confocal focusing system (DCFS) to measure the variation in position of DCFS focus with and without a measured ultra-long focal-length lens (UFL), uses the distance between the two focuses to obtain the UFL focal-length, and thereby achieving the precise measurement of ultra-long focal-length. The method has a high focusing precision, a strong anti-interference capability and a short measurement light-path. The theoretical analyses and preliminary experimental results indicate that the relative measurement error is about 0.01% when the method is used for the measurement of back-focus-distance (BFD).

  12. Anabaena cell ageing monitored with confocal fluorescence spectroscopy. (United States)

    Ke, Shan; Bindokas, Vytas; Haselkorn, Robert


    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.

  13. Classification of billiard motions in domains bounded by confocal parabolas

    Energy Technology Data Exchange (ETDEWEB)

    Fokicheva, V V [M. V. Lomonosov Moscow State University, Faculty of Mechanics and Mathematics, Moscow (Russian Federation)


    We consider the billiard dynamical system in a domain bounded by confocal parabolas. We describe such domains in which the billiard problem can be correctly stated. In each such domain we prove the integrability for the system, analyse the arising Liouville foliation, and calculate the invariant of Liouville equivalence--the so-called marked molecule. It turns out that billiard systems in certain parabolic domains have the same closures of solutions (integral trajectories) as the systems of Goryachev-Chaplygin-Sretenskii and Joukowski at suitable energy levels. We also describe the billiard motion in noncompact domains bounded by confocal parabolas, namely, we describe the topology of the Liouville foliation in terms of rough molecules. Bibliography: 16 titles.

  14. Optomechatronics Design and Control for Confocal Laser Scanning Microscopy


    Yoo, H W


    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 processes and aberration-corrected imaging to localize the targeted biomolecule precisely through optical disturbances by specimen. In this thesis, optomechatronics design and control are discussed for...

  15. Enlightening the Pink: Use of Confocal Microscopy in Pink Lesions. (United States)

    Gill, Melissa; González, Salvador


    Solitary pink lesions can pose a particular challenge to dermatologists because they may be almost or completely featureless clinically and dermoscopically, previously requiring biopsy to exclude malignancy. However, these lesions usually are not particularly challenging histopathologically. Thus, the incorporation of in vivo reflectance confocal microscopy into the clinical practice, which allows for noninvasive examination of the skin at the cellular level revealing features previously seen only on histopathology, is particularly useful for this subset of clinically difficult lesions.

  16. Multi-spectral confocal microendoscope for in-vivo imaging (United States)

    Rouse, Andrew Robert

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

  17. 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


    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.

  18. In-vivo multi-spectral confocal microscopy (United States)

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


    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.

  19. 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


    @@ 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.

  20. Adaptive optics parallel near-confocal scanning ophthalmoscopy. (United States)

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


    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.

  1. Aerial wetting contact angle measurement using confocal microscopy


    Chesna, Jacob W.; Wiedmaier, Bob F.; Wang, Jinlin; Samara, Ayman; Leach, Richard K.; Her, Tsing-Hua; Smith, Stuart T.


    A method is presented in which the wetting contact angle of a sessile drop is acquired aerially using confocal techniques to measure the radius and the height of a droplet deposited on a planar surface. The repeatability of this method is typically less than 0.25°, and often less than 0.1°, for droplet diameters less than 1 mm. To evaluate accuracy of this method, an instrument uncertainty budget is developed, which predicts a combined uncertainty of 0.91° for a 1 mm diameter water droplet wi...

  2. UV laser mediated cell selective destruction by confocal microscopy

    Directory of Open Access Journals (Sweden)

    Giangrande Angela


    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


    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.


    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 (United States)

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


    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))


    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]. (United States)

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


    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. Quantification and confocal imaging of protein specific molecularly imprinted polymers


    Hawkins, DM; Trache, A; Ellis, EA; Stevenson, D.; Holzenburg, A.; Meininger, GA; Reddy, Subrayal M


    We have employed FITC-albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labelled template is reported, with subsequent characterisation of the smart material to show that the HydroMIP possess a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC-albumin imprinted HydroMIP using confocal microscopy is described, with t...

  9. 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.


    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.

  10. 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


    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

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

    Institute of Scientific and Technical Information of China (English)


    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.

  12. Confocal Raman-AFM, A New Tool for Materials Research (United States)

    Schmidt, Ute


    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 (United States)

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


    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. (United States)

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


    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. Use of confocal microscopy for nanoparticle drug delivery through skin (United States)

    Zhang, Leshuai W.; Monteiro-Riviere, Nancy A.


    Confocal laser scanning microscopy (CLSM) is a well-used microscopic tool that provides valuable morphological and functional information within cells and tissues. The application of CLSM to skin and the topical penetration of nanoparticles (NP) will be addressed. First, we describe the advantages of confocal microscopy compared to other techniques and its use relative to skin research. Second, we discuss the ability of CLSM to detect single NP. Regarding their interaction with skin, the appropriate method to retain nanoparticle localization in the tissue with minimal fixation is critically important. Also, the interaction of several different types of NP (quantum dots, fullerene and dendrimers) and their interaction with skin detected by CLSM under various conditions (flexed, tape stripped and abraded skin) is reviewed. Finally, human epidermal keratinocytes and dendritic cells that serve as appropriate in vitro models for skin cell interactions and cellular uptake of NP are also discussed. In conclusion, the unique functions of CLSM such as the ability to detect fluorescence, optical sectioning, three dimensional remodeling, as well as its use in the reflection mode in tandem with other methods, provides great promise with broad applications regarding the interactions of nanomaterials with skin.

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

    Directory of Open Access Journals (Sweden)

    Hongda Mao


    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.

  17. Emission characteristics of light-emitting diodes by confocal microscopy (United States)

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


    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.

  18. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    Directory of Open Access Journals (Sweden)

    Beaudet A.


    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.

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

  20. High contrast, depth-resolved thermoreflectance imaging using a Nipkow disk confocal microscope. (United States)

    Summers, J A; Yang, T; Tuominen, M T; Hudgings, J A


    We have developed a depth-resolved confocal thermal imaging technique that is capable of measuring the temperature distribution of an encapsulated or semi-obstructed device. The technique employs lock-in charge coupled device-based thermoreflectance imaging via a Nipkow disk confocal microscope, which is used to eliminate extraneous reflections from above or below the imaging plane. We use the confocal microscope to predict the decrease in contrast and dynamic range due to an obstruction for widefield thermoreflectance, and we demonstrate the ability of confocal thermoreflectance to maintain a high contrast and thermal sensitivity in the presence of large reflecting obstructions in the optical path.

  1. 3D imaging of neutron tracks using confocal microscopy (United States)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan


    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. Materials and corrosion characterization using the confocal resonator

    Energy Technology Data Exchange (ETDEWEB)

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


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

  3. Clinical results with acridine orange using a novel confocal laparoscope (United States)

    Tanbakuchi, Anthony A.; Rouse, Andrew R.; Hatch, Kenneth D.; Gmitro, Arthur F.


    We previously reported on the development of a multi-spectral confocal laparoscope for clinical imaging. In this paper we present current results using the system to image ovaries with a new laparoscope design using the contrast agent acridine orange. This new laparoscope integrates computer controlled systems for focus, depth scans, and localized contrast agent delivery. Precise axial position control is accomplished with tiny stepper motors integrated inside the laparoscope handle. Ergonomic handle controls allow for data acquisition, deliver of contrast agents, and adjustment of imaging depth during procedures by the surgeon. We have approval to use acridine orange in our clinical trials to image ovaries in vivo during oophorectomies. We present in vivo results using both acridine orange and fluorescein as the topically administered contrast agent.

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

    CERN Document Server

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


    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...

  5. Error analysis for a laser differential confocal radius measurement system. (United States)

    Wang, Xu; Qiu, Lirong; Zhao, Weiqian; Xiao, Yang; Wang, Zhongyu


    In order to further improve the measurement accuracy of the laser differential confocal radius measurement system (DCRMS) developed previously, a DCRMS error compensation model is established for the error sources, including laser source offset, test sphere position adjustment offset, test sphere figure, and motion error, based on analyzing the influences of these errors on the measurement accuracy of radius of curvature. Theoretical analyses and experiments indicate that the expanded uncertainty of the DCRMS is reduced to U=0.13  μm+0.9  ppm·R (k=2) through the error compensation model. The error analysis and compensation model established in this study can provide the theoretical foundation for improving the measurement accuracy of the DCRMS.

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

    Energy Technology Data Exchange (ETDEWEB)

    De Stefano, Luca [Institute for Microelectronics and Microsystems, Department of Naples, National Council of Research, Via P Castellino 111, 80131 Naples (Italy); D' Auria, Sabato [Institute of Protein Biochemistry, National Council of Research, Via P Castellino 111, 80131 Naples (Italy)


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

  7. 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: [Sensor Electronic Technology, Inc., 1195 Atlas Road, Columbia, SC (United States)


    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.

  8. Characterization of Developing Cotton Fibers by Confocal Raman Microscopy

    Directory of Open Access Journals (Sweden)

    Luis Cabrales


    Full Text Available Cellulose deposition in developing cotton fibers has been studied previously with analytical techniques, such as Fourier transform infrared spectroscopy (FTIR, High-performance liquid chromatography (HPLC and Thermogravimetric analysis (TGA. Recent technological developments in instrumentation have made Raman microscopy emerge as an extraordinary analytical tool in biological and plant research. The advantage of using confocal Raman microscopy (CRM resides in the lateral spatial resolution and in the fact that Raman spectroscopy provides not only chemical composition information, but also structural information. Cross-sections of cotton fibers harvested at different developmental stages were studied with CRM. The Raman bands assigned to cellulose were analyzed. The results of this study indicate that CRM can be used as a tool to study cellulose deposition in cotton fibers and could provide useful information on cellulose deposition during cotton fiber development.

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

    Directory of Open Access Journals (Sweden)

    Jaikrit Bhutani


    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.

  10. Aerial wetting contact angle measurement using confocal microscopy (United States)

    Chesna, Jacob W.; Wiedmaier, Bob F.; Wang, Jinlin; Samara, Ayman; Leach, Richard K.; Her, Tsing-Hua; Smith, Stuart T.


    A method is presented in which the wetting contact angle of a sessile drop is acquired aerially using confocal techniques to measure the radius and the height of a droplet deposited on a planar surface. The repeatability of this method is typically less than 0.25°, and often less than 0.1°, for droplet diameters less than 1 mm. To evaluate accuracy of this method, an instrument uncertainty budget is developed, which predicts a combined uncertainty of 0.91° for a 1 mm diameter water droplet with a contact angle of 110°. For droplets having diameters less than 1 mm and contact angles between 15° and 160°, these droplets approach spherical shape and their contact angles can be computed analytically with less than 1% error. For larger droplets, gravitational deformation needs to be considered.

  11. Reflectance confocal microscopy for cutaneous infections and infestations. (United States)

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


    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.


    Directory of Open Access Journals (Sweden)

    Barbora Radochová


    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.

  13. In vivo reflectance confocal microscopy in a typical case of melasma Microscopia confocal reflectante in vivo em um caso típico de melasma


    Mariana Carvalho Costa; Hernando Vega Eljaiek; Leonardo Spagnol Abraham; Luna Azulay-Abulafia; Marco Ardigo


    Melasma is a common disorder of hypermelanosis that affects mainly young and middle-aged women of Fitzpatrick's phototypes III-V. The disease significantly impacts their lives. In vivo reflectance confocal microscopy, a spreading technology for the noninvasive evaluation of the skin up to the papillary dermis, provides real-time en face images with cellular resolution. We present a case of melasma with in vivo reflectance confocal microscopy findings closely correlated to the histopathologica...

  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)


    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. (United States)

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


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

    Directory of Open Access Journals (Sweden)

    Guoliang Huang


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

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

    Directory of Open Access Journals (Sweden)

    Sandro Sbordone


    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.

  18. Development of in vivo confocal microscope for reflection and fluorescence imaging simultaneously (United States)

    Ahn, MyoungKi; Chun, ByungSeon; Song, Cheol; Gweon, DaeGab


    In-vivo confocal microscope technology can be applied to the medical imaging diagnosis and new drug development. We present an in-vivo confocal microscope that can acquire a reflection image and a fluorescence image simultaneously and independently. To obtain reflection confocal images, we used a linearly polarized diode laser with the wavelength of 830 nm. To acquire fluorescence confocal images, we used two diode lasers with the wavelength of 488 nm and 660 nm, respectively. Because of a broad wavelength bandwidth from visible (488 nm) to near-IR (830 nm), we designed and optimized the optical system to reduce various optical aberrations. With the developed in-vivo confocal microscope, we performed ex-vivo cell imaging and in-vivo imaging of the human skin.

  19. Confocal microscopy indentation system for studying in situ chondrocyte mechanics. (United States)

    Han, Sang-Kuy; Colarusso, Pina; Herzog, Walter


    Chondrocytes synthesize extracellular matrix molecules, thus they are essential for the development, adaptation and maintenance of articular cartilage. Furthermore, it is well accepted that the biosynthetic activity of chondrocytes is influenced by the mechanical environment. Therefore, their response to mechanical stimuli has been studied extensively. Much of the knowledge in this area of research has been derived from testing of isolated cells, cartilage explants, and fixed cartilage specimens: systems that differ in important aspects from chondrocytes embedded in articular cartilage and observed during loading conditions. In this study, current model systems have been improved by working with the intact cartilage in real time. An indentation system was designed on a confocal microscope that allows for simultaneous loading and observation of chondrocytes in their native environment. Cell mechanics were then measured under precisely controlled loading conditions. The indentation system is based on a light transmissible cylindrical glass indentor of 0.17 mm thickness and 1.64 mm diameter that is aligned along the focal axis of the microscope and allows for real time observation of live cells in their native environment. The system can be used to study cell deformation and biological responses, such as calcium sparks, while applying prescribed loads on the cartilage surface. It can also provide novel information on the relationship between cell loading and cartilage adaptive/degenerative processes in the intact tissue.

  20. Application of Reflectance Confocal Microscopy in Dermatology Practice

    Directory of Open Access Journals (Sweden)

    Ayşe Esra Koku Aksu


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

  1. Quantitative analysis of in vivo confocal microscopy images: a review. (United States)

    Patel, Dipika V; McGhee, Charles N


    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.

  2. Axial scanning in confocal microscopy employing adaptive lenses (CAL). (United States)

    Koukourakis, Nektarios; Finkeldey, Markus; Stürmer, Moritz; Leithold, Christoph; Gerhardt, Nils C; Hofmann, Martin R; Wallrabe, Ulrike; Czarske, Jürgen W; Fischer, Andreas


    In this paper we analyze the capability of adaptive lenses to replace mechanical axial scanning in confocal microscopy. The adaptive approach promises to achieve high scan rates in a rather simple implementation. This may open up new applications in biomedical imaging or surface analysis in micro- and nanoelectronics, where currently the axial scan rates and the flexibility at the scan process are the limiting factors. The results show that fast and adaptive axial scanning is possible using electrically tunable lenses but the performance degrades during the scan. This is due to defocus and spherical aberrations introduced to the system by tuning of the adaptive lens. These detune the observation plane away from the best focus which strongly deteriorates the axial resolution by a factor of ~2.4. Introducing balancing aberrations allows addressing these influences. The presented approach is based on the employment of a second adaptive lens, located in the detection path. It enables shifting the observation plane back to the best focus position and thus creating axial scans with homogeneous axial resolution. We present simulated and experimental proof-of-principle results.

  3. Backscattered light confocal imaging of intracellular MTT-formazan crystals. (United States)

    Bernas, Tytus; Dobrucki, Jurek W


    Metabolically active animal and plant cells reduce MTT tetrazolium salt to a corresponding nonfluorescent formazan. Reduction of MTT by viable cells is exploited in a number of tests widely used in biological research. The aim of this study was to optimize a microscopy method of detecting small crystals of MTT-formazan formed in intact cells maintained in in vitro cultures. We examined scattering properties of small intracellular crystals of MTT formazan and found that the efficiency of light scattering was dependent on wavelength. Small (formazan, formed in viable cells, scattered red, but not blue, light. Large crystals, which are formed later at a stage when cells begin to lose viability, scattered both red and blue light. We conclude that optimal detection of early stages of crystallization of MTT-formazan in living cells is possible using confocal microscopy of red, but not blue, scattered light. High contrast and resolution of images can be achieved by filtering out interference effects in the frequency domain.

  4. 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


    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.

  5. Adaptive optics in digital micromirror based confocal microscopy (United States)

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


    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.

  6. Improvement of spatial resolution in confocal microscope with shifted-focus phase filter (United States)

    Huang, Xiangdong; Xiang, Xiaoyan; Wang, Chongyang


    A spatial super-resolution method is proposed based on the multiplicative character of confocal microscope's amplitude point-spread functions. The axial resolution can be greatly improved by introducing a shifted-focus phase filters in illumination part of a confocal microscope. However, this improvement is accompanied by a decrease of transversal resolution. Thus, a super-Gaussian phase filter is optimized to control the focal shift and transversal intensity distribution in a confocal microscope. Numerical simulation results indicate that the proposed method is useful to obtain a significant improvement in the optical sectioning capacity.

