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Sample records for confocal laser scanning

  1. Spectrally encoded confocal scanning laser ophthalmoscope

    NASA Astrophysics Data System (ADS)

    Tao, Yuankai K.; Izatt, Joseph A.

    2010-02-01

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

  2. Optimization of confocal scanning laser ophthalmoscope design

    PubMed Central

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

    2013-01-01

    Abstract. 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. PMID:23864013

  3. Three-dimensional scanning confocal laser microscope

    DOEpatents

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

    1999-01-01

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

  4. CONFOCAL LASER SCANNING MICROSCOPY OF RAT FOLLICLE DEVELOPMENT

    EPA Science Inventory

    This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pr...

  5. Confocal scanning beam laser microscope/macroscope: applications in fluorescence

    NASA Astrophysics Data System (ADS)

    Dixon, Arthur E.; Damaskinos, Savvas; Ribes, Alfonso

    1996-03-01

    A new confocal scanning beam laser microscope/macroscope is described that combines the rapid scan of a scanning beam laser microscope with the large specimen capability of a scanning stage microscope. This instrument combines an infinity-corrected confocal scanning laser microscope with a scanning laser macroscope that uses a telecentric f*(Theta) laser scan lens to produce a confocal imaging system with a resolution of 0.25 microns at a field of view of 25 microns and 5 microns at a field of view of 75,000 microns. The frame rate is 5 seconds per frame for a 512 by 512 pixel image, and 25 seconds for a 2048 by 2048 pixel image. Applications in fluorescence are discussed that focus on two important advantages of the instrument over a confocal scanning laser microscope: an extremely wide range of magnification, and the ability to image very large specimens. Examples are presented of fluorescence and reflected-light images of high quality printing, fluorescence images of latent fingerprints, packaging foam, and confocal autofluorescence images of a cricket.

  6. A New Multichannel Spectral Imaging Laser Scanning Confocal Microscope

    PubMed Central

    Zhang, Yunhai; Hu, Bian; Dai, Yakang; Yang, Haomin; Huang, Wei; Xue, Xiaojun; Li, Fazhi; Zhang, Xin; Jiang, Chenyu; Gao, Fei; Chang, Jian

    2013-01-01

    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. PMID:23585775

  7. A handheld laser scanning confocal reflectance imaging–confocal Raman microspectroscopy system

    PubMed Central

    Patil, Chetan A.; Arrasmith, Christopher L.; Mackanos, Mark A.; Dickensheets, David L.; Mahadevan-Jansen, Anita

    2012-01-01

    Confocal reflectance microscopy and confocal Raman spectroscopy have shown potential for non-destructive analysis of samples at micron-scale resolutions. Current studies utilizing these techniques often employ large bench-top microscopes, and are not suited for use outside of laboratory settings. We have developed a microscope which combines laser scanning confocal reflectance imaging and confocal Raman spectroscopy into a compact handheld probe that is capable of high-resolution imaging and spectroscopy in a variety of settings. The compact size of the probe is largely due to the use of a MEMS mirror for beam scanning. The probe is capable of axial resolutions of up to 4 μm for the confocal imaging channel and 10 μm for the confocal Raman spectroscopy channel. Here, we report instrument design, characterize optical performance, and provide images and spectra from normal skin to demonstrate the instrument’s capabilities for clinical diagnostics. PMID:22435097

  8. FOOD SURFACE TEXTURE MEASUREMENT USING REFLECTIVE CONFOCAL LASER SCANNING MICROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Confocal laser scanning microscopy (CLSM) was used in the reflection mode to characterize the surface texture (roughness) of sliced food surfaces. Sandpapers of grit size between 150 and 600 were used as the height reference to standardize the CLSM hardware settings. Sandpaper particle sizes were v...

  9. Imaging retinal densitometry with a confocal Scanning Laser Ophthalmoscope.

    PubMed

    van Norren, D; van de Kraats, J

    1989-01-01

    We describe a novel use of the Scanning Laser Ophthalmoscope (SLO), viz. as an imaging retinal densitometer. In our SLO a helium-neon or an argon laser beam is moved in a raster pattern over the retina; the reflected light is descanned (confocal SLO) and collected by a photomultiplier. Images of the fundus subtending 22 by 18 deg are displayed on a TV monitor. Single frames taken with 514 nm light were stored in a computer in arrays of 256 by 256 pixels and density differences between dark adapted and bleached images were calculated. With a full bleach density differences of about 0.35 were found in the center of the fovea; at retinal eccentricities of 15-20 deg we found 0.15. After selective bleaching with 633 nm light substantial density differences were only seen in the foveal area. We conclude that the confocal SLO is a very suitable instrument for imaging fundus reflectometry. PMID:2631402

  10. Photobleaching property of confocal laser scanning microscopy with masked illumination

    NASA Astrophysics Data System (ADS)

    Kim, DongUk; Moon, Sucbei; Song, Hoseong; Yang, Wenzhong; Kim, Dug Y.

    2010-02-01

    Confocal laser scanning microscopy (CLSM) has become the tool of choice for high-contrast fluorescence imaging in the study of the three-dimensional and dynamic properties of biological system. However, the high cost and complexity of commercial CLSMs urges many researchers to individually develop low cost and flexible confocal microscopy systems. The high speed scanner is an influential factor in terms of cost and system complexity. Resonant galvo scanners at several kHz have been commonly used in custom-built CLSMs. However, during the repeated illumination for live cell imaging or 3D image formation, photobleaching and image distortion occurred at the edges of the scan field may be more serious than the center due to an inherent property (e.g. sinusoidal angular velocity) of the scan mirror. Usually, no data is acquired at the edges due to large image distortion but the excitation beam is still illuminated. Here, we present the photobleaching property of CLSM with masked illumination, a simple and low cost method, to exclude the unintended excitation illumination at the edges. The mask with a square hole in its center is disposed at the image plane between the scan lens and the tube lens in order to decrease photobleaching and image distortion at the edges. The excluded illumination section is used as the black level of the detected signals for a signal quantizing step. Finally, we demonstrated the reduced photobleaching at the edges on a single layer of fluorescent beads and real-time image acquisition without a standard composite video signal by using a frame grabber.

  11. Confocal laser scanning microscopy with spatiotemporal structured illumination.

    PubMed

    Gao, Peng; Nienhaus, G Ulrich

    2016-03-15

    Confocal laser scanning microscopy (CLSM), which is widely utilized in the biological and biomedical sciences, is limited in spatial resolution due to diffraction to about half the light wavelength. Here we have combined structured illumination with CLSM to enhance its spatial resolution. To this end, we have used a spatial light modulator (SLM) to generate fringe patterns of different orientations and phase shifts in the excitation spot without any mechanical movement. We have achieved 1.8 and 1.7 times enhanced lateral and axial resolutions, respectively, by synthesizing the object spectrum along different illumination directions. This technique is thus a promising tool for high-resolution morphological or fluorescence imaging, especially in deep tissue. PMID:26977667

  12. Diffusion of photoacid generators by laser scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

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

    1998-06-01

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

  13. Automatic analysis for neuron by confocal laser scanning microscope

    NASA Astrophysics Data System (ADS)

    Satou, Kouhei; Aoki, Yoshimitsu; Mataga, Nobuko; Hensh, Takao K.; Taki, Katuhiko

    2005-12-01

    The aim of this study is to develop a system that recognizes both the macro- and microscopic configurations of nerve cells and automatically performs the necessary 3-D measurements and functional classification of spines. The acquisition of 3-D images of cranial nerves has been enabled by the use of a confocal laser scanning microscope, although the highly accurate 3-D measurements of the microscopic structures of cranial nerves and their classification based on their configurations have not yet been accomplished. In this study, in order to obtain highly accurate measurements of the microscopic structures of cranial nerves, existing positions of spines were predicted by the 2-D image processing of tomographic images. Next, based on the positions that were predicted on the 2-D images, the positions and configurations of the spines were determined more accurately by 3-D image processing of the volume data. We report the successful construction of an automatic analysis system that uses a coarse-to-fine technique to analyze the microscopic structures of cranial nerves with high speed and accuracy by combining 2-D and 3-D image analyses.