  7. 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


    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.

  8. Confocal laser scanning microscopy in study of bone calcification

    Energy Technology Data Exchange (ETDEWEB)

    Nishikawa, Tetsunari, E-mail: [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)


    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.

  9. Laser ablation of basal cell carcinomas guided by confocal microscopy (United States)

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


    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.

  10. 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


    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.

  11. A confocal scanning laser ophthalmoscope for retinal vessel oximetry (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.

  12. Anti-translational research: from the bedside back to the bench for reflectance confocal microscopy (United States)

    Gareau, Daniel


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

  13. Semi-automated confocal imaging of fungal pathogenesis on plants: microscopic analysis of macroscopic specimens (United States)

    Contextualizing natural genetic variation in plant disease resistance in terms of pathogenesis can provide information about the function of causal genes. Cellular mechanisms associated with pathogenesis can be elucidated with confocal microscopy, but systematic phenotyping platforms—from sample pro...

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

    Directory of Open Access Journals (Sweden)

    Babu Kalpana


    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.

  15. In vivo reflectance confocal microscopy in a typical case of melasma


    Costa,Mariana Carvalho; Eljaiek,Hernando Vega; Abraham,Leonardo Spagnol; Azulay-Abulafia,Luna; Ardigo, Marco


    Melasma is a common disorder of hypermelanosis that affects mainly young and middle-aged women of Fitzpatrick's phototypes III-V. The disease significantly impacts their lives. In vivo reflectance confocal microscopy, a spreading technology for the noninvasive evaluation of the skin up to the papillary dermis, provides real-time en face images with cellular resolution. We present a case of melasma with in vivo reflectance confocal microscopy findings closely correlated to the histopathologica...

  16. Laparoscopic manipulation of a probe-based confocal laser endomicroscope using a steerable intravascular catheter. (United States)

    Schneider, Crispin; Desjardins, Adrien E; Gurusamy, Kurinchi; Hawkes, David J; Davidson, Brian R


    Probe-based confocal laser endomicroscopy is an emerging imaging modality that enables visualization of histologic details during endoscopy and surgery. A method of guiding the probe with millimeter accuracy is required to enable imaging in all regions of the abdomen accessed during laparoscopy. On the basis of a porcine model of laparoscopic liver resection, we report our experience of using a steerable intravascular catheter to guide a probe-based confocal laser endomicroscope.

  17. Development of fibre-optic confocal microscopy for detection and diagnosis of dental caries. (United States)

    Rousseau, C; Poland, S; Girkin, J M; Hall, A F; Whitters, C J


    We report on the development of a fibre-optics-based confocal imaging system for the detection and potential diagnosis of early dental caries. A novel optical instrument, capable of recording axial profiles through caries lesions using single-mode optical fibres, has been developed. The practical study illustrates that miniature confocal devices based around single-mode optical fibres may provide additional diagnostic information for the general dental practitioner.

  18. Confocal Bioluminescence Imaging for Living Tissues with a Caged Substrate of Luciferin. (United States)

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


    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.

  19. Analysis of reactive oxygen species in the guard cell of wheat stoma with confocal microscope. (United States)

    Liu, Dongwu; Chen, Zhiwei; Shi, Peiguo; Wang, Xue; Cai, Weiwei


    Recently, the laser-scanning confocal microscope has become a routine technique and indispensable tool for cell biological studies. Previous studies indicated that reactive oxygen species (ROS) were generated in tobacco epidermal cells with confocal microscope. In the present studies, the probe 2',7'-dichlorof luorescein diacetate (H₂DCF-DA) was used to research the change of ROS in the guard cell of wheat stoma, and catalase (CAT) was used to demonstrate that ROS had been labeled. The laser-scanning mode of confocal microscope was XYT, and the time interval between two sections was 1.6351 s. Sixty optical sections were acquired with the laser-scanning confocal microscope, and CAT (60,000 U mg⁻¹) was added after four optical sections were scanned. Furthermore, the region of interest (ROI) was circled and the fluorescence intensity of ROS was quantified with Leica Confocal Software. The quantitative data were exported and the trend chart was made with software Excell. The results indicated that ROS were produced intracellularly in stomatal guard cells, and the quantified fluorescence intensity of ROS was declined with CAT added. It is a good method to research the instantaneous change of ROS in plant cells with confocal microscope and fluorescence probe H₂DCF-DA.

  20. Optimizing the acquisition and analysis of confocal images for quantitative single-mobile-particle detection. (United States)

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


    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.

  1. In vivo confocal microscopy of meibomian glands in primary blepharospasm (United States)

    Lin, Tong; Gong, Lan


    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 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 correlated to MG orifice diameter (P = 0.008). The LSCM provided a noninvasive

  2. Clinical applications of in vivo fluorescence confocal laser scanning microscopy (United States)

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


    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

  3. 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


    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.

  4. In vivo reflectance confocal microscopy in a typical case of melasma Microscopia confocal reflectante in vivo em um caso típico de melasma

    Directory of Open Access Journals (Sweden)

    Mariana Carvalho Costa


    Full Text Available Melasma is a common disorder of hypermelanosis that affects mainly young and middle-aged women of Fitzpatrick's phototypes III-V. The disease significantly impacts their lives. In vivo reflectance confocal microscopy, a spreading technology for the noninvasive evaluation of the skin up to the papillary dermis, provides real-time en face images with cellular resolution. We present a case of melasma with in vivo reflectance confocal microscopy findings closely correlated to the histopathological features described in the literature.O melasma é um distúrbio pigmentar caracterizado por hipermelanose, que afeta principalmente mulheres jovens e de meia-idade com fototipos III-V de Fitzpatrick e acarreta em impacto significativo na qualidade de vida das mesmas. A microscopia confocal reflectante in vivo, uma tecnologia em expansão voltada para análise da pele até a derme superior, proporciona imagens en face em tempo real com resolução celular. Apresentamos um caso de melasma com achados na microscopia confocal reflectante in vivo fortemente correlacionados com as características histopatológicas descritas na literatura.

  5. 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


    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.

  6. Clinical applications of a real-time scanning-slit confocal microscope designed for real-time observations of the in-vivo human cornea (United States)

    Masters, Barry R.


    We describe a new, real-time, flying slit confocal microscope, that has unique features and imaging characteristics for in vivo human ocular imaging. In vivo real-time confocal microscopy is currently used to investigate the tear film, renewal of the ocular surface, the role of epithelial innervation in epithelial cell proliferation, wound healing, kinetics of drug penetration, the effects of laser refractive surgery on the keratocyte activation and distribution in the stroma, and the nature of endothelial defects. The following clinical examples will be presented and discussed: confocal microscopy of normal human basal and wing cells in the epithelium, confocal microscopy of lamellar and penetrating corneal grafts, confocal microscopy of corneal ulcer, confocal microscopy of scar formation after herpes keratitis, and confocal microscopy of corneal innervation. The use of scanning slit confocal microscopes has unique advantages over other instrumental systems based on pinhole-containing Nipkow disks (tandem-scanning confocal microscopes) for clinical in vivo confocal microscopy.

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

    Directory of Open Access Journals (Sweden)

    Saubaméa Bruno


    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

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

    Directory of Open Access Journals (Sweden)

    Strandh Christer


    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.

  9. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Nohra E. Beltran


    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.

  11. Portable oral cancer detection using a miniature confocal imaging probe with a large field of view (United States)

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


    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.

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

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


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

  13. Reflectance confocal microscopy for scarring and non-scarring alopecia real-time assessment. (United States)

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


    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.

  14. Improving Resolution of Confocal Laser Induced Fluorescence in Argon Helicon Plasma (United States)

    Soderholm, Mark; Vandervort, Robert; Scime, Earl; McKee, John; McCarren, Dustin


    Laser Induced Fluorescence (LIF) provides measurements of flow speed, temperature and when absolutely calibrated, density of ions or neutrals in a plasma. Traditionally, laser induced fluorescence requires two ports on a plasma device. One port is used for laser injection and the other is used for fluorescence emission collection. Traditional LIF is tedious and time consuming to align. These difficulties motivate the development of an optical configuration that requires a single port and remains fully aligned at all times; confocal LIF. Our confocal optical design employs a single two inch diameter lens to both inject the laser light and collect the stimulated emission from an argon plasma. A dichroic mirror is used to separate the injected laser light from the collected emission. The measurement location is scanned radially by manually adjusting the final focusing lens position. In the initial version of the confocal optical system, measurements were poorly resolved radially because they were integrated over a fairly large path length (~4 cm) centered at the focal point. Here we present collected data from a modified configuration that significantly improves the special resolution of confocal measurements. The confocal measurements are compared to traditional, two-port, LIF measurements over the same radial range.

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

    KAUST Repository

    Yong Wan,


    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.

  16. 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: [Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Egerlandstraße 3, 91058 Erlangen (Germany); Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Egerlandstraße 3, 91058 Erlangen (Germany)


    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.

  17. 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


    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.

  18. Fiber-optic confocal microscopy using a miniaturized needle-compatible imaging probe (United States)

    Pillai, Rajesh S.; Lorenser, Dirk; Sampson, David D.


    We report on the design and implementation of a 350 μm-diameter confocal imaging probe based on gradient-index (GRIN) optics and a fiber-based scanning arrangement. The form factor of the probe is such that it can potentially be inserted into a 22-gauge hypodermic needle to perform high-resolution confocal fluorescence imaging in solid tissues. We introduce a simple scanning arrangement based on lensed fiber, which eliminates off-axis aberrations induced by conventional scanning optics and is suitable for integration into a compact hand-held unit. We present the details of the optical design and experimental verification of the performance of the optical system. The measured lateral resolution of ~700 nm is in agreement with the optical design and is the highest resolution reported for a confocal fluorescence imaging probe of this size. Further, we demonstrate the imaging capability of the probe by obtaining high-resolution images of fluorescently labeled muscle fibers.

  19. 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


    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.

  20. 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


    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.

  1. Confocal detection of Rayleigh scattering for residual stress measurement in chemically tempered glass (United States)

    Hödemann, S.; Möls, P.; Kiisk, V.; Murata, T.; Saar, R.; Kikas, J.


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

  2. Super-resolution spinning-disk confocal microscopy using optical photon reassignment. (United States)

    Azuma, Takuya; Kei, Takayuki


    Spinning-disk confocal microscopy is a proven technology for investigating 3D structures of biological specimens. Here we report a super-resolution method based on spinning-disk confocal microscopy that optically improves lateral resolution by a factor of 1.37 with a single exposure. Moreover, deconvolution yields twofold improvement over the diffraction limit. With the help of newly modified Nipkow disk which comprises pinholes and micro-lenses on the front and back respectively, emitted photons from specimen can be optically reassigned to the most probable locations they originate from. Consequently, the improvement in resolution is achieved preserving inherent sectioning capabilities of confocal microscopy. This extremely simple implementation will enable reliable observations at super high resolution in biomedical routine research.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hödemann, S., E-mail:; 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)


    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.

  4. Determination of Nanogram Microparticles from Explosives after Real Open-Air Explosions by Confocal Raman Microscopy. (United States)

    Zapata, Félix; García-Ruiz, Carmen


    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.

  5. 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)


    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}.

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

    Directory of Open Access Journals (Sweden)

    Radenović Lidija


    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.

  7. Agminated cellular blue naevi of the penis: dermoscopic, confocal and histopathological correlation of two cases. (United States)

    Collgros, H; Vicente, A; Díaz, A M; Rodríguez-Carunchio, L; Malvehy, J; Puig, S


    Blue naevi may present rarely as multiple lesions grouped in a circumscribed area, described as agminated blue naevi. This clinical presentation may mimic metastatic malignant melanoma. We present two cases of agminated cellular blue naevi of the penis, with dermoscopy, reflectance confocal microscopy and histopathological correlation. Dermoscopy of the area showed multiple grouped lesions of homogeneous dark-brown to blue colour. Using reflectance confocal microscopy, focusing on the bluish areas, predominantly bright dendritic cells were visible at the dermoepidermal junction and papillary dermis, while in the brownish areas the presence of dendritic and bright cells predominated in the basal layer. Our patients are of special interest as they are the first cases, to our knowledge, reported of agminated blue naevi on the penis, studied by both dermoscopy and confocal microscopy, confirming the diagnosis with histopathological correlation. Moreover, one case represented a divided or 'kissing' blue naevus of the penis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lafond, Maxime, E-mail:; 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)


    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

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

    Lunter, Dominique; Daniels, Rolf


    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.

  10. Confocal microphotoluminescence of InGaN-based light-emitting diodes


    Okamoto, K.; Kaneta, A; Kawakami, Y.; Fujita, S; Choi, J.; Terazima, M; Mukai, T


    Spatially resolved photoluminescence (PL) of InGaN/GaN/AlGaN-based quantum-well-structured light-emitting diodes (LEDs) with a yellow-green light (530 nm) and an amber light (600 nm) was measured by using confocal microscopy. Submicron-scale spatial inhomogeneities of both PL intensities and spectra were found in confocal micro-PL images. We also found clear correlations between PL intensities and peak wavelength for both LEDs. Such correlations for yellow-green and amber LEDs were differen...

  11. 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


    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.

  12. 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


    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.

  13. Three-dimensional measurement and visualization of internal flow of a moving droplet using confocal micro-PIV. (United States)

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


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

  14. 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


    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.

  15. Fast imaging with inelastically scattered electrons by off-axis chromatic confocal electron microscopy. (United States)

    Zheng, Changlin; Zhu, Ye; Lazar, Sorin; Etheridge, Joanne


    We introduce off-axis chromatic scanning confocal electron microscopy, a technique for fast mapping of inelastically scattered electrons in a scanning transmission electron microscope without a spectrometer. The off-axis confocal mode enables the inelastically scattered electrons to be chromatically dispersed both parallel and perpendicular to the optic axis. This enables electrons with different energy losses to be separated and detected in the image plane, enabling efficient energy filtering in a confocal mode with an integrating detector. We describe the experimental configuration and demonstrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@840  eV) signal in LaB6. Scan rates up to 2 orders of magnitude faster than conventional methods were used, enabling a corresponding reduction in radiation dose and increase in the field of view. If coupled with the enhanced depth and lateral resolution of the incoherent confocal configuration, this offers an approach for nanoscale three-dimensional chemical mapping.

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

  18. 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


    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.

  19. Effects of photon reabsorption phenomena in confocal micro-photoluminescence measurements in crystalline silicon (United States)

    Roigé, A.; Alvarez, J.; Jaffré, A.; Desrues, T.; Muñoz, D.; Martín, I.; Alcubilla, R.; Kleider, J.-P.


    Confocal micro-photoluminescence (PL) spectroscopy has become a powerful characterization technique for studying novel photovoltaic (PV) materials and structures at the micrometer level. In this work, we present a comprehensive study about the effects and implications of photon reabsorption phenomena on confocal micro-PL measurements in crystalline silicon (c-Si), the workhorse material of the PV industry. First, supported by theoretical calculations, we show that the level of reabsorption is intrinsically linked to the selected experimental parameters, i.e., focusing lens, pinhole aperture, and excitation wavelength, as they define the spatial extension of the confocal detection volume, and therefore, the effective photon traveling distance before collection. Second, we also show that certain sample properties such as the reflectance and/or the surface recombination velocity can also have a relevant impact on reabsorption. Due to the direct relationship between the reabsorption level and the spectral line shape of the resulting PL emission signal, reabsorption phenomena play a paramount role in certain types of micro-PL measurements. This is demonstrated by means of two practical and current examples studied using confocal PL, namely, the estimation of doping densities in c-Si and the study of back-surface and/or back-contacted Si devices such as interdigitated back contact solar cells, where reabsorption processes should be taken into account for the proper interpretation and quantification of the obtained PL data.

  20. 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


    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

  1. Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification (United States)

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

  2. Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool (United States)

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


    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. 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.


    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

  4. Handheld multispectral dual-axis confocal microscope for cervical cancer screening (United States)

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


    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.

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

    DEFF Research Database (Denmark)

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


    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 fermente...


    NARCIS (Netherlands)



    This paper describes a non-invasive method for the study of mitochondrial morphology in cultured human skin fibroblasts by confocal scanning laser microscopy after staining the mitochondria with 2-[4-(dimethyl-aminostyryl]-1-methylpyridinium iodide. This method is applied to compare mitochondria in

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

    Energy Technology Data Exchange (ETDEWEB)

    Schoonjans, Tom, E-mail: [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: [X-ray Microspectroscopy and Imaging Research Group (XMI), Department of Analytical Chemistry, Ghent University, Krijgslaan 281 S12, B-9000 Ghent (Belgium)


    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.