  14. Managing multiple image stacks from confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Zerbe, Joerg; Goetze, Christian H.; Zuschratter, Werner

    1999-05-01

    A major goal in neuroanatomy is to obtain precise information about the functional organization of neuronal assemblies and their interconnections. Therefore, the analysis of histological sections frequently requires high resolution images in combination with an overview about the structure. To overcome this conflict we have previously introduced a software for the automatic acquisition of multiple image stacks (3D-MISA) in confocal laser scanning microscopy. Here, we describe a Windows NT based software for fast and easy navigation through the multiple images stacks (MIS-browser), the visualization of individual channels and layers and the selection of user defined subregions. In addition, the MIS browser provides useful tools for the visualization and evaluation of the datavolume, as for instance brightness and contrast corrections of individual layers and channels. Moreover, it includes a maximum intensity projection, panning and zoom in/out functions within selected channels or focal planes (x/y) and tracking along the z-axis. The import module accepts any tiff-format and reconstructs the original image arrangement after the user has defined the sequence of images in x/y and z and the number of channels. The implemented export module allows storage of user defined subregions (new single image stacks) for further 3D-reconstruction and evaluation.

  15. Confocal laser scanning microscopy in study of bone calcification

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  16. The use of laser scanning confocal microscopy (LSCM) in materials science.

    PubMed

    Hovis, D B; Heuer, A H

    2010-12-01

    Laser scanning confocal microscopes are essential and ubiquitous tools in the biological, biochemical and biomedical sciences, and play a similar role to scanning electron microscopes in materials science. However, modern laser scanning confocal microscopes have a number of advantages for the study of materials, in addition to their obvious uses for high resolution reflected and transmitted light optical microscopy. In this paper, we provide several examples that exploit the laser scanning confocal microscope's capabilities of pseudo-infinite depth of field imaging, topographic imaging, photo-stimulated luminescence imaging and Raman spectroscopic imaging. PMID:21077878

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1996-02-01

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

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

    SciTech Connect

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

    2008-11-17

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

  20. Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

    PubMed

    Paddock, Stephen W; Eliceiri, Kevin W

    2014-01-01

    Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques. PMID:24052346

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES USING CONFOCAL LASER SCANNING MICROSCOPY

    EPA Science Inventory

    MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES USING CONFOCAL LASER SCANNING MICROSCOPY

    Robert M. Zucker Susan C. Jeffery and Sally D. Perreault

    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Prot...

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

    EPA Science Inventory

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

  5. Visualization and quantification of dentin structure using confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Kimura, Yuichi; Wilder-Smith, Petra B.; Krasieva, Tatiana B.; Arrastia-Jitosho, Anna-Marie A.; Liaw, Lih-Huei L.; Matsumoto, Koukichi

    1997-07-01

    Dentin was visualized using a new fluorescence technique and confocal laser scanning microscopy. Thirty extracted human teeth showing no clinical signs of caries were investigated. All teeth were horizontally sectioned to approximately 200 micrometers thickness and sections were subjected to different pretreatment conditions as follows: vacuum only, ultrasonication only, sodium hypochlorite only, sodium hypochlorite and vacuum, sodium hypochlorite and ultrasonication, and a combination of sodium hypochlorite, vacuum, and ultrasonication. Some samples were left untreated to serve as control. Following pretreatment, rhodamine 123 fluorescent dye was used for staining at concentrations ranging from 10-3 to 10-7 M for 1 to 24 h at pH 6.0, 6.5, or 7.4. Optical staining occurred at pH 7.4 and concentrations >= 10-5 M over 3 h or longer. Surface images obtained using confocal laser scanning microscopy were similar to those observed by scanning electron microscopy without the need for sample- altering conventional scanning electron microscope preparation techniques. Subsurface imaging to a depth of approximately 60 micrometers was achieved using confocal laser microscope techniques. This fluorescence technique offers a useful new alternative for visualization and quantification of dentin.

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

    SciTech Connect

    White, T

    2007-05-08

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

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

    PubMed

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

    2006-04-01

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

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

    PubMed

    Wedekind, P; Kubitscheck, U; Peters, R

    1994-10-01

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

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

    PubMed

    Meller, Karl; Theiss, Carsten

    2006-03-01

    We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 degrees C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton. PMID:16360280

  10. 3-D reconstruction of neurons from multichannel confocal laser scanning image series.

    PubMed

    Wouterlood, Floris G

    2014-01-01

    A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. Scanning of a specimen, with stepwise axial (Z-) movement of the stage in between each scan, produces Z-series of confocal images of a tissue volume, which then can be used to 3-D reconstruct structures of interest. The operator first configures separate channels (e.g., laser, filters, and detector settings) for each applied fluorochrome and then acquires Z-series of confocal images: one series per channel. Channel signal separation is extremely important. Measures to avoid bleaching are vital. Post-acquisition deconvolution of the image series is often performed to increase resolution before 3-D reconstruction takes place. In the 3-D reconstruction programs described in this unit, reconstructions can be inspected in real time from any viewing angle. By altering viewing angles and by switching channels off and on, the spatial relationships of 3-D-reconstructed structures with respect to structures visualized in other channels can be studied. Since each brand of CLSM, computer program, and 3-D reconstruction package has its own proprietary set of procedures, a general approach is provided in this protocol wherever possible. PMID:24723320

  11. Confocal Laser Microscope Scanning Applied To Three-Dimensional Studies Of Biological Specimens.

    NASA Astrophysics Data System (ADS)

    Franksson, Olof; Liljeborg, Anders; Carlsson, Kjell; Forsgren, Per-Ola

    1987-08-01

    The depth-discriminating property of confocal laser microscope scanners can be used to record the three-dimensional structure of specimens. A number of thin sections (approx. 1 μm thick) can be recorded by a repeated process of image scanning and refocusing of the microscope. We have used a confocal microscope scanner in a number of feasibility studies to investigate its possibilities and limitations. It has proved to be well suited for examining fluorescent specimens with a complicated three-dimensional structure, such as nerve cells. It has also been used to study orchid seeds, as well as cell colonies, greatly facilitating evaluation of such specimens. Scanning of the specimens is performed by a focused laser beam that is deflected by rotating mirrors, and the reflected or fluorescent light from the specimen is detected. The specimen thus remains stationary during image scanning, and is only moved stepwise in the vertical direction for refocusing between successive sections. The scanned images consist of 256*256 or 512*512 pixels, each pixel containing 8 bits of data. After a scanning session a large number of digital images, representing consecutive sections of the specimen, are stored on a disk memory. In a typical case 200 such 256*256 images are stored. To display and process this information in a meaningful way requires both appropriate software and a powerful computer. The computer used is a 32-bits minicomputer equipped with an array processor (FPS 100). The necessary software was developed at our department.

  12. Investigation of phosphatidylcholine enhancing FITC-insulin across buccal mucosa by confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Tian, Weiqun; Su, Li; Zeng, Shaoqun; Luo, Qingming; Gao, Qiuhua; Xu, Huibi

    2002-04-01

    The aim was to characterize the transport of fluorescein isothiocyanate (FITC)-labeled dextran and insulin with different resoluble compounds for peptides and proteins through buccal mucosa. The penetration rate of insulin molecules through porcine buccal mucosa (a nonkeratinized epithelium, comparable to human buccal mucosa) was investigated by measuring transbuccal fluxes and by analyzing the distribution of the fluorescent probe in the rabbit buccal mucosa epithelium, using confocal laser scanning microscopy for visualizing permeation pathways. The confocal images of the distribution pattern of FITC-dextran and FITC-insulin showed that the paracellular route is the major pathway of FITC-dextran through buccal mucosa epithelium, the intra-cellular route is the major pathway of FITC-insulin through buccal mucosa epithelium. The permeation rate can be increased by co-administration of soybean phosphatidylcholine (SPC).