  8. Computer Aided Diagnosis for Confocal Laser Endomicroscopy in Advanced Colorectal Adenocarcinoma

    DEFF Research Database (Denmark)

    Ştefănescu, Daniela; Streba, Costin; Cârţână, Elena Tatiana;


    INTRODUCTION: Confocal laser endomicroscopy (CLE) is becoming a popular method for optical biopsy of digestive mucosa for both diagnostic and therapeutic procedures. Computer aided diagnosis of CLE images, using image processing and fractal analysis can be used to quantify the histological struct...

  9. 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.


    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

  10. Visualizing the Tumor Microenvironment of Liver Metastasis by Spinning Disk Confocal Microscopy. (United States)

    Babes, Liane; Kubes, Paul


    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.

  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.


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

    DEFF Research Database (Denmark)

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


    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...

  13. 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


    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

  14. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case. (United States)

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


    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.

  15. Experimental demonstration of ray-optical refraction with confocal lenslet arrays. (United States)

    Courtial, Johannes; Kirkpatrick, Blair C; Logean, Eric; Scharf, Toralf


    We observe imaging through windows comprising pairs of confocal lenslet arrays that have different focal lengths but that are otherwise identical. Image space is stretched in the longitudinal direction only. Such windows are examples of METATOYs, optical components that can change light-ray direction in ways that appear wave-optically forbidden.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  17. Confocal restricted-height imaging of suspension cells (CRISC) in a PDMS microdevice during apoptosis

    NARCIS (Netherlands)

    Munoz-Pinedo, Cristina; Green, Douglas R.; Berg, van den Albert


    We have monitored and imaged cell death induced in human leukemic U937 cells over time using three-color confocal imaging. Three different apoptotic inducers, anti-Fas, TNF- and Etoposide were used. Individual cascaded events such as loss of mitochondrial transmembrane potential, exposure of phospha

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

    Directory of Open Access Journals (Sweden)

    Emine Kaya


    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

  19. Confocal Raman microscopy for in depth analysis in the field of cultural heritage (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Kobayashi A


    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

  1. In vivo confocal microscopy for the oral cavity: Current state of the field and future potential. (United States)

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


    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.

  2. 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.

  3. Near-infrared-excited confocal Raman spectroscopy advances in vivo diagnosis of cervical precancer. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    D Shyam Prasad Reddy


    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

  5. Evaluation of human sclera after femtosecond laser ablation using two photon and confocal microscopy (United States)

    Sun, Hui; Kurtz, Ronald; Juhasz, Tibor


    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial thickness intrascleral channels can be created with a femtosecond laser operating at a wavelength of 1700 nm. Such channels have the potential to increase outflow facility and reduce elevated IOP. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in human cadaver eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such channels. This demonstrates that concept of integrating femtosecond laser surgery, and two-photon and confocal imaging has the future potential for image-guided high-precision surgery in transparent and translucent tissue.

  6. OCT and in vivo confocal microscopy of a pigmented corneal tumor-like lesion. (United States)

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


    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.

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

    KAUST Repository

    Lin, Neil Y. C.


    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.

  8. In vivo observation of papillae of the human tongue using confocal laser scanning microscopy. (United States)

    Just, Tino; Stave, Joachim; Pau, Hans Wilhelm; Guthoff, Rudolf


    The aim of this investigation was to visualize the epithelial structures of the tongue using confocal laser scanning microscopy (LSM). The human tongue epithelium of 28 healthy subjects, aged 21-67 years, mean age 38 years, 14 women and 14 men, was examined in vivo by LSM. Using LSM, a combination of the Heidelberg Retina Tomograph HRT II and the Rostock Cornea Module, up to 800-fold magnifications were obtained. On the tongue surface both filiform and fungiform papillae and their taste pores were easily identified. The epithelium of the tongue with its subcellular structures could be observed up to a depth of 50 microm, cellular structures up to 150 microm and subepithelial vessels up to 300 microm. Additionally the papillary crests and blood flow were visible. Confocal LSM seems suitable for noninvasive in vivo examination of the tongue. The hydraulic z scan, the manual start setting and the measurement of the depth allow a clear classification of the observed structures.

  9. The role of confocal microscopy in the dermato-oncology practice. (United States)

    Diaconeasa, A; Boda, D; Neagu, M; Constantin, C; Căruntu, C; Vlădău, L; Guţu, D


    Reflectance-mode confocal microscopy (RCM) is a new in vivo skin imaging technique. We present our one-year experience in RCM examinations in skin tumors and the retrospective analysis of patients enrolled in the Dermatological Department of 'N. Paulescu' Institute using the Fotofinder Dermoscope IIŴ for the dermatoscopy analysis and VivaScope 1500Ŵ for in vivo RCM. We established the rank of RCM in the complex algorithm of skin cancer diagnose, showing that the presented experience can open new possibilities to implement this automated image analyzing system in the routine practice. Our analyzed cases clearly showed that confocal microscopy, therefore, optical biopsy, could guide the clinician towards an accurate diagnosis before surgical removal. Moreover, we emphasized that the development of this technique increases the potential of future teledermatologic applications.

  10. Dental pulp stem cells (DPSCs) differentiation study by confocal Raman microscopy (United States)

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


    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.

  11. 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


    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...

  12. Measuring the lens focal length by laser reflection-confocal technology. (United States)

    Yang, Jiamiao; Qiu, Lirong; Zhao, Weiqian; Shao, Rongjun; Li, Zhigang


    A laser reflection-confocal focal-length measurement (LRCFM) is proposed for the high-accuracy measurement of lens focal length. LRCFM uses the peak points of confocal response curves to precisely identify the lens focus and vertex of the lens last surface. LRCFM then accurately measures the distance between the two positions to determine the lens focal length. LRCFM uses conic fitting, which significantly enhances measurement accuracy by inhibiting the influence of environmental disturbance and system noise on the measurement results. The experimental results indicate that LRCFM has a relative expanded uncertainty of less than 0.0015%. Compared with existing measurement methods, LRCFM has high accuracy and a concise structure. Thus, LRCFM is a feasible method for high-accuracy focal-length measurements.

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

    Directory of Open Access Journals (Sweden)

    Youngjae Won


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

  14. 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


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

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

    Directory of Open Access Journals (Sweden)

    Truernit Elisabeth


    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.

  16. 3-D Confocal microscopy track analysis: a promising tool for determining CR-39 response function

    Energy Technology Data Exchange (ETDEWEB)

    Vaginay, F. E-mail:; Fromm, M.; Pusset, D.; Meesen, G.; Chambaudet, A.; Poffijn, A


    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.

  17. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy (United States)

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


    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

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

    Directory of Open Access Journals (Sweden)

    Ramla Gary


    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.

  19. Selective Bioparticle Retention and Characterization in a Chip-Integrated Confocal Ultrasonic Cavity

    DEFF Research Database (Denmark)

    Svennebring, J.; Manneberg, O.; Skafte-Pedersen, Peder;


    We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field at the c......We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field...... sample feeding, a set of several manipulation functions performed in series is demonstrated: sample bypass-injection-aggregation and retention-positioning. Finally, we demonstrate transillumination microscopy imaging Of Ultrasonically trapped COS-7 cell aggregates. Biotechnol. Bioeng. 2009;103: 323-328....

  20. 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


    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.

  1. Compensation of phase aberration by using a virtual confocal scheme in digital holographic microscopy. (United States)

    Chew, Yang-Kun; Shiu, Min-Tzung; Wang, Je-Chung; Chang, Chi-Ching


    This work presents cost-effective, simple arbitrary phase-step digital holographic microscopy to suppress both zero-order and twin-image terms. A virtual confocal offset lens under in-line configuration is also used to compensate for the introduced quadratic phase by using a microscope objective lens. In addition to reducing the difficulties of physical confocal configurations, the proposed method significantly increases the magnification power, ultimately achieving the purposes of an optical zoom. An attempt is also made to reduce the noise interference of a high magnification system by developing a long focal lens to reduce light detection size, subsequently gaining an approximately plane wave light source to illuminate the object within the effective depth of focus. Experimental results indicate that the proposed high magnification system can be elevated with low noise interference, and image reconstruction without quadratic phase terms.

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

    Directory of Open Access Journals (Sweden)

    Francesca Salvatori


    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.

  3. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Markus Piwko


    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.

  5. Coherent confocal microscope with a phase-only filter in its extended source

    Institute of Scientific and Technical Information of China (English)

    YANG Chu-ping


    The phase information of an extended source is reconstructed by use of a two-zone (annular) phase-only filter in a coherent confocal scanning optical microscope.The dependence of its resolution on its source size is investigated theoretically by its three-dimensional optical transfer function (3D OTF).The results show that the resolution is improved, even though the source size is enlarged.

  6. 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: [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)


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

  7. 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

  8. Errors in confocal fluorescence ratiometric imaging microscopy due to chromatic aberration. (United States)

    Lin, Yuxiang; Gmitro, Arthur F


    Confocal fluorescence ratiometric imaging is an optical technique used to measure a variety of important biological parameters. A small amount of chromatic aberration in the microscope system can introduce a variation in the signal ratio dependent on the fluorophore concentration gradient along the optical axis and lead to bias in the measurement. We present a theoretical model of this effect. Experimental results and simulations clearly demonstrate that this error can be significant and should not be ignored.

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

    Institute of Scientific and Technical Information of China (English)


    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 ...

  10. Full-field transmission x-ray imaging with confocal polycapillary x-ray optics. (United States)

    Sun, Tianxi; Macdonald, C A


    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.


    Institute of Scientific and Technical Information of China (English)



    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.

  12. 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)


    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.


    Energy Technology Data Exchange (ETDEWEB)

    White, T


    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.

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

    Directory of Open Access Journals (Sweden)

    Altmeyer Peter


    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


    Directory of Open Access Journals (Sweden)

    O. L. Pustovalova


    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. 

  16. Single beam optical trapping integrated in a confocal microscope for biological applications. (United States)

    Visscher, K; Brakenhoff, G J


    Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Application of chromatic confocal displacement sensor in measurement of tip clearance (United States)

    Bi, Chao; Li, Di; Fang, Jianguo; Zhang, Bin


    In the field of aeronautics, the tip clearance of rotor exerts a crucial influence on the performance of the aero engine. As defined as the radial distance between the top of the blade and the inner wall of the casing, the tip clearance of too large or small size will adversely affect the normal running of the engine. In order to realize accurate measurement of the tip clearance in a simple way, a non-contact measuring method by the chromatic confocal displacement sensor is proposed in the paper. The sensor possesses the advantages such as small volume, good signal-to-noise ratio, high accuracy and response frequency etc., which make it be widely used in engineering and industry. For testing the performance and potential application of the sensor, a simulation testing platform is established. In the platform, a simulation blisk is installed on the air bearing spindle and a chromatic confocal displacement sensor is fixed on the platform to measure the displacement variation of the blade tip, which can be used to characterize the variation of the tip clearance. In the simulation experiments, both of single and continuous measurement of the tip clearance of the 36 blades on the blisk is executed. As the results of experiments show, the chromatic confocal displacement sensor can meet the requirements of measuring task, in which both of high measuring efficiency and accuracy could be achieved. Therefore, the measuring method proposed in the paper can be utilized in the actual assembling sites of the aero engine.

  18. Confocal reflectance quantitative phase microscopy system for cell biology studies (Conference Presentation) (United States)

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


    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.

  19. Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope. (United States)

    Kim, Dong Uk; Moon, Sucbei; Song, Hoseong; Kwon, Hyuk-Sang; Kim, Dug Young


    High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.

  20. Simultaneous confocal fluorescence microscopy and optical coherence tomography for drug distribution and tissue integrity assessment (United States)

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


    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.

  1. Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions (United States)

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


    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.

  2. The application of dermal papillary rings in dermatology by in vivo confocal laser scanning microscopy (United States)

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


    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.

  3. Laser confocal measurement system for curvature radius of lenses based on grating ruler (United States)

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


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

  4. 3-D reconstruction of neurons from multichannel confocal laser scanning image series. (United States)

    Wouterlood, Floris G


    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.

  5. Confocal Laser Endomicroscopy in Neurosurgery: A New Technique with Much Potential

    Directory of Open Access Journals (Sweden)

    David Breuskin


    Full Text Available Technical innovations in brain tumour diagnostic and therapy have led to significant improvements of patient outcome and recurrence free interval. The use of technical devices such as surgical microscopes as well as neuronavigational systems have helped localising tumours as much as fluorescent agents, such as 5-aminolaevulinic acid, have helped visualizing pathologically altered tissue. Nonetheless, intraoperative instantaneous frozen sections and histological diagnosis remain the only method of gaining certainty of the nature of the resected tissue. This technique is time consuming and does not provide close-to-real-time information. In gastroenterology, confocal endoscopy closed the gap between tissue resection and histological examination, providing an almost real-time histological diagnosis. The potential of this technique using a confocal laser endoscope EndoMAG1 by Karl Storz Company was evaluated by our group on pig brains, tumour tissue cell cultures, and fresh human tumour specimen. Here, the authors report for the first time on the results of applying this new technique and provide first confocal endoscopic images of various brain and tumour structures. In all, the technique harbours a very promising potential to provide almost real-time intraoperative diagnosis, but further studies are needed to provide evidence for the technique’s potential.

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

    Directory of Open Access Journals (Sweden)

    Safak Korkmaz


    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.

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

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    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)


    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)

  8. 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


    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...

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

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Ryou Watanabe


    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

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

    Directory of Open Access Journals (Sweden)

    Elif Demirkilinc Biler


    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.

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

    Directory of Open Access Journals (Sweden)

    Cotarla Ion


    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.

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

    Directory of Open Access Journals (Sweden)

    Hayyam Kiratli


    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.

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

    Directory of Open Access Journals (Sweden)

    Cosimo Mazzotta


    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.

  15. Dual-detection confocal microscopy: high-speed surface profiling without depth scanning (United States)

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


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Apedo, K.L., E-mail: [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)


    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.

  17. Confocal soft X-ray scanning transmission microscopy: setup, alignment procedure and limitations. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Haidekker Mark A


    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

  19. Label-free detection of tumor markers in a colon carcinoma tumor progression model by confocal Raman microspectroscopy (United States)

    Scalfi-Happ, Claudia; Rück, Angelika; Udart, Martin; Hauser, Carmen; Dürr, Christine; Kriebel, Martin


    Living colon carcinoma cells were investigated by confocal Raman microspectroscopy. An in vitro model of tumor progression was established. Evaluation of data sets by cluster analysis reveals that lipid bodies might be a valuable diagnostic parameter for early carcinogenesis.

  20. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy. (United States)

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


    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.

  1. Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy (United States)

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


    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.

  2. Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications. (United States)

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


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

  3. Mobile large area confocal scanner for imaging tumor margins: initial testing in the pathology department (United States)

    Abeytunge, Sanjee; Li, Yongbiao; Larson, Bjorg; Peterson, Gary; Toledo-Crow, Ricardo; Rajadhyaksha, Milind


    Surgical oncology is guided by examining pathology that is prepared during or after surgery. The preparation time for Mohs surgery in skin is 20-45 minutes, for head-and-neck and breast cancer surgery is hours to days. Often this results in incomplete tumor removal such that positive margins remain. However, high resolution images of excised tissue taken within few minutes can provide a way to assess the margins for residual tumor. Current high resolution imaging methods such as confocal microscopy are limited to small fields of view and require assembling a mosaic of images in two dimensions (2D) to cover a large area, which requires long acquisition times and produces artifacts. To overcome this limitation we developed a confocal microscope that scans strips of images with high aspect ratios and stitches the acquired strip-images in one dimension (1D). Our "Strip Scanner" can image a 10 x 10 mm2 area of excised tissue with sub-cellular detail in about one minute. The strip scanner was tested on 17 Mohs excisions and the mosaics were read by a Mohs surgeon blinded to the pathology. After this initial trial, we built a mobile strip scanner that can be moved into different surgical settings. A tissue fixture capable of scanning up to 6 x 6 cm2 of tissue was also built. Freshly excised breast and head-and-neck tissues were imaged in the pathology lab. The strip-images were registered and displayed simultaneously with image acquisition resulting in large, high-resolution confocal mosaics of fresh surgical tissue in a clinical setting.

  4. Cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells. (United States)

    Castiel, Asher; Visochek, Leonid; Mittelman, Leonid; Zilberstein, Yael; Dantzer, Francoise; Izraeli, Shai; Cohen-Armon, Malka


    Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

  5. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy (United States)

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


    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

  6. 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.

  7. One shot confocal microscopy based on wavelength/space conversion by use of multichannel spectrometer (United States)

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


    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.

  8. A Piezoelectric Screw Dislocation Interacting with an Elliptical Piezoelectric Inhomogeneity Containing a Confocal Elliptical Rigid Core

    Institute of Scientific and Technical Information of China (English)

    蒋纯志; 谢超; 刘又文


    The electro-elastic interaction between a piezoelectric screw dislocation and an elliptical piezoelectric inhomogeneity, which contains an electrically conductive confocal elliptical rigid core under remote anti-plane shear stresses and in-plane electrical load is dealt with. The anaJytical solutions to the elastic field and the electric field, the interracial stress fields of inhomogeneity and matrix under longitudinal shear and the image force acting on the dislocation are derived by means of complex method. The effect of material properties and geometric configurations of the rigid core on interracial stresses generated by a remote uniform load, rigid core and material electroelastic properties on the image force is discussed.