  13. Use of the confocal laser scanning microscope in studies on the developmental biology of marine crustaceans.

    PubMed

    Buttino, Isabella; Ianora, Adrianna; Carotenuto, Ylenia; Zupo, Valerio; Miralto, Antonio

    2003-03-01

    Confocal Laser Scanning Microscope techniques have been applied to study the developmental biology of marine copepods and decapod larvae. The lipophylic probes DiI and DiOC(6) were used to study both the external and internal morphology of these crustaceans, whereas the same DiOC(6) and the specific nuclear probe Hoechst 33342 were used to study embryonic development of copepods in vivo. To distinguish viable from non-viable copepod embryos, the vital dye dichlorodihydrofluorescein diacetate (H(2)DCFDA) was used. Major advantages and difficulties in the use of these non-invasive techniques in studies of the reproductive biology of marine crustaceans are discussed. PMID:12567403

  14. Ti-6Al-4V electron beam weld qualification using laser scanning confocal microscopy

    SciTech Connect

    Wanjara, P. . E-mail: priti.wanjara@cnrc-nrc.gc.ca; Brochu, M.; Jahazi, M.

    2005-03-15

    Processing conditions for manufacturing Ti-6Al-4V components by welding using an electron beam source are known to influence the transformation microstructure in the narrow fusion and heat-affected zones of the weld region. This work examined the effect of multiple-sequence welding on the characteristics of the transformed beta microstructure, using laser scanning confocal microscopy to resolve the Widmanstaetten alpha-beta structure in the fusion zone. The evolution in the alpha interlamellar spacing and plate thickness with processing was then related to microhardness measurements in the weld region.

  15. Observation of dendritic cell morphology under light, phase-contrast or confocal laser scanning microscopy.

    PubMed

    Tan, Yuen-Fen; Leong, Chooi-Fun; Cheong, Soon-Keng

    2010-12-01

    Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs. PMID:21329180

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

    PubMed Central

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

    2016-01-01

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

  17. Scanning computed confocal imager

    DOEpatents

    George, John S.

    2000-03-14

    There is provided a confocal imager comprising a light source emitting a light, with a light modulator in optical communication with the light source for varying the spatial and temporal pattern of the light. A beam splitter receives the scanned light and direct the scanned light onto a target and pass light reflected from the target to a video capturing device for receiving the reflected light and transferring a digital image of the reflected light to a computer for creating a virtual aperture and outputting the digital image. In a transmissive mode of operation the invention omits the beam splitter means and captures light passed through the target.

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

    NASA Astrophysics Data System (ADS)

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

    2010-08-01

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

  19. Plasmon resonance and the imaging of metal-impregnated neurons with the laser scanning confocal microscope

    PubMed Central

    Thompson, Karen J; Harley, Cynthia M; Barthel, Grant M; Sanders, Mark A; Mesce, Karen A

    2015-01-01

    The staining of neurons with silver began in the 1800s, but until now the great resolving power of the laser scanning confocal microscope has not been utilized to capture the in-focus and three-dimensional cytoarchitecture of metal-impregnated cells. Here, we demonstrate how spectral confocal microscopy, typically reserved for fluorescent imaging, can be used to visualize metal-labeled tissues. This imaging does not involve the reflectance of metal particles, but rather the excitation of silver (or gold) nanoparticles and their putative surface plasmon resonance. To induce such resonance, silver or gold particles were excited with visible-wavelength laser lines (561 or 640 nm), and the maximal emission signal was collected at a shorter wavelength (i.e., higher energy state). Because the surface plasmon resonances of noble metal nanoparticles offer a superior optical signal and do not photobleach, our novel protocol holds enormous promise of a rebirth and further development of silver- and gold-based cell labeling protocols. DOI: http://dx.doi.org/10.7554/eLife.09388.001 PMID:26670545

  20. Real time confocal laser scanning microscopy: Potential applications in space medicine and cell biology

    NASA Astrophysics Data System (ADS)

    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.

  1. Further study of trichosanthin's effect on mouse embryos with confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Hui; Zhang, Chunyang; Ma, Hui; Chen, Die Yan

    2001-09-01

    Trichosanthin(TCS), a ribosome inactivating protein extracted from the root tuber of a traditional Chinese medicine herb Tian Huo Fen(THF), possessed abortifacient, anti-tumor and anti-human immunodeficiency virus(HIV) activities. For centuries in China, THF has been used as an effective folk medicine to terminate early and midtrimester pregnancies and to treat ectopic pregnancies, hydatidiform moles and trophoblastic tumor. We observed the changes in reactive oxygen species and intracellular calcium in mouse embryos induced by TCS with confocal laser scanning microscopy in combination with the fluorescene diacetate (DCFHDA) and Fluo-3-AM. The results indicated that TCS induced increase in intracellular calcium and production of reactive oxygen species in mouse embryos , and TCS inhibited the development of mouse embryos effectively. Mouse embryos of different developmental stages before implantation are used in the experiments. This provides new insight into mechanism for abortifacient activity of TCS.

  2. Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopy.

    PubMed

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

    2010-03-01

    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. PMID:19725070

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

    PubMed Central

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

    2016-01-01

    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. PMID:26950118

  4. Starch/carrageenan/milk proteins interactions studied using multiple staining and Confocal Laser Scanning Microscopy.

    PubMed

    Matignon, A; Moulin, G; Barey, P; Desprairies, M; Mauduit, S; Sieffermann, J M; Michon, C

    2014-01-01

    This study focused on the effects of the interactions between modified waxy maize starch, kappa carrageenan and skim milk on the microstructure of their mixed systems using Confocal Laser Scanning Microscopy (CLSM). A multiple staining of the components was set up with a view to improving starch covalent staining. In starch/carrageenan pasted mixtures, carrageenan was found to adsorb on and penetrate slightly into the starch granules, whereas no interactions were observed between starch and milk proteins. In ternary mixtures, interactions between starch granules and carrageenan were no longer observed, even when milk proteins were added after starch swelling in the carrageenan solution, thus showing preferential interactions between carrageenan/milk proteins in comparison to carrageenan/starch granules. Modifying the blending order of the components led to microstructure differences depending on several parameters such as starch/carrageenan interactions, carrageenan/milk proteins network structure, level of starch granules disruption and amylopectin contribution to the microstructure. PMID:24274517

  5. An alternative method of promoter assessment by confocal laser scanning microscopy.

    PubMed

    Sahoo, Dipak K; Ranjan, Rajiv; Kumar, Deepak; Kumar, Alok; Sahoo, Bhabani S; Raha, Sumita; Maiti, Indu B; Dey, Nrisingha

    2009-10-01

    A rapid and useful method of promoter activity analysis using techniques of confocal laser scanning microscopy (CLSM) is described in the present study. The activities of some pararetroviral promoters such as CaMV35S (Cauliflower mosaic virus), FMVSgt3 (Figwort mosaic virus sub-genomic transcript) and MMVFLt12 (Mirabilis mosaic virus full-length transcript) coupled to GFP (green fluorescent protein) and GUS (beta-glucuronidase) reporter genes were determined simultaneously by the CLSM technique and other available conventional methods for reporter gene assay based on relevant biochemical and molecular approaches. Consistent and comparable results obtained by CLSM as well as by other conventional assay methods confirm the effectiveness of the CLSM approach for assessment of promoter activity. Hence the CLSM method can be suggested as an alternative way for promoter analysis on the basis of high throughput. PMID:19540268

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

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

    SciTech Connect

    Onishi, Celia T.; Shimizu, Ichiko

    2004-01-02

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

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

    PubMed

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

    2016-01-01

    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. PMID:26950118

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

    NASA Astrophysics Data System (ADS)

    Leue, Martin; Gerke, Horst H.