  9. New method for lens thickness measurement by the frequency-shifted confocal feedback (United States)

    Tan, Yidong; Zhu, Kaiyi; Zhang, Shulian


    We describe a new method for lens thickness and air gap measurement based on the frequency-shifted confocal feedback. The light intensity fluctuation is eliminated by the heterodyne modulation and the detection sensitivity is improved prominently by the frequency-shifted feedback effect. The measurement results for different materials and kinds of lenses are presented in the paper, including K9 plain glasses, fused silica plain glass, and K9 biconvex lens. The uncertainty of the axial positioning is better than 0.0005 mm and the accuracy reaches micron range. It is promising to be applied in the multi-layer interface positioning and measurement area.

  10. 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: [IFEG-CONICET, (X5016LAE) Ciudad Universitaria, Córdoba (Argentina); FAMAF, Universidad Nacional de Córdoba, (X5016LAE) Ciudad Universitaria, Córdoba (Argentina)


    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.

  11. In Situ Confocal Raman Mapping Study of a Single Ti-Assisted ZnO Nanowire

    Directory of Open Access Journals (Sweden)

    Gandhi Ashish


    Full Text Available Abstract In this work, we succeeded in preparing in-plane zinc oxide nanowires using a Ti-grid assisted by the chemical vapor deposition method. Optical spatial mapping of the Confocal Raman spectra was used to investigate the phonon and geometric properties of a single ZnO nanowire. The local optical results reveal a red shift in the non-polar E 2 high frequency mode and width broadening along the growth direction, reflecting quantum-confinement in the radial direction.

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

    Institute of Scientific and Technical Information of China (English)


    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.

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




    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...

  14. Confocal and dermoscopic features of basal cell carcinoma in Gorlin-Goltz syndrome: A case report. (United States)

    Casari, Alice; Argenziano, Giuseppe; Moscarella, Elvira; Lallas, Aimilios; Longo, Caterina


    Gorlin-Goltz (GS) syndrome is an autosomal dominant disease linked to a mutation in the PTCH gene. Major criteria include the onset of multiple basal cell carcinoma (BCC), keratocystic odontogenic tumours in the jaws and bifid ribs. Dermoscopy and reflectance confocal microscopy represent imaging tools that are able to increase the diagnostic accuracy of skin cancer in a totally noninvasive manner, without performing punch biopsies. Here we present a case of a young woman in whom the combined approach of dermoscopy and RCM led to the identification of multiple small inconspicuous lesions as BCC and thus to the diagnosis of GS syndrome.

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

    CERN Document Server

    Papageorge, Alexander T; Lev, Benjamin L


    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.


    Institute of Scientific and Technical Information of China (English)

    Kecheng Li; Douglas W. Reeve


    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.


    Institute of Scientific and Technical Information of China (English)

    KechengLi; DouglasW.Reeve


    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.

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

    Directory of Open Access Journals (Sweden)

    Attila Bokros


    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.

  19. 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: [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)


    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.

  20. 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.


    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.

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

    Directory of Open Access Journals (Sweden)

    Samet James M


    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

  2. 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)


    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.

  3. Insights into esophagus tissue architecture using two-photon confocal microscopy (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Pang A


    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

  5. 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.


    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.

  6. Confocal microscopy using variable-focal-length microlenses and an optical fiber bundle


    Yang, Lisong; Mac Raighne, Aaron; McCabe, Eithne M.; Dunbar, L. Andrea; Scharf, Toralf


    The use of variable-focal-length (VFL) microlenses can provide a way to axially scan the foci across a sample by electronic control. We demonstrate an approach to coupling VFL microlenses individually to a fiber bundle as a way to create a high-throughput aperture array with a controllable aperture pattern. It would potentially be applied in real-time confocal imaging in vivo for biological specimens. The VFL microlenses that we used consist of a liquid-crystal film sandwiched between a pair ...

  7. 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


    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

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


    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.

  9. Stacking illumination of a confocal reflector light emitting diode automobile headlamp with an asymmetric triangular prism. (United States)

    Chen, Hsi-Chao; Zhou, Jia-Hao; Zhou, Yang


    A confocal reflector lamp with an asymmetric triangular prism was designed for a stacking illumination of a light emitting diode (LED) automobile headlamp fitting ECE R112 asymmetrical regulation. The optical system includes three 1st elliptic reflectors, three 2nd parabolic reflectors, and one asymmetric triangular prism. Three elliptic and parabolic reflectors were assembled with three confocal reflector modules; two modules projected the cut-off line of a 0° angle, and the other module projected the cut-off line of a 15° angle using of an asymmetric triangular prism. The ray tracing, optical simulation, and mockup experiment results exhibited that the illumination distribution met the regulation of ECE R112 class B, and the ideal efficiency could reach 96.8% in theory. The tolerance analysis showed the efficiency remained above 98% under the error values of ±0.2  mm of the position of the LED light source, and the y direction of the up-down movement was more sensitive than the x and z directions. The measurement results of the mockup sample safety factor were all larger than 1.15 and supported the regulation of the ECE R112 Class B.

  10. Surgical imaging catheter for confocal microendoscopy with advanced contrast delivery and focus systems (United States)

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


    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.

  11. Quantification of whey in fluid milk using confocal Raman microscopy and artificial neural network. (United States)

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


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

  12. A preliminary assessment of using a white light confocal imaging profiler for cut mark analysis. (United States)

    Schmidt, Christopher W; Moore, Christopher R; Leifheit, Randell


    White light confocal microscopy creates detailed 3D representations of microsurfaces that can be qualitatively and quantitatively analyzed. The study describes its application to the analysis of cut marks on bone, particularly when discerning cuts made by steel tools from those made by stone. The process described comes from a study where cuts were manually made on a cow rib with seven cutting tools, four stone (an unmodified chert flake, a chert biface, a bifacially ground slate fragment, and an unsharpened piece of slate), and three steel (a Swiss Army Knife, a serrate steak knife, and a serrate saw). Kerfs were magnified ×20 and 3D data clouds were generated using a Sensofar(®) White Light Confocal Profiler (WLCP). Kerf profiles and surface areas, volumes, mean depths, and maximum depths were calculated with proprietary software (SensoScan(®) and SolarMap(®)). For the most part, the stone tools make shallower and wider cuts. Kerf floors can be studied at higher magnifications; they were viewed at ×100. When comparing the kerf floors of the unsharpened slate and the serrate steak knife it was found that the slate floor was more uneven, but the serrate steak knife generated more overall relief. Although preliminary, the approach described here successfully distinguishes stone and steel tools; the authors conclude that the WLCP is a promising technology for cut mark analysis because of the very detailed 3D representations it creates and the numerous avenues of analysis it provides.

  13. Dye-enhanced multimodal confocal microscopy for noninvasive detection of skin cancers in mouse models (United States)

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


    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.

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

    CERN Document Server

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


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

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

    Directory of Open Access Journals (Sweden)

    Mozhgan Rezaei Kanavi


    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.

  16. Simultaneous OCT/confocal-OCT/ICG system for imaging the eye (United States)

    Podoleanu, Adrian G.; Rosen, Richard B.; Dobre, George; Rogers, John A.; Garcia, Patricia; Pedro, Justin; Dunne, Shane; Jackson, David A.; Weitz, Rishard


    En-face OCT acquired simultaneously with paired confocal ophthalmoscopic (CO) images provides unprecedented point-to-point correlation between surface and subsurface anatomy of the retina. An advanced prototype of a dual channel OCT/CO instrument was developed in terms of signal to noise ratio and image size. The system can operate in A, B and C-scan regimes. The design is such that there is a strict pixel to pixel correspondence between the OCT and confocal images. An extensive array of clinic pathologies were studied including macular degeneration, central serous retinopathy (CSR), macular hole, macular pucker, cystoid macular edema (CME), diabetic maculopathy, and macular trauma. We report observation of reoccurring patterns in the en-face OCT images which could be identified with different diseases. The system can also simultaneously produce en-face OCT and indocyanine green (ICG) fluorescence images where the same source is used to produce the OCT image and excite the ICG. The system is compact and assembled on a chin rest and it enables the clinician to visualise the same area of the eye fundus in terms of both en face OCT slices and ICG angiograms, displayed side by side. The images are collected by fast en-face scanning (T-scan) followed by slower scanning along a transverse direction and depth scanning. The system is capable of providing chosen OCT B-scans at selected points from the ICG image.

  17. Chromatic confocal microscopy for multi-depth imaging of epithelial tissue. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Vineeta Nikhil


    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.

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

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    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:; 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)


    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. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy (United States)

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


    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.

  1. Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy. (United States)

    Crossman, David J; Ruygrok, Peter N; Hou, Yu Feng; Soeller, Christian


    Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.

  2. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy. (United States)

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


    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.

  3. [Clinical forms of acanthamoeba keratitis as viewed from the standpoint of biomicroscopy and confocal microscopy]. (United States)

    Maĭchuk, Iu F; Maĭchuk, D Iu


    Clinical cases of 60 patients with acanthamebic keratitis examined by biomicroscopy and of 22 patients largely examined by confocal microscopy are generalized. Acanthamebic keratitis is a slowly progressing infectious lesion of the cornea, which is caused by acanthamebas freely residing in soil and water. Contaminated contact lenses are the key risk factor. The main clinical features of acanthamebic keratitis are defined; they are presence of risk factors; a unilateral lesion in young, healthy and immune-competent persons; a typical clinical pattern of surface keratitis mainly of the ring shape; corneal neuritis without corneal neovascularization but with a severe pain in the eye; and a slow chronic clinical course, i.e. lasting for several weeks and months. Confocal microscopy is the most effective and fast diagnostic tool because it ensures the detection of acanthamebic cysts and trophozoids in all strata of the corneal stroma. The authors isolate, within the clinical course of acanthamebic keratitis, 5 stages; they are surface epithelial keratitis; surface epithelial punctate keratitis; stromal ring-shaped keratitis; ulcerous keratitis; and keratoscleritis.

  4. Laser multi-reflection differential confocal long focal-length measurement. (United States)

    Li, Zhigang; Qiu, Lirong; Zhao, Weiqian; Zhao, Qi


    We propose a new laser multi-reflection differential confocal focal-length measurement (LDCFM) method to meet the requirements of high-precision measurements of long focal lengths. An optical flat and a reflector are placed behind a test lens for reflecting the measuring beam repeatedly. Then, LDCFM uses the property that the null points of differential confocal response curves precisely correspond to the convergence points of the multi-reflected measuring beam to exactly determine the positions of the convergence points accurately. Subsequently, the position variation of the reflector is measured with different reflection times by using a distance-measuring instrument, and thereby the long focal length is measured precisely. Theoretical analyses and preliminary experimental results indicate that the LDCFM method has a relative expanded standard uncertainty (k=2) of 0.04% for the test lens with a focal length of 9.76 m. The LDCFM method can provide a novel approach for high-precision focal-length measurements.

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

    Directory of Open Access Journals (Sweden)

    Elizabeth Kolos


    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.

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

    Directory of Open Access Journals (Sweden)

    S V Ravi


    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.

  7. Morphological study of adult male worms of Schistosoma mansoni Sambon, 1907 by confocal laser scanning microscopy

    Directory of Open Access Journals (Sweden)

    Machado-Silva José Roberto


    Full Text Available Aiming to detail data obtained through brightfield microscopy (BM on reproductive, excretory and digestive system, specimens of Schistosoma mansoni eight weeks old, were recovered from SW mice, stained with Langeron's carmine and analyzed under a confocal laser scanning microscope CLSM 410 (Carl Zeiss. The reproductive system presented a single and lobate testis, with intercommunications between the lobes without efferent duct. Supernumerary testicular lobe was amorphous and isolated from the normal ones. Collecting tubules (excretory ducts, followed by the excretory bladder, opening to the external media through the excretory pore, were observed at the posterior extremity of the body. In the digestive tract, a cecal swelling was noted at the junction that originates the single cecum. It was concluded that through confocal laser scanning microscopy, new interpretations of morphological structures of S. mansoni worms could be achieved, modifying adopted and current descriptions. The gonad consists of a single lobed testis, similar to that observed in some trematode species. Moreover, the same specimens can be observed either by BM or CLSM, considering that the latter causes only focal and limited damage in tissue structures.

  8. Micron-scale resolution optical tomography of entire mouse brains with confocal light sheet microscopy. (United States)

    Silvestri, Ludovico; Bria, Alessandro; Costantini, Irene; Sacconi, Leonardo; Peng, Hanchuan; Iannello, Giulio; Pavone, Francesco Saverio


    Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.


    Directory of Open Access Journals (Sweden)

    Karsten Rodenacker


    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.

  10. Combining microtomy and confocal laser scanning microscopy for structural analyses of plant-fungus associations. (United States)

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


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

  11. Demonstration of the Protein Involvement in Cell Electropermeabilization using Confocal Raman Microspectroscopy (United States)

    Azan, Antoine; Untereiner, Valérie; Gobinet, Cyril; Sockalingum, Ganesh D.; Breton, Marie; Piot, Olivier; Mir, Lluis M.


    Confocal Raman microspectroscopy was used to study the interaction between pulsed electric fields and live cells from a molecular point of view in a non-invasive and label-free manner. Raman signatures of live human adipose-derived mesenchymal stem cells exposed or not to pulsed electric fields (8 pulses, 1 000 V/cm, 100 μs, 1 Hz) were acquired at two cellular locations (nucleus and cytoplasm) and two spectral bands (600–1 800 cm−1 and 2 800–3 100 cm−1). Vibrational modes of proteins (phenylalanine and amide I) and lipids were found to be modified by the electropermeabilization process with a statistically significant difference. The relative magnitude of four phenylalanine peaks decreased in the spectra of the pulsed group. On the contrary, the relative magnitude of the amide I band at 1658 cm−1 increased by 40% when comparing pulsed and control group. No difference was found between the control and the pulsed group in the high wavenumber spectral band. Our results reveal the modification of proteins in living cells exposed to pulsed electric fields by means of confocal Raman microspectroscopy. PMID:28102326

  12. Correlated confocal and super-resolution imaging by VividSTORM. (United States)

    Barna, László; Dudok, Barna; Miczán, Vivien; Horváth, András; László, Zsófia I; Katona, István


    Single-molecule localization microscopy (SMLM) is rapidly gaining popularity in the life sciences as an efficient approach to visualize molecular distribution with nanoscale precision. However, it has been challenging to obtain and analyze such data within a cellular context in tissue preparations. Here we describe a 5-d tissue processing and immunostaining procedure that is optimized for SMLM, and we provide example applications to fixed mouse brain, heart and kidney tissues. We then describe how to perform correlated confocal and 3D-superresolution imaging on these sections, which allows the visualization of nanoscale protein localization within labeled subcellular compartments of identified target cells in a few minutes. Finally, we describe the use of VividSTORM (, an open-source software for correlated confocal and SMLM image analysis, which facilitates the measurement of molecular abundance, clustering, internalization, surface density and intermolecular distances in a cell-specific and subcellular compartment-restricted manner. The protocol requires only basic skills in tissue staining and microscopy.

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

    Directory of Open Access Journals (Sweden)

    Massimiliano eCardinale


    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.

  14. Design of 220 GHz electronically scanned reflectarrays for confocal imaging systems (United States)

    Hedden, Abigail S.; Dietlein, Charles R.; Wikner, David A.


    The authors analyze properties of a 220 GHz imaging system that uses a scanned reflectarray to perform electronic beam scanning of a confocal imager for applications including imaging meter-sized fields of view at 50 m standoff. Designs incorporating reflectarrays with confocal imagers have not been examined previously at these frequencies. We examine tradeoffs between array size, overall system size, and number of achievable image pixels resulting in a realistic architecture capable of meeting the needs of our application. Impacts to imaging performance are assessed through encircled energy calculations, beam pointing accuracy, and examining the number and intensity of quantization lobes that appear over the scan ranges of interest. Over the desired scan range, arrays with 1 and 2-bit phase quantization showed similar array main beam energy efficiencies. Two-bit phase quantization is advantageous in terms of pointing angle error, resulting in errors of at most 15% of the diffraction-limited beam size. However, both phase quantization cases considered resulted in spurious returns over the scan range of interest and other array layouts should be examined to eliminate potential imaging artifacts.

  15. Laser Scanning In Vivo Confocal Microscopy of Clear Grafts after Penetrating Keratoplasty (United States)

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


    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

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

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    Giulio eFerrari


    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.

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

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    Inceu Georgeta


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

  18. Corneal confocal microscopy reveals trigeminal small sensory fiber neuropathy in amyotrophic lateral sclerosis (United States)

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


    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

  19. In vivo corneal confocal microscopy in diabetes: Where we are and where we can get. (United States)

    Maddaloni, Ernesto; Sabatino, Francesco


    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.

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

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    Dai Wang


    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.

  1. Confocal Microscopy–Guided Laser Ablation for Superficial and Early Nodular Basal Cell Carcinoma (United States)

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


    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

  2. Novel Method for Differentiating Histological Types of Gastric Adenocarcinoma by Using Confocal Raman Microspectroscopy. (United States)

    Hsu, Chih-Wei; Huang, Chia-Chi; Sheu, Jeng-Horng; Lin, Chia-Wen; Lin, Lien-Fu; Jin, Jong-Shiaw; Chau, Lai-Kwan; Chen, Wenlung


    Gastric adenocarcinoma, a single heterogeneous disease with multiple epidemiological and histopathological characteristics, accounts for approximately 10% of cancers worldwide. It is categorized into four histological types: papillary adenocarcinoma (PAC), tubular adenocarcinoma (TAC), mucinous adenocarcinoma (MAC), and signet ring cell adenocarcinoma (SRC). Effective differentiation of the four types of adenocarcinoma will greatly improve the treatment of gastric adenocarcinoma to increase its five-year survival rate. We reported here the differentiation of the four histological types of gastric adenocarcinoma from the molecularly structural viewpoint of confocal Raman microspectroscopy. In total, 79 patients underwent laparoscopic or open radical gastrectomy during 2008-2011: 21 for signet ring cell carcinoma, 21 for tubular adenocarcinoma, 14 for papillary adenocarcinoma, 6 for mucinous carcinoma, and 17 for normal gastric mucosas obtained from patients underwent operation for other benign lesions. Clinical data were retrospectively reviewed from medical charts, and Raman data were processed and analyzed by using principal component analysis (PCA) and linear discriminant analysis (LDA). Two-dimensional plots of PCA and LDA clearly demonstrated that the four histological types of gastric adenocarcinoma could be differentiated, and confocal Raman microspectroscopy provides potentially a rapid and effective method for differentiating SRC and MAC from TAC or PAC.

  3. Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques. (United States)

    Chakraborty, Nilay; Wang, Mian; Solocinski, Jason; Kim, Wonsuk; Argento, Alan


    This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera's collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.

  4. Analysis of the in vivo confocal Raman spectral variability in human skin (United States)

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


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

  5. A fully automatic framework for cell segmentation on non-confocal adaptive optics images (United States)

    Liu, Jianfei; Dubra, Alfredo; Tam, Johnny


    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.

  6. Two-photon fluorescence and confocal reflected light imaging of thick tissue structures (United States)

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


    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.

  7. High speed velocimetry and concentration measurements in a microfluidic mixer using fluorescence confocal microscopy (United States)

    Inguva, Venkatesh; Perot, Blair; Kathuria, Sagar; Rothstein, Jonathan; Bilsel, Osman


    This work experimentally examines the performance of a quasi-turbulent micro-mixer that was designed to produce rapid mixing for protein-folding experiments. The original design of the mixer was performed using Direct Numerical Simulation (DNS) of the flow field and LES of the high Sc number scalar field representing the protein. The experimental work is designed to validate the DNS results. Both the velocity field and the protein concentration require validation. Different experiments were carried out to measure these two quantities. Concentration measurements are performed using a 488nm continuous wave laser coupled with a confocal microscope to measure fluorescence intensity during mixing. This is calibrated using the case where no mixing occurs. The velocity measurements use a novel high speed velocimetry technique capable of measuring speeds on the order of 10 m/s in a micro channel. The technique involves creating a pulsed confocal volume from a Ti-Sapphire laser with a pulse width of 260ns and observing the decay of fluorescence due to the fluid motion. Results from both experiments will be presented along with a comparison to the DNS results. The work is supported by NSF IDBR Award No. 1353942.

  8. Automated Tracing and Segmentation Tool for Migrating Neurons in 4D Confocal Imagery

    Energy Technology Data Exchange (ETDEWEB)

    Karakaya, Mahmut [ORNL; Kerekes, Ryan A [ORNL; Solecki, Dr. David [St. Jude Children' s Research Hospital


    Accurate tracing and segmentation of subcellular components of migrating neurons in confocal image sequences are prerequisite steps in many neurobiology studies to understand the biological machinery behind the movement of developing neurons. In this paper, we present an automated tracking, tracing, and segmentation tool for soma, leading, and trailing process of migrating neurons in time-lapse image stacks acquired with a confocal fluorescence microscope. In our approach, we first localize each neuron in the maximum intensity projection of the first frame using manual labeling of the soma and end points of the leading and trailing process. By using each soma position at the first frame, we automatically track the somas in rest of the frames. Then, leading and trailing process are traced in each frame from the soma center to the labeled end tip of the process by using fast marching algorithm. Finally, the soma, leading and trailing processes of each neuron are segmented by using the soma center and traces as seed points, and their boundaries are separated from each other. Based on qualitative results, we demonstrate the capability to automatically track, trace, and segment the soma, leading, and trailing processes of a migrating neuron with minimal user input.

  9. Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging (United States)

    Chen, Wei-Liang; Chou, Chen-Kuan; Lin, Ming-Gu; Chen, Yang-Fang; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Tsai, Tsung-Hua; Kim, Ki-Hean; Kim, Daekeun; So, Peter T. C.; Lin, Sung-Jan; Dong, Chen-Yuan


    Both reflected confocal and multiphoton microscopy can have clinical diagnostic applications. The successful combination of both modalities in tissue imaging enables unique image contrast to be achieved, especially if a single laser excitation wavelength is used. We apply this approach for skin and corneal imaging using the 780-nm output of a femtosecond, titanium-sapphire laser. We find that the near-IR, reflected confocal (RC) signal is useful in characterizing refractive index varying boundaries in bovine cornea and porcine skin, while the multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) intensities can be used to image cytoplasm and connective tissues (collagen), respectively. In addition, quantitative analysis shows that we are able to detect MAF from greater imaging depths than with the near-IR RC signal. Furthermore, by performing RC imaging at 488, 543, and 633 nm, we find that a longer wavelength leads to better image contrast for deeper imaging of the bovine cornea and porcine skin tissue. Finally, by varying power of the 780-nm source, we find that comparable RC image quality was achieved in the 2.7 to 10.7-mW range.

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

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


    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.

  11. Confocal laser scanning microscopy for detection of Schistosoma mansoni eggs in the gut of mice.

    Directory of Open Access Journals (Sweden)

    Martha Charlotte Holtfreter

    Full Text Available BACKGROUND: The gold standard for diagnosing Schistosoma mansoni infections is the detection of eggs from stool or biopsy specimens. The viability of collected eggs can be tested by the miracidium hatching procedure. Direct detection methods are often limited in patients with light or early infections, whereas serological tests and PCR methods fail to differentiate between an inactive and persistent infection and between schistosomal species. Recently, confocal laser scanning microscopy (CLSM has been introduced as a diagnostic tool in several fields of medicine. In this study we evaluated CLSM for the detection of viable eggs of S. mansoni directly within the gut of infected mice. METHODOLOGY/PRINCIPAL FINDINGS: The confocal laser scanning microscope used in this study is based on the Heidelberg Retina Tomograph II scanning laser system in combination with the Rostock Cornea Module (image modality 1 or a rigid endoscope (image modality 2. Colon sections of five infected mice were examined with image modalities 1 and 2 for schistosomal eggs. Afterwards a biopsy specimen was taken from each colon section and examined by bright-field microscopy. Visualised eggs were counted and classified in terms of viability status. CONCLUSIONS/SIGNIFICANCE: We were able to show that CLSM visualises eggs directly within the gut and permits discrimination of schistosomal species and determination of egg viability. Thus, CLSM may be a suitable non-invasive tool for the diagnosis of schistosomiasis in humans.

  12. The surface morphology analysis based on progressive approximation method using confocal three-dimensional micro X-ray fluorescence (United States)

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


    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.


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


    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.

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

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    Ranno S


    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

  15. [Studies on Effect of Alkali Pretreatment on Anaerobic Digestion of Rice Straw with Confocal Raman Microscopy]. (United States)

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


    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

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

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    Vanathi Murugesan


    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. 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


    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.

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

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    Babu Kalpana


    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.

  19. 1KW Power Transmission Using Wireless Acoustic-Electric Feed-Through (WAEF) (United States)

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


    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.

  20. Three-dimensional imaging of DNA fragments during electrophoresis using a confocal detector (United States)

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


    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.

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


    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. Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies (United States)

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


    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.

  3. Method and apparatus for a high-resolution three dimensional confocal scanning transmission electron microscope (United States)

    de Jonge, Niels [Oak Ridge, TN


    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.

  4. Confocal laser scanning microscopy detection of chlorophylls and carotenoids in chloroplasts and chromoplasts of tomato fruit. (United States)

    D'Andrea, Lucio; Amenós, Montse; Rodríguez-Concepción, Manuel


    Plant cells are unique among eukaryotic cells because of the presence of plastids, including chloroplasts and chromoplasts. Chloroplasts are found in green tissues and harbor the photosynthetic machinery (including chlorophyll molecules), while chromoplasts are present in non-photosynthetic tissues and accumulate large amounts of carotenoids. During tomato fruit development, chloroplasts are converted into chromoplasts that accumulate high levels of lycopene, a linear carotenoid responsible for the characteristic red color of ripe fruit. Here, we describe a simple and fast method to detect both types of fully differentiated plastids (chloroplasts and chromoplasts), as well as intermediate stages, in fresh tomato fruits. The method is based on the differential autofluorescence of chlorophylls and carotenoids (lycopene) detected by Confocal Laser Scanning Microscopy.

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

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    Natallia Eduarda Uzunbajakava


    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.

  6. Confocal Raman spectroscopy of island nuclei formed at the initial stage of quartz glass crystallization (United States)

    Pankin, D. V.; Zolotarev, V. M.; Colas, M.; Cornette, J.; Evdokimova, M. G.


    Island nuclei formed on a polished quartz-glass surface upon heating to 1100°C have been investigated by confocal Raman spectroscopy. The structural and chemical composition of islands is shown to be a central nucleus, a shell around the nucleus, and a thin peripheral ring closing this shell. The formation and growth of individual regions of an island nucleus are found to occur in several stages. The shell around the nucleus is mainly formed by α-SiO2 and α-cristobalite nanoparticles with a size ≥40 nm, whereas the α-SiO2 nanoparticles in the nucleus and peripheral ring are 2-15 nm in size.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  8. In vivo confocal Raman spectroscopy study of the vitamin A derivative perfusion through human skin (United States)

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


    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.

  9. Investigating Effects of Proteasome Inhibitor on Multiple Myeloma Cells Using Confocal Raman Microscopy

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    Jeon Woong Kang


    Full Text Available Due to its label-free and non-destructive nature, applications of Raman spectroscopic imaging in monitoring therapeutic responses at the cellular level are growing. We have recently developed a high-speed confocal Raman microscopy system to image living biological specimens with high spatial resolution and sensitivity. In the present study, we have applied this system to monitor the effects of Bortezomib, a proteasome inhibitor drug, on multiple myeloma cells. Cluster imaging followed by spectral profiling suggest major differences in the nuclear and cytoplasmic contents of cells due to drug treatment that can be monitored with Raman spectroscopy. Spectra were also acquired from group of cells and feasibility of discrimination among treated and untreated cells using principal component analysis (PCA was accessed. Findings support the feasibility of Raman technologies as an alternate, novel method for monitoring live cell dynamics with minimal external perturbation.

  10. Consistency and distribution of reflectance confocal microscopy features for diagnosis of cutaneous T cell lymphoma (United States)

    Lange-Asschenfeldt, Susanne; Babilli, Jasmin; Beyer, Marc; Ríus-Diaz, Francisca; González, Salvador; Stockfleth, Eggert; Ulrich, Martina


    Reflectance confocal microscopy (RCM) represents a noninvasive imaging technique that has previously been used for characterization of mycosis fungoides (MF) in a pilot study. We aimed to test the applicability of RCM for diagnosis and differential diagnosis of MF in a clinical study. A total of 39 test sites of 15 patients with a biopsy-proven diagnosis of either MF, parapsoriasis, Sézary syndrome, or lymphomatoid papulosis were analyzed for presence and absence of RCM features of MF. Cochran and Chi2 analysis were applied to test the concordance between investigators and the distribution of RCM features, respectively. For selected parameters, the Cochran analysis showed good concordance between investigators. Inter-observer reproducibility was highest for junctional atypical lymphocytes, architectural disarray, and spongiosis. Similarly, Chi2 analysis demonstrated that selected features were present at particularly high frequency in individual skin diseases, with values ranging from 73% to 100% of all examined cases.

  11. Identification of different bacterial species in biofilms using confocal Raman microscopy (United States)

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


    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.

  12. Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope. (United States)

    Wang, Peng; Behan, Gavin; Kirkland, Angus I; Nellist, Peter D; Cosgriff, Eireann C; D'Alfonso, Adrian J; Morgan, Andrew J; Allen, Leslie J; Hashimoto, Ayako; Takeguchi, Masaki; Mitsuishi, Kazutaka; Shimojo, Masayuki


    Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored.

  13. Trypan blue as a fluorochrome for confocal laser scanning microscopy of arbuscular mycorrhizae in three mangroves. (United States)

    Kumar, T; Majumdar, A; Das, P; Sarafis, V; Ghose, M


    Roots of three mangroves, Acanthus ilicifolius, Ceriops tagal and Excoecaria agallocha, collected from forests of the Sundarbans of India were stained with trypan blue to observe arbuscular mycorrhizal colonization. Spores of arbuscular mycorrhizal fungi isolated from rhizospheric soil, collected together with the root samples, also were stained for testing the suitability of the dye as a fluorochrome. Confocal laser scanning microscopy images were constructed. A. ilicifolius and E. agallocha exhibited "Arum" type colonization with highly branched arbuscules, whereas C. tagal showed "Paris" type association with clumped and collapsed arbuscules. We demonstrated that trypan blue is a suitable fluorochrome for staining arbuscular mycorrhizal fungal spores, fungal hyphae, arbuscules and vesicles, which presumably have a considerable amount of surface chitin. It appears that as the integration of chitin into the fungal cell wall changes, its accessibility to trypan blue dye also changes.

  14. In vivo reflectance-mode confocal microscopy in clinical dermatology and cosmetology. (United States)

    González, S; Gilaberte-Calzada, Y


    In vivo reflectance confocal microscopy (RCM) is a non-invasive imaging tool that allows real-time visualization of cells and structures in living skin with near histological resolution. RCM has been used for the assessment of benign and malignant lesions, showing great potential for applications in basic skin research and clinical dermatology. RCM also reveals dynamic changes in the skin over time and in response to specific stimuli, like ultraviolet exposure, which makes it a promising tool in cosmetology, as it allows repetitive sampling without biopsy collection, causing no further damage to the areas under investigation. This review summarizes the latest advances in RCM, and its applications in the characterization of both normal and pathological skin.

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

    KAUST Repository

    Nappini, Silvia


    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.

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

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim; Sternberg, Claus


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

  17. Imaging rat esophagus using combination of reflectance confocal and multiphoton microscopy (United States)

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


    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.

  18. Characterization of acoustic lenses with the Foucault test by confocal laser scanning microscopy (United States)

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


    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.

  19. 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.

  20. A two dimensional optical input to one dimensional serial pulse transformation using confocal reflectors. (United States)

    Hulse, George


    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.

  1. Evaluation of the Cytotoxic Behavior of Fungal Extracellular Synthesized Ag Nanoparticles Using Confocal Laser Scanning Microscope. (United States)

    Salaheldin, Taher A; Husseiny, Sherif M; Al-Enizi, Abdullah M; Elzatahry, Ahmed; Cowley, Alan H


    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.

  2. Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Yin Liu


    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.

  4. High numerical aperture microendoscope objective for a fiber confocal reflectance microscope (United States)

    Kester, Robert T.; Tkaczyk, Tomasz S.; Descour, Michael R.; Christenson, Todd; Richards-Kortum, Rebecca


    A disposable high numerical aperture microendoscope objective has been designed, fabricated, and tested for use with a fiber confocal reflectance microscope. The objective uses high precision LIGA fabricated components to integrate imaging components and hydraulic suction lines into a housing that measures only 3.85 mm in outer diameter and 14.65 mm in length. The hydraulics are used to translate tissue through the focal plane for three dimensional imaging. This device is diffraction limited for λ = 850 nm, has a numerical aperture of 1.0, a field of view of 250 µm, and a working distance of 450 µm. The objective is intended for in vivo imaging of precancerous cells.

  5. Pharmaceutical applications of confocal laser scanning microscopy: the physical characterisation of pharmaceutical systems. (United States)

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


    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.

  6. In vivo amyloid aggregation kinetics tracked by time-lapse confocal microscopy in real-time. (United States)

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


    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.