    2016-04-01

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

  10. Handheld histology-equivalent sectioning laser-scanning confocal optical microscope for interventional imaging.

    PubMed

    Kumar, Karthik; Avritscher, Rony; Wang, Youmin; Lane, Nancy; Madoff, David C; Yu, Tse-Kuan; Uhr, Jonathan W; Zhang, Xiaojing

    2010-04-01

    A handheld, forward-imaging, laser-scanning confocal microscope (LSCM) demonstrating optical sectioning comparable with microtome slice thicknesses in conventional histology, targeted towards interventional imaging, is reported. Fast raster scanning (approximately 2.5 kHz line scan rate, 3.0-5.0 frames per second) was provided by a 2-axis microelectromechanical system (MEMS) scanning mirror fabricated by a method compatible with complementary metal-oxide-semiconductor (CMOS) processing. Cost-effective rapid-prototyped packaging combined the MEMS mirror with micro-optical components into a probe with 18 mm outer diameter and 54 mm rigid length. ZEMAX optical design simulations indicate the ability of the handheld optical system to obtain lateral resolution of 0.31 and axial resolution of 2.85 microm. Lateral and axial resolutions are experimentally measured at 0.5 microm and 4.2 microm respectively, with field of view of 200 x 125 microm. Results of reflectance imaging of ex vivo swine liver, and fluorescence imaging of the expression of cytokeratin and mammaglobin tumor biomarkers in epithelial human breast tissue from metastatic breast cancer patients are presented. The results indicate that inexpensive, portable handheld optical microscopy tools based on silicon micromirror technologies could be important in interventional imaging, complementing existing coarse-resolution techniques to improve the efficacy of disease diagnosis, image-guided excisional microsurgery, and monitored photodynamic therapy. PMID:20012209

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

    PubMed Central

    Cardinale, Massimiliano

    2014-01-01

    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. PMID:24639675

  12. Spectral imaging technique for retinal perfusion detection using confocal scanning laser ophthalmoscopy

    NASA Astrophysics Data System (ADS)

    Rasta, Seyed Hossein; Manivannan, Ayyakkannu; Sharp, Peter F.

    2012-11-01

    To evaluate retinal perfusion in the human eye, a dual-wavelength confocal scanning laser ophthalmoscope (cSLO) was developed that provides spectral imaging of the fundus using a combination of red (670 nm) and near-infrared (810 nm) wavelengths. The image of the ocular fundus was analyzed to find out if quantitative measurements of the reflectivity of tissue permit assessment of the oxygen perfusion of tissue. We explored problems that affect the reproducibility of patient measurements such as non-uniformity errors on the image. For the first time, an image processing technique was designed and used to minimize the errors of oxygen saturation measurements by illumination correction in retina wide field by increasing SNR. Retinal images were taken from healthy and diabetic retinopathy eyes using the cSLO with a confocal aperture of 100 μm. The ratio image (RI) of red/IR, as oxygen saturation (SO2) index, was calculated for normal eyes. The image correction technique improved the reproducibility of the measurements. Average RI intensity variation of healthy retina tissue was determined within a range of about 5.5%. The capability of the new technique to discriminate oxygenation levels of retinal artery and vein was successfully demonstrated and showed good promise in the diagnosis of the perfused retina.

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

    PubMed Central

    Nikhil, Vineeta; Singh, Renuka

    2013-01-01

    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. PMID:23956528

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

    PubMed

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

    2014-05-01

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

  15. Evaluation of confocal laser scanning microscopy for enumeration of virus-like particles in aquatic systems

    PubMed Central

    Agis, Martin; Luef, Birgit

    2016-01-01

    Abstract Abundances of virus-like particles (VLPs, mostly bacteriophages) are high in aquatic environments; therefore, techniques for precise enumeration are essential in ecological monitoring. VLPs were determined after staining with SYBR Gold by conventional epifluorescence microscopy and compared to enumerations performed by confocal laser scanning microscopy (CLSM). In order to assess the potential of CLSM for viral direct counts (VDCs), we processed samples from different freshwater and marine systems. Optical sectioning by CLSM and production of an overlay picture of multiple scans enables the often uneven whole investigated filter area to be brought to the plane of focus. This allows for subsequent image analysis of digitally created high-quality images. Another advantage using the CLSM was that the short spot excitation of the stain via laser beam minimized fading of the stain. The VDC results show that there is no significant difference between the two methods. Regarding the known difficulties of viral abundance estimates on particulate material, CLSM was further applied to enumerate VLPs on a small set of marine transparent exopolymeric particles sampled from the Atlantic Ocean. Our data suggest that CLSM is a useful tool to count viruses in water samples as well as attached to certain types of aquatic aggregates. PMID:23108709

  16. Three-dimensional reconstruction of paramecium primaurelia oral apparatus through confocal laser scanning optical microscopy

    NASA Astrophysics Data System (ADS)

    Beltrame, Francesco; Ramoino, Paola; Fato, Marco; Delmonte Corrado, Maria U.; Marcenaro, Giampiero; Crippa Franceschi, Tina

    1992-06-01

    Studies on the complementary mating types of Paramecium primaurelia (Protozoa, Ciliates) have shown that cell lines which differ from each other in mating type expression are characterized by different cell contents, organization, and physiology. Referring to these differences and to the differential rates of food vacuole formation, oral apparatuses of the two mating type cells are assumed to possibly differ from each other in some traits, such as, for instance, in their lengths. In our work, the highly organized oral structures are analyzed by means of a laser scanning confocal optical microscope (CLSM), which provides their 3-D visualization and measurement. The extraction of the 3-D intrinsic information related to the biological objects under investigation can be in turn related to their functional state, according to the classical paradigm of structure to function relationships identification. In our experiments, we acquired different data sets. These are optical slices of the biological sample under investigation, acquired in a confocal situation, through epi-illumination, in reflection, and, for comparison with conventional microscopy, 2-D images acquired via a standard TV camera coupled to the microscope itself. Our CLSM system is equipped with a laser beam at 488 and 514 nm and the data have been acquired with various steps of optical slicing, ranging from .04 to .25 micrometers. The volumes obtained by piling-up the slices are rendered through different techniques, some of them directly implemented on the workstation controlling the CLSM system, some of them on a SUN SPARC station 1, where the original data were transferred via an Ethernet link. In this last instance, original software has been developed for the visualization and animation of the 3-D structures, running under UNIX and X-Window, according to a ray-tracing algorithm.

  17. Comparison between optical-resolution photoacoustic microscopy and confocal laser scanning microscopy for turbid sample imaging

    NASA Astrophysics Data System (ADS)

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

    Optical-resolution photoacoustic microscopy (ORPAM) in theory provides lateral resolution equivalent to the optical diffraction limit. Scattering media, such as biological turbid media, attenuates the optical signal and also alters the diffraction-limited spot size of the focused beam. The ORPAM signal is generated only from a small voxel in scattering media with dimensions equivalent to the laser spot size after passing through scattering layers and is detected by an acoustic transducer, which is not affected by optical scattering. Thus, both ORPAM and confocal laser scanning microscopy (CLSM) reject scattered light. A multimodal optical microscopy platform that includes ORPAM and CLSM was constructed, and the lateral resolution of both modes was measured using patterned thin metal film with and without a scattering barrier. The effect of scattering media on the lateral resolution was studied using different scattering coefficients and was compared to computational results based on Monte Carlo simulations. It was found that degradation of lateral resolution due to optical scattering was not significant for either ORPAM or CLSM. The depth discrimination capability of ORPAM and CLSM was measured using microfiber embedded in a light scattering phantom material. ORPAM images demonstrated higher contrast compared to CLSM images partly due to reduced acoustic signal scattering.

  18. Comparison between optical-resolution photoacoustic microscopy and confocal laser scanning microscopy for turbid sample imaging.