  7. Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopy. (United States)

    Pawliczek, Piotr; Romanowska-Pawliczek, Anna; Soltys, Zbigniew


    Various deconvolution algorithms are often used for restoration of digital images. Image deconvolution is especially needed for the correction of three-dimensional images obtained by confocal laser scanning microscopy. Such images suffer from distortions, particularly in the Z dimension. As a result, reliable automatic segmentation of these images may be difficult or even impossible. Effective deconvolution algorithms are memory-intensive and time-consuming. In this work, we propose a parallel version of the well-known Richardson-Lucy deconvolution algorithm developed for a system with distributed memory and implemented with the use of Message Passing Interface (MPI). It enables significantly more rapid deconvolution of two-dimensional and three-dimensional images by efficiently splitting the computation across multiple computers. The implementation of this algorithm can be used on professional clusters provided by computing centers as well as on simple networks of ordinary PC machines.

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

    CERN Document Server

    Gasser, U; Weitz, D A


    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


    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)


    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


    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. Confocal laser endomicroscopy for diagnosis of Barrett´s esophagus

    Directory of Open Access Journals (Sweden)

    Helmut eNeumann


    Full Text Available Barrett´s esophagus (BE is established as a premalignant condition in the distal esophagus. Current surveillance guidelines recommend random biopsies every 1-2 cm at intervals of 3-5 years. Advanced endoscopic imaging of BE underwent several technical revolutions within the last decade including broad-field (red-flag techniques (e.g. chromoendoscopy and small-field techniques with confocal laser endomicroscopy (CLE at the forefront. In this review we will focus on advanced endoscopic imaging using CLE for the diagnosis and characterization of BE and associated neoplasia. In addition, we will critically discuss the technique of CLE and provide some tricks and hints for the daily routine practice of CLE for diagnosis of BE.

  13. Technology insight: Laser-scanning confocal microscopy and endocytoscopy for cellular observation of the gastrointestinal tract. (United States)

    Inoue, Haruhiro; Kudo, Shin-ei; Shiokawa, Akira


    Recent advances in endoscopic imaging technology have enabled the visualization of early-stage cancer and its precursors in the gastrointestinal tract. Chromoendoscopy, magnifying endoscopy, endoscopic optical coherent tomography, spectroscopy, and various combinations of these technologies, are all important for the recognition of small and unclear lesions. To observe cancer cells in vivo, two types of ultra-high magnifying endoscope--'laser-scanning confocal endoscopy series' and 'contact endoscopy series'--that have a maximum of more than 1,000x magnifying power have been developed. These endoscopes can generate high-quality images of both living cancer cells and normal cells in the gastrointestinal tract, with a quality comparable to that possible with conventional cytology. These novel imaging technologies may make in vivo histological diagnosis by virtual histology possible.

  14. Confocal restricted-height imaging of suspension cells (CRISC) in a PDMS microdevice during apoptosis. (United States)

    Muñoz-Pinedo, Cristina; Green, Douglas R; van den Berg, Albert


    We have monitored and imaged cell death induced in human leukemic U937 cells over time using three-color confocal imaging. Three different apoptotic inducers, anti-Fas, TNF-alpha and Etoposide were used. Individual cascaded events such as loss of mitochondrial transmembrane potential, exposure of phosphatidyl-serine, membrane blebbing and permeabilization of the cell membrane have been observed in real time with different individual cells. From the results, an interesting heterogeneicity in the apoptotic phenotype has been observed. The CRISC method is easy to use and provides biologist with a powerful additional tool to study in real-time processes of several hours of duration such as apoptosis. We predict that the period of cell viability obtained after protein coating of the PDMS devices (>80 h) will also allow monitoring of other biological processes of longer duration or long onset time, such as mitosis, phagocytosis and differentiation.

  15. Transdermal delivery of betahistine hydrochloride using microemulsions: physical characterization, biophysical assessment, confocal imaging and permeation studies. (United States)

    Hathout, Rania M; Nasr, Maha


    Transdermal delivery of betahistine hydrochloride encapsulated in various ethyl oleate, Capryol 90(®), Transcutol(®) and water microemulsion formulations was studied. Two different kinds of phase diagrams were constructed for the investigated microemulsion system. Pseudoplastic flow that is preferable for skin delivery was recorded for the investigated microemulsions. A balanced and bicontinuous microemulsion formulation was suggested and showed the highest permeation flux (0.50±0.030mgcm(-2)h(-1)). The effect of the investigated microemulsions on the skin electrical resistance was used to explain the high permeation fluxes obtained. Confocal laser scanning microscopy was used to confirm the permeation enhancement and to reveal the penetration pathways. The results obtained suggest that the proposed microemulsion system highlighted in the current work can serve as a promising alternative delivery means for betahistine hydrochloride.

  16. A confocal view of the calcifying odontogenic cyst: Report of two cases

    Directory of Open Access Journals (Sweden)

    Soumya Makarla


    Full Text Available The calcifying odontogenic cyst (COC is a rare odontogenic lesion representing about 1% of all jaw cysts. There is a wide age range from 1 to 82 years with a first peak in the second decade and the second in the sixth/seventh decade. This lesion is characterized by the presence of "ghost" cells. The pathogenesis of the lesion is from the reduced enamel epithelium or the remnants of odontogenic epithelium. Here, we report two cases, both in 18-year-old male patients; previously diagnosed as dentigerous cyst and residual cyst respectively. But histologically, both the cases turned out to be COCs. Confocal laser scanning microscopy (CLSM has become an invaluable tool for a wide range of investigations in the medical sciences for imaging thin optical sections of tissues. The COCs were evaluated using CLSM to analyze the properties of the cystic lining and the ghost cells.

  17. 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


    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.

  18. Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope.

    Directory of Open Access Journals (Sweden)

    André Klauss

    Full Text Available By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as "easy-STED", achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.

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

    Directory of Open Access Journals (Sweden)

    Chao eHuang


    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.

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


    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.

  1. Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer. (United States)

    Rappaz, Benjamin; Barbul, Alexander; Emery, Yves; Korenstein, Rafi; Depeursinge, Christian; Magistretti, Pierre J; Marquet, Pierre


    Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label-free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (lambda = 663 nm) was used to record holograms in an off-axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n = 1.418 +/- 0.012, volume 83 +/- 14 fl, MCH = 29.9 pg, and MCHC 362 +/- 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 +/- 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single-living RBCs with a high accuracy.

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


    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

  3. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart. (United States)

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


    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.

  4. Assessing strain mapping by electron backscatter diffraction and confocal Raman microscopy using wedge-indented Si

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    Friedman, Lawrence H.; Vaudin, Mark D.; Stranick, Stephan J.; Stan, Gheorghe; Gerbig, Yvonne B.; Osborn, William; Cook, Robert F., E-mail:


    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{sup −4} in strain. CRM was similarly precise, but was limited in accuracy to several times this value. - Highlights: • We map strain by electron backscatter diffraction and confocal Raman microscopy. • The test vehicle is the multi-axial strain field of wedge-indented silicon. • Strain accuracy is assessed by direct experimental intercomparison. • Accuracy is also assessed by atomic force microscopy and finite element analyses. • Electron diffraction measurements are accurate; Raman measurements need refinement.

  5. Depth-profiling by confocal Raman microscopy (CRM): data correction by numerical techniques. (United States)

    Tomba, J Pablo; Eliçabe, Guillermo E; Miguel, María de la Paz; Perez, Claudio J


    The data obtained in confocal Raman microscopy (CRM) depth profiling experiments with dry optics are subjected to significant distortions, including an artificial compression of the depth scale, due to the combined influence of diffraction, refraction, and instrumental effects that operate on the measurement. This work explores the use of (1) regularized deconvolution and (2) the application of simple rescaling of the depth scale as methodologies to obtain an improved, more precise, confocal response. The deconvolution scheme is based on a simple predictive model for depth resolution and the use of regularization techniques to minimize the dramatic oscillations in the recovered response typical of problem inversion. That scheme is first evaluated using computer simulations on situations that reproduce smooth and sharp sample transitions between two materials and finally it is applied to correct genuine experimental data, obtained in this case from a sharp transition (planar interface) between two polymeric materials. It is shown that the methodology recovers very well most of the lost profile features in all the analyzed situations. The use of simple rescaling appears to be only useful for correcting smooth transitions, particularly those extended over distances larger than those spanned by the operative depth resolution, which limits the strategy to the study of profiles near the sample surface. However, through computer simulations, it is shown that the use of water immersion objectives may help to reduce optical distortions and to expand the application window of this simple methodology, which could be useful, for instance, to safely monitor Fickean sorption/desorption of penetrants in polymer films/coatings in a nearly noninvasive way.

  6. Selective presynaptic terminal remodeling induced by spatial, but not cued, learning: a quantitative confocal study. (United States)

    McGonigal, R; Tabatadze, N; Routtenberg, A


    The hippocampal mossy fibers (MFs) are capable of behaviorally selective, use-dependent structural remodeling. Indeed, we previously observed a new layer of Timm's staining induced in the stratum oriens (SO) in CA3 after spatial but not cued water maze learning (Rekart et al., (2007) Learn Mem 14:416-421). This led to the prediction that there is a learning-specific induction of presynaptic terminal plasticity of MF axons. This study confirms this prediction demonstrating, at the confocal level of analysis, terminal-specific, and behavior-selective presynaptic structural plasticity linked to long-term memory. Male adult Wistar rats were trained for 5 days to locate a hidden or visible platform in a water maze and a retention test was performed 7 days later. MF terminal subtypes, specifically identified by an antibody to zinc transporter 3 (ZnT3), were counted from confocal z-stacks in the stratum lucidum (SL) and the SO. In hidden platform trained rats, there was a significant increase in the number of large MF terminals (LMTs, 2.5-10 μm diameter, >2 μm(2) area) compared to controls both in the proximal SL (P CA3 pyramidal neurons, while SMTs are known to target inhibitory interneurons. The present findings highlight the pivotal role in memory of presynaptic structural plasticity. Because the "sprouting" observed is specific to the LMT, with no detectable change in the number of the SMT, learning may enhance net excitatory input to CA3 pyramidal neurons. Given the sparse coding of the MF-CA3 connection, and the role that granule cells play in pattern separation, the remodeling observed here may be expected to have a major impact on the long-term integration of spatial context into memory.

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

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    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.

  8. Oscillating optical tweezer-based 3-D confocal microrheometer for investigating the intracellular micromechanics and structures (United States)

    Ou-Yang, H. D.; Rickter, E. A.; Pu, C.; Latinovic, O.; Kumar, A.; Mengistu, M.; Lowe-Krentz, L.; Chien, S.


    Mechanical properties of living biological cells are important for cells to maintain their shapes, support mechanical stresses and move through tissue matrix. The use of optical tweezers to measure micromechanical properties of cells has recently made significant progresses. This paper presents a new approach, the oscillating optical tweezer cytorheometer (OOTC), which takes advantage of the coherent detection of harmonically modulated particle motions by a lock-in amplifier to increase sensitivity, temporal resolution and simplicity. We demonstrate that OOTC can measure the dynamic mechanical modulus in the frequency range of 0.1-6,000 Hz at a rate as fast as 1 data point per second with submicron spatial resolution. More importantly, OOTC is capable of distinguishing the intrinsic non-random temporal variations from random fluctuations due to Brownian motion; this capability, not achievable by conventional approaches, is particular useful because living systems are highly dynamic and often exhibit non-thermal, rhythmic behavior in a broad time scale from a fraction of a second to hours or days. Although OOTC is effective in measuring the intracellular micromechanical properties, unless we can visualize the cytoskeleton in situ, the mechanical property data would only be as informative as that of "Blind men and the Elephant". To solve this problem, we take two steps, the first, to use of fluorescent imaging to identify the granular structures trapped by optical tweezers, and second, to integrate OOTC with 3-D confocal microscopy so we can take simultaneous, in situ measurements of the micromechanics and intracellular structure in living cells. In this paper, we discuss examples of applying the oscillating tweezer-based cytorheometer for investigating cultured bovine endothelial cells, the identification of caveolae as some of the granular structures in the cell as well as our approach to integrate optical tweezers with a spinning disk confocal microscope.

  9. Selective presynaptic terminal remodeling induced by spatial, but not cued, learning: a quantitative confocal study (United States)

    McGonigal, R.; Tabatadze, N.; Routtenberg, A.


    The hippocampal mossy fibers (MFs) are capable of behaviorally-selective, use-dependent structural remodeling. Indeed, we previously observed a new layer of Timm’s staining induced in the stratum oriens (SO) in CA3 after spatial but not cued water maze learning (Rekart et al., Learn. Mem. 2007; 14:416–421). This led to the prediction that there is a learning-specific induction of presynaptic terminal plasticity of MF axons. The present study confirms this prediction demonstrating, at the confocal level of analysis, terminal-specific and behavior-selective presynaptic structural plasticity linked to long-term memory. Male adult Wistar rats were trained for 5d to locate a hidden or visible platform in a water maze and a retention test was performed 7d later. MF terminal subtypes, specifically identified by an antibody to zinc transporter 3 (ZnT3), were counted from confocal z-stacks in the stratum lucidum (SL) and the SO. In hidden platform trained rats there was a significant increase in the number of large MF terminals (LMTs, 2.5–10µm diameter, >2µm2 area) compared to controls both in the proximal SL (p CA3 pyramidal neurons, while SMTs are known to target inhibitory interneurons. The present findings highlight the pivotal role in memory of presynaptic structural plasticity. Because the ‘sprouting’ observed is specific to the LMT, with no detectable change in the number of the SMT, learning may enhance net excitatory input to CA3 pyramidal neurons. Given the sparse coding of the MF-CA3 connection, and the role that granule cells play in pattern separation, the remodeling observed here may be expected to have a major impact on the long-term integration of spatial context into memory. PMID:22180136

  10. Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures (United States)

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


    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.

  11. Modeling of fibrin gels based on confocal microscopy and light-scattering data. (United States)

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


    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.

  12. Effects of contact lens wearing on keratoconus: a confocal microscopy observation (United States)

    Ghosh, Somnath; Mutalib, Haliza A; Sharanjeet-Kaur; Ghoshal, Rituparna; Retnasabapathy, Shamala


    AIM To evaluate the corneal cell morphology of new keratoconus patients wearing two different types of rigid gas-permeable (RGP) contact lenses for 1y. METHODS Thirty nine eyes of 39 new keratoconus patients were selected and randomly fitted with two types of RGP contact lenses. Group 1 had 21 eyes with regular rigid gas-permeable (RRGP) contact lens and rest 18 eyes were in group 2 with specially designed rigid gas-permeable (SRGP) contact lens. Corneal cell morphology was evaluated using a slit scanning confocal microscope at no-lens wear and after 1y of contact lens wearing. RESULTS After 1y of contact lens wearing in group 1, the mean anterior and posterior stromal keratocyte density were significantly less (P=0.006 and P=0.001, respectively) compared to no-lens wear. The mean cell area of anterior and posterior stromal keratocyte were also significantly different (P=0.005 and P=0.001) from no-lens wear. The anterior and posterior stromal haze increased by 18.74% and 23.81%, respectively after 1y of contact lens wearing. Whereas in group 2, statistically significant changes were observed only in cell density & area of anterior stroma (P=0.001 and P=0.001, respectively) after 1y. While, level of anterior and posterior stromal haze increased by 16.67% and 11.11% after 1y of contact lens wearing. Polymegathism and pleomorphism also increased after 1y of contact lens wearing in both the contact lens groups. CONCLUSION Confocal microscopy observation shows the significant alterations in corneal cell morphology of keratoconic corneas wearing contact lenses especially in group 1. The type of contact lens must be carefully selected to minimize changes in corneal cell morphology. PMID:28251081

  13. Localization and movement of mineral oil in plants by fluorescence and confocal microscopy. (United States)

    Tan, B L; Sarafis, V; Beattie, G A C; White, R; Darley, E M; Spooner-Hart, R


    Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.

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

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    Soumya Mitra, Oleg Mironov, Thomas H. Foster


    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.

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


    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.

  16. An in vitro Comparative Evaluation of Three Remineralizing Agents using Confocal Microscopy (United States)

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


    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

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

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    Chung, D.S.; Alkire, R.C. [Univ. of Illinois, Urbana, IL (United States)


    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.

  18. Spectrally Encoded Confocal Microscopy (SECM) for Diagnosing of Breast Cancer in Excision and Margin Specimens (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


    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

  19. 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.

  20. Automated Microscopy: Macro Language Controlling a Confocal Microscope and its External Illumination: Adaptation for Photosynthetic Organisms. (United States)

    Steinbach, Gábor; Kaňa, Radek


    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 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.

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

    Directory of Open Access Journals (Sweden)

    Ferrari Giulio


    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.

  2. 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.


    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

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


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


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

  4. Toward three-dimensional virtual biopsy of oral lesions through the development of a confocal endomicroscope interfaced with embedded computing (United States)

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


    Oral lesions are conventionally diagnosed using white light endoscopy and histopathology of biopsy samples. Oral lesions are often flat and difficult to visualize under white light illumination. Moreover, histopathology is timeconsuming and there is a need to develop minimally invasive optical biopsy techniques to complement current techniques. Confocal laser endomicroscopy holds promise for virtual biopsy in disease diagnosis. This technique enables fluorescence imaging of tissue structures at microscopic resolution. We have developed a prototype real-time 3- dimensional (3D) imaging system using a laser endomicroscope interfaced with embedded computing. A Field- Programmable Gate Array computing platform has been programmed to synchronize cross-sectional image grabbing and Z-depth scanning, as well as automate acquisition of confocal image stacks. A PC was used for real-time volume rendering of the confocal image stacks. We conducted pre-clinical and pilot clinical studies to image the murine and human oral cavity. High quality volume renderings of the confocal image stacks were generated using 3D texture slicing. Tissue morphology and 3D structures could be visualized. The results demonstrate the potential of the system for diagnostic imaging of the oral cavity. This paves the way toward real-time virtual biopsy of oral lesions, with the aim to achieve same-day diagnosis in a clinical setting.

  5. Toward real-time virtual biopsy of oral lesions using confocal laser endomicroscopy interfaced with embedded computing (United States)

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


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

  6. 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


    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.

  7. 3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

    Energy Technology Data Exchange (ETDEWEB)

    Fredrich, J.T.


    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.

  8. 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.


    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

  9. 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.


    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

  10. 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


    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...

  11. Spatial inhomogeneity in spectra and exciton dynamics in porphyrin micro-rods and micro-brushes: Confocal microscopy

    Indian Academy of Sciences (India)



    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.

  12. A new wide field-of-view confocal imaging system and its applications in drug discovery and pathology (United States)

    Li, Gang; Damaskinos, Savvas; Dixon, Arthur E.; Lee, Lucy E. J.


    Conventional widefield light microscopy and confocal scanning microscopy have been indispensable for pathology and drug discovery research. Clinical specimens from diseased tissues are examined, new drug candidates are tested on drug targets, and the morphological and molecular biological changes of cells and tissues are observed. High throughput screening of drug candidates requires highly efficient screening instruments. A standard biomedical slide is 1 by 3 inches (25.4 by 76.2 mm) in size. A typical tissue specimen is 10 mm in diameter. To form a high resolution image of the entire specimen, a conventional widefield light microscope must acquire a large number of small images of the specimen, and then tile them together, which is tedious, inefficient and error-prone. A patented new wide field-of-view confocal scanning laser imaging system has been developed for tissue imaging, which is capable of imaging an entire microscope slide without tiling. It is capable of operating in brightfield, reflection and epi-fluorescence imaging modes. Three (red, green and blue (RGB)) lasers are used to produce brightfield and reflection images, and to excite various fluorophores. This new confocal system makes examination of large biomedical specimens more efficient, and makes fluorescence examination of large specimens possible for the first time without tiling. Description of the new confocal technology and applications of the imaging system in pathology and drug discovery research, for example, imaging large tissue specimens, tissue microarrays, and zebrafish sections, are reported in this paper.

  13. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells. (United States)

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


    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.

  14. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Vyacheslav M Shkryl

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

  16. Development of 3D Chromatin Texture Analysis Using Confocal Laser Scanning Microscopy

    Directory of Open Access Journals (Sweden)

    André Huisman


    Full Text Available Introduction: Analysis of nuclear texture features as a measure of nuclear chromatin changes has been proven to be useful when measured on thin (5–6 μm tissue sections using conventional 2D bright field microscopy. The drawback of this approach is that most nuclei are not intact because of those thin sections. Confocal laser scanning microscopy (CLSM allows measurements of texture in 3D reconstructed nuclei. The aim of this study was to develop 3D texture features that quantitatively describe changes in chromatin architecture associated with malignancy using CLSM images. Methods: Thirty-five features thoughtfully chosen from 4 categories of 3D texture features (discrete texture features, Markovian features, fractal features, grey value distribution features were selected and tested for invariance properties (rotation and scaling using artificial images with a known grey value distribution. The discriminative power of the 3D texture features was tested on artificially constructed benign and malignant 3D nuclei with increasing nucleolar size and advancing chromatin margination towards the periphery of the nucleus. As a clinical proof of principle, the discriminative power of the texture features was assessed on 10 benign and 10 malignant human prostate nuclei, evaluating also whether there was more texture information in 3D whole nuclei compared to a single 2D plane from the middle of the nucleus. Results: All texture features showed the expected invariance properties. Almost all features were sensitive to variations in the nucleolar size and to the degree of margination of chromatin. Fourteen texture features from different categories had high discriminative power for separating the benign and malignant nuclei. The discrete texture features performed less than expected. There was more information on nuclear texture in 3D than in 2D. Conclusion: A set of 35 3D nuclear texture features was used successfully to assess nuclear chromatin patterns

  17. Confocal Raman Microscopy of Hybrid-Supported Phospholipid Bilayers within Individual C18-Functionalized Chromatographic Particles. (United States)

    Kitt, Jay P; Harris, Joel M


    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

  18. Implementation of Accurate and Fast DNA Cytometry by Confocal Microscopy in 3D

    Directory of Open Access Journals (Sweden)

    Lennert S. Ploeger


    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

  19. Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles (United States)

    Guggenheim, Emily J.; Khan, Abdullah; Pike, Jeremy; Chang, Lynne; Lynch, Iseult; Rappoport, Joshua Z.


    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

  20. Influences of edges and steep slopes in 3D interference and confocal microscopy (United States)

    Xie, Weichang; Hagemeier, Sebastian; Woidt, Carsten; Hillmer, Harmut; Lehmann, Peter


    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

  1. 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)



    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.

  2. Differentiating gastrointestinal stromal tumors from gastric adenocarcinomas and normal mucosae using confocal Raman microspectroscopy (United States)

    Hsu, Chih-Wei; Huang, Chia-Chi; Sheu, Jeng-Horng; Lin, Chia-Wen; Lin, Lien-Fu; Jin, Jong-Shiaw; Chen, Wenlung


    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.

  3. Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy (United States)

    Hardman, P.; Spooner, B. S.


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

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

    Directory of Open Access Journals (Sweden)

    Marco Antonio Botelho


    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. 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.

  5. Quantification of Mesenchymal Stem Cell (MSC delivery to a target site using in vivo confocal microscopy.

    Directory of Open Access Journals (Sweden)

    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.

  6. Visualization of Golgia apparatus as an intracellular calcium store by laser scanning confocal microscope

    Institute of Scientific and Technical Information of China (English)



    Using laser scanning confocal microscopy,we have found that the in cells loaded with fluo-3/AM,highest intracellular Ca2+ in the perinuclear region is associated with the Golgi apparatus.The spatiotemporal subcellular distribution of Ca2+ in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated.PDGF,which releases Ca2+ from intracellular stores by inositol(1,4,5)-trisphosphate pathway ,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the golgi apparatus.Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin,an inhibitor of the Ca2+ transport ATPase of intracellular calcium store,Permeablizing the plasma membrane by 10μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store.These results suggest that the Golgi apparatus plays a role in Ca2+ regulation in signal transduction.

  7. 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


    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.

  8. Implementation of fluorescence confocal mosaicing microscopy by "early adopter" Mohs surgeons: a review of recent progress (United States)

    Jain, Manu; Rajadhyaksha, Milind; Nehal, Kishwer


    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.

  9. Penetration of resin-based materials into initial erosion lesion: A confocal microscopic study. (United States)

    Ionta, Franciny Querobim; Boteon, Ana Paula; Moretto, Marcelo Juliano; Júnior, Odair Bim; Honório, Heitor Marques; Silva, Thiago Cruvinel; Wang, Linda; Rios, Daniela


    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.

  10. Descemetic and Predescemetic DALK in Keratoconus Patients: A Clinical and Confocal Perspective Study

    Directory of Open Access Journals (Sweden)

    Domenico Schiano-Lomoriello


    Full Text Available Purpose. To evaluate the clinical outcomes and in vivo confocal microscopy (IVCM features of keratoconus patients who underwent deep anterior lamellar keratoplasty (DALK. Methods. DALK was performed using the big bubble technique in all the patients. If the bubble was not successful to bare the descemet membrane, a manual dissection layer-by layer was performed to expose a deep stromal plane close to the DM. The patients were divided in two groups depending on the intraoperative baring of the descemet membrane: predescemetic DALK (PD-DALK and descemetic DALK (D-DALK group. Results. One month after surgery the D-DALK patients show an increase of mean BCVA. In the PD-DALK group mean BCVA did not show significant improvement as compared to preoperative values. At 6 months after surgery mean BCVA was found to be similar in both groups. At 1 month IVCM the peak of reflectivity of the interface was lower in D-DALK group compared to PD-DALK. At 6 months the values of reflectivity were comparable. Conclusions. At 1 month D-DALK seems to lead to a minor interface reflectivity and to a better BCVA; these differences disappear after 6 months and the values of interface reflectivity and BCVA are comparable between D-DALK and PD-DALK.

  11. Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo. (United States)

    Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean


    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.

  12. High temperature monitoring of silicon carbide ceramics by confocal energy dispersive X-ray fluorescence spectrometry (United States)

    Li, Fangzuo; Liu, Zhiguo; Sun, Tianxi


    In the present work, we presented an alternative method for monitoring of the oxidation situation of silicon carbide (SiC) ceramics at various high temperatures in air by measuring the Compton-to-Rayleigh intensity ratios (ICo/IRa) and effective atomic numbers (Zeff) of SiC ceramics with the confocal energy dispersive X-ray fluorescence (EDXRF) spectrometer. A calibration curve of the relationship between ICo/IRa and Zeff was established by using a set of 8 SiC calibration samples. The sensitivity of this approach is so high that it can be easily distinguished samples of Zeff differing from each other by only 0.01. The linear relationship between the variation of Zeff and the variations of contents of C, Si and O of SiC ceramics were found, and the corresponding calculation model of the relationship between the ΔZ and the ΔCC, ΔCSi, and ΔCO were established. The variation of contents of components of the tested SiC ceramics after oxidation at high temperature was quantitatively calculated based on the model. It was shown that the results of contents of carbon, silicon and oxygen obtained by this method were in good agreement with the results obtained by XPS, giving values of relative deviation less than 1%. It was concluded that the practicality of this proposed method for monitoring of the oxidation situation of SiC ceramics at high temperatures was acceptable.

  13. Real time confocal laser scanning microscopy: Potential applications in space medicine and cell biology (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 (United States)

    Hutcheson, Joshua A.; Khan, Foysal Z.; Powless, Amy J.; Benson, Devin; Hunter, Courtney; Fritsch, Ingrid; Muldoon, Timothy J.


    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. Characterization of tumor microvascular structure and permeability: comparison between magnetic resonance imaging and intravital confocal imaging (United States)

    Reitan, Nina Kristine; Thuen, Marte; Goa, Pa˚L. Erik; de Lange Davies, Catharina


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

  16. A confocal microscopy-based atlas of tissue architecture in the tapeworm Hymenolepis diminuta. (United States)

    Rozario, Tania; Newmark, Phillip A


    Tapeworms are pervasive and globally distributed parasites that infect millions of humans and livestock every year, and are the causative agents of two of the 17 neglected tropical diseases prioritized by the World Health Organization. Studies of tapeworm biology and pathology are often encumbered by the complex life cycles of disease-relevant tapeworm species that infect hosts such as foxes, dogs, cattle, pigs, and humans. Thus, studies of laboratory models can help overcome the practical, ethical, and cost-related difficulties faced by tapeworm parasitologists. The rat intestinal tapeworm Hymenolepis diminuta is easily reared in the laboratory and has the potential to enable modern molecular-based experiments that will greatly contribute to our understanding of multiple aspects of tapeworm biology, such as growth and reproduction. As part of our efforts to develop molecular tools for experiments on H. diminuta, we have characterized a battery of lectins, antibodies, and common stains that label different tapeworm tissues and organ structures. Using confocal microscopy, we have assembled an "atlas" of H. diminuta organ architecture that will be a useful resource for helminthologists. The methodologies we describe will facilitate characterization of loss-of-function perturbations using H. diminuta. This toolkit will enable a greater understanding of fundamental tapeworm biology that may elucidate new therapeutic targets toward the eradication of these parasites.

  17. Efficacy of photodynamic therapy against larvae of Aedes aegypti: confocal microscopy and fluorescence-lifetime imaging (United States)

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


    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.

  18. Progress in reflectance confocal microscopy for imaging oral tissues in vivo (United States)

    Peterson, Gary; Zanoni, Daniella K.; Migliacci, Jocelyn; Cordova, Miguel; Rajadhyaksha, Milind; Patel, Snehal


    We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and "video-mosaicing", extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.

  19. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Rajesh Kumar


    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.

  1. 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.

  2. Analysis of mitochondrial mechanical dynamics using a confocal fluorescence microscope with a bent optical fibre. (United States)

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


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

  3. Confocal microscopy for astrocyte in vivo imaging: Recycle and reuse in microscopy (United States)

    Pérez-Alvarez, Alberto; Araque, Alfonso; Martín, Eduardo D.


    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

  4. Pseudoxanthoma elasticum and reflectance confocal microscopy: report of two affected young sisters

    Directory of Open Access Journals (Sweden)

    Victor Desmond Mandel


    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.

  5. Statistical strategies to reveal potential vibrational markers for in vivo analysis by confocal Raman spectroscopy (United States)

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


    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.

  6. Spatial Gradients in Particle Reinforced Polymers Characterized by X-Ray Attenuation and Laser Confocal Microscopy

    Energy Technology Data Exchange (ETDEWEB)



    The goal of this work is to develop techniques for measuring gradients in particle concentration within filled polymers, such as encapsulant. A high concentration of filler particles is added to such materials to tailor physical properties such as thermal expansion coefficient. Sedimentation and flow-induced migration of particles can produce concentration gradients that are most severe near material boundaries. Therefore, techniques for measuring local particle concentration should be accurate near boundaries. Particle gradients in an alumina-filled epoxy resin are measured with a spatial resolution of 0.2 mm using an x-ray beam attenuation technique, but an artifact related to the finite diameter of the beam reduces accuracy near the specimen's edge. Local particle concentration near an edge can be measured more reliably using microscopy coupled with image analysis. This is illustrated by measuring concentration profiles of glass particles having 40 {micro}m median diameter using images acquired by a confocal laser fluorescence microscope. The mean of the measured profiles of volume fraction agrees to better than 3% with the expected value, and the shape of the profiles agrees qualitatively with simple theory for sedimentation of monodisperse particles. Extending this microscopy technique to smaller, micron-scale filler particles used in encapsulant for microelectronic devices is illustrated by measuring the local concentration of an epoxy resin containing 0.41 volume fraction of silica.

  7. Confocal Raman study of aging process in diabetes mellitus human voluntaries (United States)

    Pereira, Liliane; Téllez Soto, Claudio Alberto; dos Santos, Laurita; Ali, Syed Mohammed; Fávero, Priscila Pereira; Martin, Airton A.


    Accumulation of AGEs [Advanced Glycation End - products] occurs slowly during the human aging process. However, its formation is accelerated in the presence of diabetes mellitus. In this paper, we perform a noninvasive analysis of glycation effect on human skin by in vivo confocal Raman spectroscopy. This technique uses a laser of 785 nm as excitation source and, by the inelastic scattering of light, it is possible to obtain information about the biochemical composition of the skin. Our aim in this work was to characterize the aging process resulting from the glycation process in a group of 10 Health Elderly Women (HEW) and 10 Diabetic Elderly Women (DEW). The Raman data were collected from the dermis at a depth of 70-130 microns. Through the theory of functional density (DFT) the bands positions of hydroxyproline, proline and AGEs (pentosidine and glucosepane) were calculated by using Gaussian 0.9 software. A molecular interpretation of changes in type I collagen was performed by the changes in the vibrational modes of the proline (P) and hydroxyproline (HP). The data analysis shows that the aging effects caused by glycation of proteins degrades type I collagen differently and leads to accelerated aging process.

  8. Probe-Based Confocal Laser Endomicroscopy for Indeterminate Biliary Strictures: Refinement of the Image Interpretation Classification

    Directory of Open Access Journals (Sweden)

    Michel Kahaleh


    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.

  9. Fluorescence confocal polarizing microscopy: Three-dimensional imaging of the director

    Indian Academy of Sciences (India)

    O D Lavrentovich


    Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by fluorescence confocal polarizing microscopy (FCPM). The technique employs the property of LC to orient the fluorescent dye molecules of anisometric shape, added in small quantities to the LC. In LC, smooth director deformations do not alter mass density of the material. Thus the density of dye is also uniform across the sample, except, perhaps, near the surfaces or at the cores of topological defects. In polarized light, the measured fluorescence signal is determined by the spatial orientation of the molecules rather than by dye concentration (as in regular biological samples stained with tissue-specific dyes). The contrast is enhanced when both excitation and detection of fluorescence light are performed in polarized light. This short review describes the essence of FCPM technique and illustrates some of its applications, including imaging of Frederiks electric-field induced effect in a nematic LC and defects such as dislocations in cholesteric LCs.