    PubMed

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

    Optical-resolution photoacoustic microscopy (ORPAM) in theory provides lateral resolution equivalent to the optical diffraction limit. Scattering media, such as biological turbid media, attenuates the optical signal and also alters the diffraction-limited spot size of the focused beam. The ORPAM signal is generated only from a small voxel in scattering media with dimensions equivalent to the laser spot size after passing through scattering layers and is detected by an acoustic transducer, which is not affected by optical scattering. Thus, both ORPAM and confocal laser scanning microscopy (CLSM) reject scattered light. A multimodal optical microscopy platform that includes ORPAM and CLSM was constructed, and the lateral resolution of both modes was measured using patterned thin metal film with and without a scattering barrier. The effect of scattering media on the lateral resolution was studied using different scattering coefficients and was compared to computational results based on Monte Carlo simulations. It was found that degradation of lateral resolution due to optical scattering was not significant for either ORPAM or CLSM. The depth discrimination capability of ORPAM and CLSM was measured using microfiber embedded in a light scattering phantom material. ORPAM images demonstrated higher contrast compared to CLSM images partly due to reduced acoustic signal scattering. PMID:26256640

  19. Modeling and simulation of protein uptake in cation exchanger visualized by confocal laser scanning microscopy.

    PubMed

    Yang, Kun; Shi, Qing-Hong; Sun, Yan

    2006-12-01

    Confocal laser scanning microscopy (CLSM) has been extensively applied in the area of protein chromatography to investigate the uptake mechanism of protein in adsorbents. However, due to the light attenuation in the deeper layers of a specimen, quantitative analysis using CLSM data is still far from reality. In this work, an attenuation equation for describing the darkening of the CLSM image in the deeper scanning layers was developed. Bovine serum albumin (BSA) adsorption to SP Sepharose FF was performed by batch adsorption and micro-column chromatography on which protein concentration in single absorbents were visualized by CLSM. The parameters in the equation were estimated by fitting it to the fluorescence intensity profiles obtained at adsorption equilibrium, and then the equation was used to simulate the effect caused by the light scattering and absorption. CLSM analysis demonstrated that BSA adsorption to SP Sepharose FF followed the shrinking core pattern and was predicted reasonably well by the pore diffusion model in combination with the attenuation equation. By comparison of the CLSM data with the simulations, it shows that the attenuation equation was useful to demonstrate the validity of an intraparticle mass transport model for the estimation of intraparticle protein concentration profiles. PMID:17034803

  20. Elastomeric photo-actuators and their investigation by confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Czaniková, Klaudia; Ilčíková, Markéta; Krupa, Igor; Mičušík, Matej; Kasák, Peter; Pavlova, Ewa; Mosnáček, Jaroslav; Chorvát, Dušan, Jr.; Omastová, Mária

    2013-10-01

    The photo-actuation behavior of nanocomposites based on ethylene-vinylacetate copolymer (EVA) and styrene-isoprene-styrene (SIS) block copolymer filled with well-dispersed and modified multiwalled carbon nanotubes (MWCNTs) is discussed in this paper. The nanocomposites were prepared by casting from solution. To improve the dispersion of the MWCNTs in EVA, the MWCNT surface was modified with a non-covalent surfactant, cholesteryl 1-pyrenecarboxylate (PyChol). To prepare SIS nanocomposites, the MWCNT surface was covalently modified with polystyrene chains. The good dispersion of the filler was confirmed by transmission electron microscopy (TEM). Special, custom-made punch/die molds were used to create a Braille element (BE)-like shape, which under shear forces induces a uniaxial orientation of the MWCNTs within the matrix. The uniaxial orientation of MWCNTs is an essential precondition to ensure the photo-actuating behavior of MWCNTs in polymeric matrices. The orientation of the MWCNTs within the matrices was examined by scanning electron microscopy (SEM). Nanocomposite BEs were illuminated from the bottom by a red light-emitting diode (LED), and the photo-actuation was investigated by confocal laser scanning microscopy (CLSM). When the BEs were exposed to light, a temporary increase in the height of the element was detected. This process was observed to be reversible: after switching off the light, the BEs returned to their original shape and height.

  1. Imaging of calcium wave propagation in guinea-pig ventricular cell pairs by confocal laser scanning microscopy.

    PubMed

    Takamatsu, T; Minamikawa, T; Kawachi, H; Fujita, S

    1991-08-01

    We describe here the use of a confocal laser scanning microscope for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca2+]i) in isolated ventricular cell pairs. The scanning apparatus of our system, paired galvanometer mirrors, can perform narrow band scanning of an area of interest at a high temporal resolution of less than 70 msec per image. The actual [Ca2+]i is obtained directly through the fluorescence intensity of injected fluo-3, which responds to changes of [Ca2+]i in optically sectioned unit volumes of the cell. Images of the calcium wave obtained during propagation between paired cells revealed that the wavefront is constant in shape and propagates at constant velocity without any delay at the cell-to-cell junction. The confocal laser scanning microscope with depth-discriminating ability is a valuable tool for taking pictures of the sequence of biological events in living cells. PMID:1782671

  2. Confocal laser scanning microscopy measurement of the morphology of vanadium pentoxide nanorods grown by electron beam irradiation or thermal oxidation

    NASA Astrophysics Data System (ADS)

    Kang, Manil; Hong, Donghyuk; Kim, Taesung; Chu, Minwoo; Kim, Sok Won

    2013-01-01

    In order to observe the morphology of nanostructures at the submicroscale, we use a confocal laser scanning (CLS) microscope built in our laboratory. The theoretical resolution of the hand-made CLS microscope is 150 nm and the performance of the microscope is evaluated by observing a USAF target. Vanadium pentoxide nanorods grown by electron beam irradiation and thermal oxidation methods are used as nanostructures and the morphologies of the nanorods observed by confocal laser scanning microscopy (CLSM) are compared with those obtained by scanning electron microscopy. The magnification and resolution of the CLSM were estimated to be approximately 1500 and 800 nm, respectively. From the results, we confirm that the CLSM can be used to measure nanostructures at the sub-micro-scale without a preconditioning process.

  3. Observation of the early stage of insulin crystallization by confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Mühlig, P.; Klupsch, Th.; Schell, U.; Hilgenfeld, R.

    2001-11-01

    It is demonstrated that high resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein crystal growth. CLSM is used to study the early crystallization stage of Des-ThrB30 human insulin in aqueous solution, under conditions known to lead to monoclinic crystals. A modified batch crystallization method for CLSM purposes is applied which allows the growth behavior of crystallites to be studied in reflected light. A few hours after the start of the experiment, microcrystallites of characteristic shapes (mainly prismatic and pyramidal) are observed, the number of which strongly depends on the concentration of higher insulin aggregates in the initial solution. From direct observation as well as from model calculations we conclude that for solute concentrations up to about 3.5-times the saturation value, growth starts from few active insulin precipitate particles while 3D nucleation is neglegible for observation times up to 24 h. The anisotropic growth rates of monoclinic, prismatic crystallites are measured along the long edge of the cover face and perpendicular to the latter. A simultaneous crossover to signifcantly higher growth rates is found when the crystallite size reaches about 2 μm. The higher growth rates are connected with the appearence of striations. We argue that this growth rate crossover is caused by an increased 2D nucleation rate at the edges and corners, which finally results in bunching of steps simultaneously spreading over adjacent crystallite faces.

  4. Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy.

    PubMed

    MacDonald, Glen H; Rubel, Edwin W

    2008-09-01

    The complex anatomy of the mammalian cochlea is most readily understood by representation in three-dimensions. However, the cochlea is often sectioned to minimize the effects of its anatomic complexity and optical properties on image acquisition by light microscopy. We have found that optical aberrations present in the decalcified cochlea can be greatly reduced by dehydration through graded ethanols followed by clearing with a mixture of five parts methyl salicylate and three parts benzyl benzoate (MSBB). Clearing the cochlea with MSBB enables acquisition of high-resolution images with multiple fluorescent labels, through the full volume of the cochlea by laser scanning confocal microscopy. The resulting images are readily applicable to three-dimensional morphometric analysis and volumetric visualizations. This method promises to be particularly useful for three-dimensional characterization of anatomy, innervation and expression of genes or proteins in the many new animal models of hearing and balance generated by genetic manipulation. Furthermore, the MSBB is compatible with most non-protein fluorophores used for histological labeling, and may be removed with traditional transitional solvents to allow subsequent epoxy embedding for sectioning. PMID:18573326

  5. In Vivo Laser Scanning Confocal Microscopy of Human Meibomian Glands in Aging and Ocular Surface Diseases

    PubMed Central

    Fasanella, Vincenzo; Mastropasqua, Rodolfo; Brescia, Lorenza; Di Staso, Federico; Ciancaglini, Marco; Mastropasqua, Leonardo