  10. PIV as a method for quantifying root cell growth and particle displacement in confocal images. (United States)

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


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

  11. Evaluation of human serum of severe rheumatoid arthritis by confocal Raman spectroscopy (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.


    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.

  12. Combining confocal laser scanning microscopy with serial section reconstruction in the study of adult neurogenesis.

    Directory of Open Access Journals (Sweden)

    Federico eLuzzati


    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.

  13. Resolution doubling using confocal microscopy via analogy with structured illumination microscopy (United States)

    Hayashi, Shinichi


    Structured illumination microscopy (SIM) is a super-resolution fluorescence microscopy with a 2-fold higher lateral resolution than conventional wide-field fluorescence (WF) microscopy. Confocal fluorescence (CF) microscopy has approximately the same optical cutoff frequency as SIM; however, the maximum theoretical increase in lateral resolution over that of WF is 1.4-fold with an infinitesimal pinhole diameter. Quantitative comparisons based on an analytical imaging formula revealed that modulation transfer functions (MTFs) of SIM reconstructed images before postprocessing are nearly identical to those of CF images recorded with an infinitesimal pinhole diameter. Here, we propose a new method using an adequate pinhole diameter combined with the use of an apodized Fourier inverse filter to increase the lateral resolution of CF images to as much as that SIM images without significant noise degradation in practice. Furthermore, the proposed method does not require a posteriori parameterization and has reproducibility. This approach can be easily applied to conventional laser scanning CF, spinning disk CF, and multiphoton microscopies.

  14. Inflammation in dry eye associated with rheumatoid arthritis: cytokine and in vivo confocal microscopy study. (United States)

    Villani, Edoardo; Galimberti, Daniela; Del Papa, Nicoletta; Nucci, Paolo; Ratiglia, Roberto


    The purpose of this research was to study ocular surface inflammation in relation to systemic disease activity in rheumatoid arthritis (RA) patients with or without secondary Sjögren's syndrome (SSII and non-SSII respectively). The study was conducted in two phases. In phase I, 12 patients with active RA SSII and 12 with active RA non-SSII were consecutively enrolled. Each completed an Ocular Surface Disease Index (OSDI) questionnaire and underwent a full eye exam and in vivo confocal microscopy examination of the cornea. Tear fluid samples were collected in sponges and analyzed for IL-1α, -6, and -8, and TNF-α. When RA activity was suppressed by systemic treatment the patients entered phase II of the study in which all of the phase I examinations were repeated. In RA SSII patients, OSDI, fluorescein staining dendritic cell density, and concentrations of IL-1α and IL-6 decreased significantly (P < 0.01) between phases I and II. Tear breakup time scores increased significantly. For RA non-SSII patients, there were no significant differences between phases I and II. Differences in the clinical, cellular and cytokine responsiveness to systemic RA treatments show that the ocular surface pathology is dissimilar for RA SSII and RA non-SSII patients.

  15. 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


    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.

  16. Visualizing epithelial expression of EGFR in vivo with distal scanning side-viewing confocal endomicroscope (United States)

    Duan, Xiyu; Li, Haijun; Zhou, Juan; Zhou, Quan; Oldham, Kenn R.; Wang, Thomas D.


    Confocal endomicroscopy is an emerging imaging technology that has recently been introduced into the clinic to instantaneously collect “optical biopsies” in vivo with histology-like quality. Here, we demonstrate a fast scanner located in the distal end of a side-viewing instrument using a compact lens assembly with numerical aperture of 0.5 to achieve a working distance of 100 μm and field-of-view of 300 × 400 μm2. The microelectromechanical systems (MEMS) mirror was designed based on the principle of parametric resonance and images at 5 frames per second. The instrument has a 4.2 mm outer diameter and 3 cm rigid length, and can pass through the biopsy channel of a medical endoscope. We achieved real time optical sections of NIR fluorescence with 0.87 μm lateral resolution, and were able to visualize in vivo binding of a Cy5.5-labeled peptide specific for EGFR to the cell surface of pre-cancerous colonocytes within the epithelium of dysplastic crypts in mouse colon. By performing targeted imaging with endomicroscopy, we can visualize molecular expression patterns in vivo that provide a biological basis for disease detection.

  17. Morphological confocal microscopy in arthropods and the enhancement of autofluorescence after proteinase K extraction. (United States)

    Valdecasas, Antonio G; Abad, Angela


    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.

  18. In vivo confocal microscopy of meibomian glands and palpebral conjunctiva in vernal keratoconjunctivitis

    Directory of Open Access Journals (Sweden)

    Qiaoling Wei


    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.

  19. Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy

    Directory of Open Access Journals (Sweden)

    Carla de Lamare Biolchini


    Full Text Available The blood flukes of mammals (Digenea: Schistosomatidae are among trematodes unique whose adult worms have separeted sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59% had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33% females had an incipient ovary (without cellular differentiation. At week four, 77.2% males presented testicular lobes with few germinative cells while 26% had developing tegumental tubercles. The immature ovary was observed in 69% females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.

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

    Directory of Open Access Journals (Sweden)

    KA. Al-Salihi


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

  1. Two-dimensional confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry (United States)

    Jun, Brian; Giarra, Matthew; Golz, Brian; Main, Russell; Vlachos, Pavlos


    We present a methodology to mitigate the major sources of error associated with two-dimensional confocal laser scanning microscopy (CLSM) images of nanoparticles flowing through a microfluidic channel. The correlation-based velocity measurements from CLSM images are subject to random error due to the Brownian motion of nanometer-sized tracer particles, and a bias error due to the formation of images by raster scanning. Here, we develop a novel ensemble phase correlation with dynamic optimal filter that maximizes the correlation strength, which diminishes the random error. In addition, we introduce an analytical model of CLSM measurement bias error correction due to two-dimensional image scanning of tracer particles. We tested our technique using both synthetic and experimental images of nanoparticles flowing through a microfluidic channel. We observed that our technique reduced the error by up to a factor of ten compared to ensemble standard cross correlation (SCC) for the images tested in the present work. Subsequently, we will assess our framework further, by interrogating nanoscale flow in the cell culture environment (transport within the lacunar-canalicular system) to demonstrate our ability to accurately resolve flow measurements in a biological system.

  2. Estimating pH at the Air/Water Interface with a Confocal Fluorescence Microscope. (United States)

    Yang, Haiya; Imanishi, Yasushi; Harata, Akira


    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.

  3. New data-driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume. (United States)

    Roselli, L; Paparella, F; Stanca, E; Basset, A


    Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex-shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex-shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data-driven formula-based approach to the calculation of biovolume of this complex-shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.

  4. Confocal ultrafast pump-probe spectroscopy: a new technique to explore nanoscale composites (United States)

    Virgili, Tersilla; Grancini, Giulia; Molotokaite, Egle; Suarez-Lopez, Inma; Rajendran, Sai Kiran; Liscio, Andrea; Palermo, Vincenzo; Lanzani, Guglielmo; Polli, Dario; Cerullo, Giulio


    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.

  5. Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing (United States)

    Lange-Asschenfeldt, Susanne; Bob, Adrienne; Terhorst, Dorothea; Ulrich, Martina; Fluhr, Joachim; Mendez, Gil; Roewert-Huber, Hans-Joachim; Stockfleth, Eggert; Lange-Asschenfeldt, Bernhard


    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.

  6. High-speed adaptive optics line scan confocal retinal imaging for human eye (United States)

    Wang, Xiaolin; Zhang, Yuhua


    Purpose Continuous and rapid eye movement causes significant intraframe distortion in adaptive optics high resolution retinal imaging. To minimize this artifact, we developed a high speed adaptive optics line scan confocal retinal imaging system. Methods A high speed line camera was employed to acquire retinal image and custom adaptive optics was developed to compensate the wave aberration of the human eye’s optics. The spatial resolution and signal to noise ratio were assessed in model eye and in living human eye. The improvement of imaging fidelity was estimated by reduction of intra-frame distortion of retinal images acquired in the living human eyes with frame rates at 30 frames/second (FPS), 100 FPS, and 200 FPS. Results The device produced retinal image with cellular level resolution at 200 FPS with a digitization of 512×512 pixels/frame in the living human eye. Cone photoreceptors in the central fovea and rod photoreceptors near the fovea were resolved in three human subjects in normal chorioretinal health. Compared with retinal images acquired at 30 FPS, the intra-frame distortion in images taken at 200 FPS was reduced by 50.9% to 79.7%. Conclusions We demonstrated the feasibility of acquiring high resolution retinal images in the living human eye at a speed that minimizes retinal motion artifact. This device may facilitate research involving subjects with nystagmus or unsteady fixation due to central vision loss. PMID:28257458

  7. Measurement of diffusion of fluorescent compounds and autofluorescence in skin in vivo using a confocal instrument (United States)

    Buttenschoen, K. K.; Sutton, E. E.; Daly, D.; Girkin, J. M.


    Using compact and affordable instrumentation based upon fluorescent confocal imaging we have tracked the movement of autofluorescent compounds through skin in near real time with high temporal and spatial resolution and sensitivity. The ability to measure the diffusion of compounds through skin with such resolution plays an important role for applications such as monitoring the penetration of pharmaceuticals applied to skin and assessing the integrity of the skin barrier. Several measurement methods exist, but they suffer from a number of problems such as being slow, expensive, non-portable and lacking sensitivity. To address these issues, we adapted a technique that we previously developed for tracking fluorescent compounds in the eye to measure the autofluorescence and the diffusion of externally applied fluorescent compounds in skin in vivo. Results are presented that show the change in autofluorescence of the volar forearm over the course of a week. We furthermore demonstrate the ability of the instrument to measure the diffusion speed and depth of externally applied fluorescent compounds both in healthy skin and after the skin barrier function has been perturbed. The instrument is currently being developed further for increased sensitivity and multi-wavelength excitation. We believe that the presented instrument is suitable for a large number of applications in fields such as assessment of damage to the skin barrier, development of topical and systemic medication and tracking the diffusion of fluorescent compounds through skin constructs as well as monitoring effects of skin products and general consumer products which may come into contact with the skin.

  8. 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)


    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.

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

    Directory of Open Access Journals (Sweden)

    Wei LI


    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.

  10. 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)


    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.)

  11. 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)


    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.

  12. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks (United States)

    Hames, Samuel C.; Ardigò, Marco; Soyer, H. Peter; Bradley, Andrew P.; Prow, Tarl W.


    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

  13. Confocal microscopy in the analysis of the etched nuclear particle tracks in polymers

    Energy Technology Data Exchange (ETDEWEB)

    Jakes, J.; Schraube, H. [GSF - Forschungszentrum fuer Umwelt und Gesundheit Neuherberg GmbH, Oberschleissheim (Germany). Inst. fuer Strahlenschutz; Gais, P. [GSF-Neuherberg (Germany). Inst. fuer Pathologie


    The possibility of the morphometric analysis of etched tracks, induced by protons and alpha particles in the organic polymer allyl diglycol carbonate (CR-39), using the confocal scanning laser microscope (CSLM), was studied. The detectors were investigated in two groups of irradiation experiments, namely: (a) irradiated with mono-energetic neutrons of energy 1.2 MeV, (b) exposed to the alpha radiation from {sup 222}Rn and its progeny. Both groups were irradiated at normal incidence. Radiation-induced latent tracks were electrochemically etched, and their morphometric parameters were investigated in the reflection mode by using the 488-nm spectral line of an argon ion laser. A constant number of up to 200 optical sections in Z-scan mode was taken through each selected etched track at vertical spacings of 0.642 {mu}m. Successive reconstructions of Z-sections were used to determine the following parameters: the mead radius of the opening channel, the maximum diameter and the length of the track, and the angle of the track wall to the surface of the sample. (author).

  14. Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy. (United States)

    Ghosh, Shirsendu; Nandi, Somen; Ghosh, Catherine; Bhattacharyya, Kankan


    Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time () of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps).

  15. Numerical descriptors for the analysis of wear surfaces using laser scanning confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Anamalay, R.V. [Dept. of Mechanical Engineering, Monash Univ., Clayton, VIC (Australia); Kirk, T.B. [Dept. of Mechanical Engineering, Monash Univ., Clayton, VIC (Australia); Panzera, D. [Dept. of Mechanical Engineering, Monash Univ., Clayton, VIC (Australia)


    Machinery wear is a major cost to industry and its minimisation would result in significant savings. In order to do this, it is important to understand the mechanisms of wear. Techniques have to be developed to enable the detailed measurement and analysis of wear surfaces. Conventional methods of surface measurement have involved profilometers. Profilometers, however, have severe limitations in terms of the surface features detectable and difficulties arise when 3D data sets of surfaces are required. Alternative methods that have been explored are stereo microscopy, reflected light interference microscopy (RLIM) and scanning electron microscopy. But these methods have proven to be severely limited either by the depth of field that can be obtained, difficulties associated with obtaining and interpreting images or the prohibitive costs involved. Laser scanning confocal microscopes (LSCM), however, have the capabilities to record surface features quickly and conveniently. LSCM techniques allow the determination and analysis of the true surface topography of a sample surface. LSCM has no depth of field limitations, is significantly cheaper than scanning electron microscopy, requires minimal sample preparation and provides images of sufficient quality for engineering purposes. Better measurement techniques facilitate the use of new surface parameters, in addition to the traditional parameters (all of which can be measured using LSCM techniques). In this paper, parameters developed for the measurement and analysis of surfaces using LSCM techniques are discussed. A comparison is made between surface analysis using LSCM techniques and conventional profilometer methods. (orig.)

  16. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study (United States)

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


    Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications. PMID:27200376

  17. Corneal Confocal Microscopy Detects Neuropathy in Subjects With Impaired Glucose Tolerance (United States)

    Asghar, Omar; Petropoulos, Ioannis N.; Alam, Uazman; Jones, Wendy; Jeziorska, Maria; Marshall, Andrew; Ponirakis, Georgios; Fadavi, Hassan; Boulton, Andrew J.M.; Tavakoli, Mitra


    OBJECTIVE Impaired glucose tolerance (IGT) represents one of the earliest stages of glucose dysregulation and is associated with macrovascular disease, retinopathy, and microalbuminuria, but whether IGT causes neuropathy is unclear. RESEARCH DESIGN AND METHODS Thirty-seven subjects with IGT and 20 age-matched control subjects underwent a comprehensive evaluation of neuropathy by assessing symptoms, neurological deficits, nerve conduction studies, quantitative sensory testing, heart rate variability deep breathing (HRVdb), skin biopsy, and corneal confocal microscopy (CCM). RESULTS Subjects with IGT had a significantly increased neuropathy symptom profile (P < 0.001), McGill pain index (P < 0.001), neuropathy disability score (P = 0.001), vibration perception threshold (P = 0.002), warm threshold (P = 0.006), and cool threshold (P = 0.03), with a reduction in intraepidermal nerve fiber density (P = 0.03), corneal nerve fiber density (P < 0.001), corneal nerve branch density (P = 0.002), and corneal nerve fiber length (P = 0.05). No significant difference was found in sensory and motor nerve amplitude and conduction velocity or HRVdb. CONCLUSIONS Subjects with IGT have evidence of neuropathy, particularly small-fiber damage, which can be detected using skin biopsy and CCM. PMID:24969581

  18. Automated Segmentation of Skin Strata in Reflectance Confocal Microscopy Depth Stacks.

    Directory of Open Access Journals (Sweden)

    Samuel C Hames

    Full Text Available 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.

  19. Compound Cellular Imaging of Laser Scanning Confocal Microscopy by Using Gold Nanoparticles and Dyes

    Directory of Open Access Journals (Sweden)

    Jiunn-Woei Liaw


    Full Text Available Combining the scattered light of gold nanoparticles (GNPs and the fluorescence of dye molecules, a compound cellular imaging of laser scanning confocal microscopy (LSCM is obtained. The human breast cancer cell line (MDA-MB-435S, BCRC 60429 is used for experiment. These cells are incubated with a glucose medium containing GNPs for 26 hours, and then are stained by Prodium Iodide (PI for their nuclei. By using a single laser to illuminate these cells and adjusting the ranges of two bandpass filters for the detection, the scattered light from the GNPs and the fluorescence of PI can be induced simultaneously, but be detected separately without crosstalk. Furthermore, a compound cellular image can be obtained by merging the two images of the expressions of GNP and PI together. From the TEM images of these cells, it is observed that GNPs are aggregated in the vesicles of the cytoplasm due to the cell’s endocytosis. The aggregation of GNPs makes the surface plasmon resonance band of GNPs broadened, so that strong scattered light from GNPs can be generated by the illumination of different-wavelength lasers (458, 488, 514, 561, and 633 nm.

  20. Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy (United States)

    Grimm, Melissa J.; D'Auria, Anthony C.; Segal, Brahm H.


    The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy. PMID:25045941