    2016-01-01

    Meibomian glands (MGs) play a crucial role in the ocular surface homeostasis by providing lipids to the superficial tear film. Their dysfunction destabilizes the tear film leading to a progressive loss of the ocular surface equilibrium and increasing the risk for dry eye. In fact, nowadays, the meibomian gland dysfunction is one of the leading causes of dry eye. Over the past decades, MGs have been mainly studied by using meibography, which, however, cannot image the glandular structure at a cellular level. The diffusion of the in vivo laser scanning confocal microscopy (LSCM) provided a new approach for the structural assessment of MGs permitting a major step in the noninvasive evaluation of these structures. LSCM is capable of showing MGs modifications during aging and in the most diffuse ocular surface diseases such as dry eye, allergy, and autoimmune conditions and in the drug-induced ocular surface disease. On the other hand, LSCM may help clinicians in monitoring the tissue response to therapy. In this review, we summarized the current knowledge about the role of in vivo LSCM in the assessment of MGs during aging and in the most diffuse ocular surface diseases. PMID:27047965

  6. In Vivo Laser Scanning Confocal Microscopy of Human Meibomian Glands in Aging and Ocular Surface Diseases.

    PubMed

    Fasanella, Vincenzo; Agnifili, Luca; Mastropasqua, Rodolfo; Brescia, Lorenza; Di Staso, Federico; Ciancaglini, Marco; Mastropasqua, Leonardo

    2016-01-01

    Meibomian glands (MGs) play a crucial role in the ocular surface homeostasis by providing lipids to the superficial tear film. Their dysfunction destabilizes the tear film leading to a progressive loss of the ocular surface equilibrium and increasing the risk for dry eye. In fact, nowadays, the meibomian gland dysfunction is one of the leading causes of dry eye. Over the past decades, MGs have been mainly studied by using meibography, which, however, cannot image the glandular structure at a cellular level. The diffusion of the in vivo laser scanning confocal microscopy (LSCM) provided a new approach for the structural assessment of MGs permitting a major step in the noninvasive evaluation of these structures. LSCM is capable of showing MGs modifications during aging and in the most diffuse ocular surface diseases such as dry eye, allergy, and autoimmune conditions and in the drug-induced ocular surface disease. On the other hand, LSCM may help clinicians in monitoring the tissue response to therapy. In this review, we summarized the current knowledge about the role of in vivo LSCM in the assessment of MGs during aging and in the most diffuse ocular surface diseases. PMID:27047965

  7. Visualization and quantification of healthy and carious dentin structure using confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Kimura, Yuichi; Wilder-Smith, Petra B. B.; Krasieva, Tatiana B.; Arrastia-Jitosho, Anna-Marie A.; Liaw, Lih-Huei L.; Matsumoto, Koukichi; Berns, Michael W.

    1996-04-01

    In this study, a fluorescence technique was developed for visualization of dentin using confocal laser scanning microscopy (CLSM). Eighteen extracted human teeth were used: 13 showing no clinical signs of caries and 5 with visually apparent decay. Preliminary study: All teeth were horizontally sectioned to approx. 200 micrometers thickness and pre-treated as follows: no pretreatment; vacuum only; ultrasonication only; sodium hypochlorite (NaOCl) only; vacuum and NaOCl; ultrasonication and NaOCl; or vacuum, ultrasonication and NaOCl. Samples were stained with Rhodamine 123 fluorescent dye at a concentration of 10-5 M in phosphate buffer saline for 1 to 24 hours. Caries study: Dentin surfaces, some with pre-existing caries, were visualized using CLSM. Most dentin tubules in sound dentin appeared open using CLSM, but most dentin tubules in carious dentin appeared closed or narrowed. Surface images obtained using CLSM were similar to those seen by SEM, but additional subsurface imaging was possible using CLSM at depth intervals of 1 micrometers to a depth of 30 - 50 micrometers . This technique shows good potential for non-invasive surface and subsurface imaging of dentin structures.

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

    PubMed

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

    2015-06-01

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

  9. In vivo assessment of the structure of skin microcirculation by reflectance confocal-laser-scanning microscopy

    NASA Astrophysics Data System (ADS)

    Sugata, Keiichi; Osanai, Osamu; Kawada, Hiromitsu

    2012-02-01

    One of the major roles of the skin microcirculation is to supply oxygen and nutrition to the surrounding tissue. Regardless of the close relationship between the microcirculation and the surrounding tissue, there are few non-invasive methods that can evaluate both the microcirculation and its surrounding tissue at the same site. We visualized microcapillary plexus structures in human skin using in vivo reflectance confocal-laser-scanning microscopy (CLSM), Vivascope 3000® (Lucid Inc., USA) and Image J software (National Institutes of Health, USA) for video image processing. CLSM is a non-invasive technique that can visualize the internal structure of the skin at the cellular level. In addition to internal morphological information such as the extracellular matrix, our method reveals capillary structures up to the depth of the subpapillary plexus at the same site without the need for additional optical systems. Video images at specific depths of the inner forearm skin were recorded. By creating frame-to-frame difference images from the video images using off-line video image processing, we obtained images that emphasize the brightness depending on changes of intensity coming from the movement of blood cells. Merging images from different depths of the skin elucidates the 3-dimensional fine line-structure of the microcirculation. Overall our results show the feasibility of a non-invasive, high-resolution imaging technique to characterize the skin microcirculation and the surrounding tissue.

  10. Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

    There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

  11. Application of Laser Scanning Confocal Microscopy to Heat and Mass Transport Modeling in Porous Microstructures

    NASA Technical Reports Server (NTRS)

    Marshall, Jochen; Milos, Frank; Fredrich, Joanne; Rasky, Daniel J. (Technical Monitor)

    1997-01-01

    Laser Scanning Confocal Microscopy (LSCM) has been used to obtain digital images of the complicated 3-D (three-dimensional) microstructures of rigid, fibrous thermal protection system (TPS) materials. These orthotropic materials are comprised of refractory ceramic fibers with diameters in the range of 1 to 10 microns and have open porosities of 0.8 or more. Algorithms are being constructed to extract quantitative microstructural information from the digital data so that it may be applied to specific heat and mass transport modeling efforts; such information includes, for example, the solid and pore volume fractions, the internal surface area per volume, fiber diameter distributions, and fiber orientation distributions. This type of information is difficult to obtain in general, yet it is directly relevant to many computational efforts which seek to model macroscopic thermophysical phenomena in terms of microscopic mechanisms or interactions. Two such computational efforts for fibrous TPS materials are: i) the calculation of radiative transport properties; ii) the modeling of gas permeabilities.

  12. Retinal Vasculature of Adult Zebrafish: In Vivo Imaging Using Confocal Scanning Laser Ophthalmoscopy

    PubMed Central

    Bell, Brent A.; Xie, Jing; Yuan, Alex; Kaul, Charles; Hollyfield, Joe G.; Anand-Apte, Bela

    2014-01-01

    Over the past 3 decades the zebrafish (Danio rerio) has become an important biomedical research species. As their use continues to grow additional techniques and tools will be required to keep pace with ongoing research using this species. In this paper we describe a novel method for in vivo imaging of the retinal vasculature in adult animals using a commercially available confocal scanning laser ophthalmoscope (SLO). With this instrumentation, we demonstrate the ability to distinguish diverse vascular phenotypes in different transgenic GFP lines. In addition this technology allows repeated visualization of the vasculature in individual zebrafish over time to document vascular leakage progression and recovery induced by intraocular delivery of proteins that induce vascular permeability. SLO of the retinal vasculature was found to be highly informative, providing images of high contrast and resolution that were capable of resolving individual vascular endothelial cells. Finally, the procedures required to acquire SLO images from zebrafish are non-invasive, simple to perform and can be achieved with low animal mortality, allowing repeated imaging of individual fish. PMID:25447564

  13. A statistical pixel intensity model for segmentation of confocal laser scanning microscopy images.

    PubMed

    Calapez, Alexandre; Rosa, Agostinho

    2010-09-01

    Confocal laser scanning microscopy (CLSM) has been widely used in the life sciences for the characterization of cell processes because it allows the recording of the distribution of fluorescence-tagged macromolecules on a section of the living cell. It is in fact the cornerstone of many molecular transport and interaction quantification techniques where the identification of regions of interest through image segmentation is usually a required step. In many situations, because of the complexity of the recorded cellular structures or because of the amounts of data involved, image segmentation either is too difficult or inefficient to be done by hand and automated segmentation procedures have to be considered. Given the nature of CLSM images, statistical segmentation methodologies appear as natural candidates. In this work we propose a model to be used for statistical unsupervised CLSM image segmentation. The model is derived from the CLSM image formation mechanics and its performance is compared to the existing alternatives. Results show that it provides a much better description of the data on classes characterized by their mean intensity, making it suitable not only for segmentation methodologies with known number of classes but also for use with schemes aiming at the estimation of the number of classes through the application of cluster selection criteria. PMID:20363677

  14. Confocal laser-scanning microscopy of capillaries in normal and psoriatic skin

    NASA Astrophysics Data System (ADS)

    Archid, Rami; Patzelt, Alexa; Lange-Asschenfeldt, Bernhard; Ahmad, Sufian S.; Ulrich, Martina; Stockfleth, Eggert; Philipp, Sandra; Sterry, Wolfram; Lademann, Juergen

    2012-10-01

    An important and most likely active role in the pathogenesis of psoriasis has been attributed to changes in cutaneous blood vessels. The purpose of this study was to use confocal laser-scanning microscopy (CLSM) to investigate dermal capillaries in psoriatic and normal skin. The structures of the capillary loops in 5 healthy participants were compared with those in affected skin of 13 psoriasis patients. The diameters of the capillaries and papillae were measured for each group with CLSM. All investigated psoriasis patients showed elongated, widened, and tortuous microvessels in the papillary dermis, whereas all healthy controls showed a single capillary loop in each dermal papilla. The capillaries of the papillary loop and the dermal papilla were significantly enlarged in the psoriatic skin lesions (diameters 24.39±2.34 and 146.46±28.52 μm, respectively) in comparison to healthy skin (diameters 9.53±1.8 and 69.48±17.16 μm, respectively) (P<0.001). CLSM appears to represent a promising noninvasive technique for evaluating dermal capillaries in patients with psoriasis. The diameter of the vessels could be seen as a well-quantifiable indicator for the state of psoriatic skin. CLSM could be useful for therapeutic monitoring to delay possible recurrences.

  15. Confocal laser-scanning microscopy for determining the structure of and keratinocyte infiltration through collagen sponges.

    PubMed

    Hanthamrongwit, M; Wilkinson, R; Osborne, C; Reid, W H; Grant, M H

    1996-03-01

    The development of artificial skin substitutes based on cultured cells and biomaterials such as collagen requires an understanding of cellular interactions with the substrate. In this study, human keratinocytes were cultured on the surface of collagen sponges, and confocal laser-scanning microscopy (CLSM) was used to assess both the microstructure of the sponge, and the cell morphology and distribution throughout the sponge. It was found that the pore size increased with increasing depth into the sponge. Both pore size and fiber thickness increased during incubation for up to 10 days at 37 degrees C in culture medium in the absence of cells. This latter effect was not observed when the sponges were incubated in distilled water. Keratinocytes penetrated into the sponge even after only 3 days in culture. By 10 days in culture, the cells had penetrated to the maximum depth that could be examined (120 microns from the sponge surface). In the presence of cells, the inner structure of the collagen sponge had altered after 10 days in culture, with the collagen fibers becoming thicker, and pore geometry less regular. The mechanism responsible for this is unknown at present. Although the presence of the keratinocytes increases distortion of the sponge structure, factors from the medium itself also contribute to this effect. CLSM is a powerful tool for assessing cellular interactions with bioimplants, providing both qualitative and quantitative information. It offers many advantages over scanning electron microscopy (SEM) and histological techniques. CLSM minimizes the time-consuming, extensive preparation of samples required with the latter two methods, and allows noninvasive serial optical sectioning of intact samples. PMID:8698696

  16. Confocal laser scanning microscopy of liesegang rings in odontogenic cysts: analysis of three-dimensional image reconstruction.

    PubMed

    Scivetti, Michele; Lucchese, Alberta; Crincoli, Vito; Pilolli, Giovanni Pietro; Favia, Gianfranco

    2009-01-01

    Liesegang rings are concentric noncellular lamellar structures, occasionally found in inflammatory tissues. They have been confused with various parasites, algas, calcification, and psammoma bodies. The authors examined Liesegang rings from oral inflammatory cysts by both optical and confocal laser scanning microscopy, and perfomed a three-dimensional reconstruction. These investigations indicate that Liesegang rings are composed of multiple birefringent concentric rings, resulting from a progressive deposition of organic substances, with an unclear pathogenesis. PMID:19274580

  17. Mobile connected dermatoscope and confocal laser scanning microscope: a useful combination applied in facial simple sensitive skin.

    PubMed

    Zha, W F; Song, W M; Ai, J J; Xu, A E

    2012-08-01

    Little is known as the effects of mobile connected dermatoscope services on diagnostic accuracy for sensitive skin. Confocal laser scanning microscope (CLSM) can non-invasively measure the thickness of epidermis. Combination of the two devices to observe sensitive skin may receive unexpected effects. To evaluate the application effect on sensitive skin with the combination of Handyscope and confocal laser scanning microscope. Twenty simple sensitive-skinned patients and 20 volunteers participated in the study. Cheek, typically, dermoscopic images were obtained from patients, and the changes in the skin texture were observed. Their epidermis thicknesses as well as the volunteers' were measured so that the thicknesses of the two groups were compared. Dermoscopic pictures of the skin texture obviously showed that dilated capillaries looked like earthworms with pigmented patches more or less floating above, and skin roughness as well as deepened dermatoglyph were also conspicuously present in some patients. The mean epidermal thickness of the patients was 79.01 μm and the volunteers' was 85.78 μm. The difference between the two groups reached 6.77 μm. There was a statistical significance (P = 0.001). Mobile connected dermatoscope and confocal laser scanning microscope might be the choice for simple sensitive skin investigation. PMID:22515509

  18. Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

    USGS Publications Warehouse

    Hackley, Paul C.; Kus, Jolanta

    2015-01-01

    We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

  19. Short fatigue crack characterization and detection using confocal scanning laser microscopy (CSLM)

    SciTech Connect

    Varvani-Farahani, A.; Topper, T.H.

    1997-12-31

    This paper presents a new technique for studying the growth and morphology of fatigue cracks. The technique allows short fatigue crack growth, crack depth, aspect ratio (crack depth/half crack length), and crack front configuration to be measured using a Confocal Scanning Laser Microscope (CSLM). CSLM measurements of the initial stage of crack growth in Al 2024-T351 revealed that microstructurally short fatigue cracks grew initially along a plane inclined to the applied stress. The angle of the inclined plane (Stage I crack growth) was found to be about 45 degrees to the axis of the applied tensile load. Aspect ratio and the angle of maximum shear plane (Mode II), obtained using the CSLM technique, showed a good agreement with those obtained using a Surface Removal (SR) technique. The aspect ratios obtained using the CSLM technique were found to remain constant with increasing crack length in Al 2024-T351 and SAE 1045 Steel at 0.83 and 0.80, respectively. Optical sectioning along the length of a crack revealed that the crack front in the interior of the materials has a semi-elliptical shape. These results are in good agreement with results obtained using the SR technique. The CSLM technique was employed to characterize the fracture surface of fatigue cracks in an SAE 1045 Steel. CSLM image processing of the fracture surface near the crack tip constructed a three dimensional profile of fracture surface asperities. The heights of asperities were obtained from this profile. Optical sectioning from a post-image-processed crack provided crack depth and crack mouth width at every point along the crack length for each load level. The crack opening stress was taken as the stress level at which the crack depth stopped increasing with increases in a lied stress. 6 refs., 9 figs., 1 tab.

  20. Toward Automated Analysis of Biofilm Architecture: Bias Caused by Extraneous Confocal Laser Scanning Microscopy Images▿

    PubMed Central

    Merod, Robin T.; Warren, Jennifer E.; McCaslin, Hope; Wuertz, Stefan

    2007-01-01

    An increasing number of studies utilize confocal laser scanning microscopy (CLSM) for in situ visualization of biofilms and rely on the use of image analysis programs to extract quantitative descriptors of architecture. Recently, designed programs have begun incorporating procedures to automatically determine threshold values for three-dimensional CLSM image stacks. We have found that the automated threshold calculation is biased when a stack contains images lacking pixels of biological significance. Consequently, we have created the novel program Auto PHLIP-ML to resolve this bias by iteratively excluding extraneous images based on their area coverage of biomass. A procedure was developed to identify the optimal percent area coverage value used for extraneous image removal (PACVEIR). The optimal PACVEIR was defined to occur when the standard deviation of mean thickness, determined from replicate image stacks, was at a maximum, because it more accurately reflected inherent structural variation. Ten monoculture biofilms of either Ralstonia eutropha JMP228n::gfp or Acinetobacter sp. strain BD413 were tested to verify the routine. All biofilms exhibited an optimal PACVEIR between 0 and 1%. Prior to the exclusion of extraneous images, JMP228n::gfp appeared to develop more homogeneous biofilms than BD413. However, after the removal of extraneous images, JMP228n::gfp biofilms were found to form more heterogeneous biofilms. Similarly, JMP228n::gfp biofilms grown on glass surfaces vis-à-vis polyethylene membranes produced significantly different architectures after extraneous images had been removed but not when such images were included in threshold calculations. This study shows that the failure to remove extraneous images skewed a seemingly objective analysis of biofilm architecture and significantly altered statistically derived conclusions. PMID:17545329

  1. Homonymous Hemianopic Hyporeflective Retinal Abnormality on Infrared Confocal Scanning Laser Photography: A Novel Sign of Optic Tract Lesion.

    PubMed

    Monteiro, Mario L R; Araújo, Rafael B; Suzuki, Ana C F; Cunha, Leonardo P; Preti, Rony C

    2016-03-01

    Infrared confocal scanning laser photography of a patient with long-standing optic tract lesion revealed a homonymous hemianopic hyporeflective image contralateral to the visual field defect. Spectral domain optical coherence tomography showed thinning of the retinal nerve fiber and retinal ganglion cell layer and thickening of the inner nuclear layer (with microcystic degeneration) in the macular area, matching the infrared image. Hyporeflective image on infrared laser photography is associated with retinal degeneration secondary to anterior visual pathway disease and, when located in homonymous hemianopic retinas, may represent a new sign of an optic tract lesion. PMID:26172159

  2. Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy.

    PubMed

    Michels, J; Gorb, S N

    2012-01-01

    Resilin is a rubber-like protein found in the exoskeleton of arthropods. It often contributes large proportions to the material of certain structures in movement systems. Accordingly, the knowledge of the presence and distribution of resilin is essential for the understanding of the functional morphology of these systems. Because of its specific autofluorescence, resilin can be effectively visualized using fluorescence microscopy. However, the respective excitation maximum is in the UV range, which is not covered by the lasers available in most of the modern commercial confocal laser scanning microscopes. The goal of this study was to test the potential of confocal laser scanning microscopy (CLSM) in combination with a 405 nm laser to visualize and analyse the presence and distribution of resilin in arthropod exoskeletons. The results clearly show that all resilin-dominated structures, which were visualized successfully using wide-field fluorescence microscopy (WFM) and a 'classical' UV excitation, could also be visualized efficiently with the proposed CLSM method. Furthermore, with the application of additional laser lines CLSM turned out to be very appropriate for studying differences in the material composition within arthropod exoskeletons in great detail. As CLSM has several advantages over WFM with respect to detailed morphological imaging, the application of the proposed CLSM method may reveal new information about the micromorphology and material composition of resilin-dominated exoskeleton structures leading to new insights into the functional morphology and biomechanics of arthropods. PMID:22142031

  3. Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope.

    PubMed

    Trepte, O; Rokahr, I; Andersson-Engels, S; Carlsson, K

    1994-12-01

    A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/delta lambda, ranges from 350 at lambda = 400 nm to 100 at lambda = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7869364

  4. 3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

    SciTech Connect

    Fredrich, J.T.

    1999-02-10

    We present advances in the application of laser scanning confocal microscopy (LSCM) to image, reconstruct, and characterize statistically the microgeometry of porous geologic and engineering materials. We discuss technical and practical aspects of this imaging technique, including both its advantages and limitations. Confocal imaging can be used to optically section a material, with sub-micron resolution possible in the lateral and axial planes. The resultant volumetric image data, consisting of fluorescence intensities for typically {approximately}50 million voxels in XYZ space, can be used to reconstruct the three-dimensional structure of the two-phase medium. We present several examples of this application, including studying pore geometry in sandstone, characterizing brittle failure processes in low-porosity rock deformed under triaxial loading conditions in the laboratory, and analyzing the microstructure of porous ceramic insulations. We then describe approaches to extract statistical microgeometric descriptions from volumetric image data, and present results derived from confocal volumetric data sets. Finally, we develop the use of confocal image data to automatically generate a three-dimensional mesh for numerical pore-scale flow simulations.

  5. Multiplex fluorescence in situ hybridization (M-FISH) and confocal laser scanning microscopy (CLSM) to analyze multispecies oral biofilms.

    PubMed

    Karygianni, Lamprini; Hellwig, Elmar; Al-Ahmad, Ali

    2014-01-01

    Multiplex fluorescence in situ hybridization (M-FISH) constitutes a favorable microbiological method for the analysis of spatial distribution of highly variable phenotypes found in multispecies oral biofilms. The combined use of confocal laser scanning microscopy (CLSM) produces high-resolution three-dimensional (3D) images of individual bacteria in their natural environment. Here, we describe the application of M-FISH on early (Streptococcus spp., Actinomyces naeslundii) and late colonizers (Fusobacterium nucleatum, Veillonella spp.) of in situ-formed oral biofilms, the acquisition of CLSM images, as well as the qualitative and quantitative analysis of these digitally obtained and processed images. PMID:24664826

  6. Study of hydroxyl carbonate apatite formation on bioactive glass coated dental ceramics by confocal laser scanning microscopy (CLSM)

    NASA Astrophysics Data System (ADS)

    Stanciu, G. A.; Savu, B.; Sandulescu, I.; Paraskevopoulos, K.; Koidis, P.

    2007-03-01

    Some dental ceramics were coated with a bioactive glass and resulted the formation of a stable and well bonded with the ceramic substrate thin layer. After immersion in a solution with ion concentrations similar to those of human blood plasma the development of hydroxy carbonate apatite layer on the surface of bioactive glass may be observed. The objective of this study was to investigate structural surface changes of bioactive glass, after exposure in a simulated body fluid for a different number of days. The roughness and topography of the hydroxyapatite surface were investigated by Confocal Scanning Laser Microscopy. The chemical composition was analyzed by Energy Dispersive Spectroscopy measurements.

  7. Inverse image alignment method for image mosaicing and video stabilization in fundus indocyanine green angiography under confocal scanning laser ophthalmoscope.

    PubMed

    Zhou, Yongjin; Xue, Hui; Wan, Mingxi

    2003-01-01

    An efficient image registration algorithm, the Inverse Compositional image alignment method based on minimization of Sum of Squared Differences of images, is applied in fundus blood vessel angiography under confocal scanning laser ophthalmoscope, to build image mosaics which have larger field of view without loss of resolution to assist diagnosis. Furthermore, based on similar technique, the angiography video stabilization algorithm is implemented for fundus documenting. The actual underlying models of motion between images and corresponding convergence criteria are also discussed. The experiment results in fundus images demonstrate the effectiveness of the registration scheme. PMID:14575786

  8. Development of a confocal laser scanning fluorescence microscope using two-photon excitation in combination with time-gated detection

    NASA Astrophysics Data System (ADS)

    Sytsma, Joost; Vroom, Jurrien; Gerritsen, Hans C.; Levine, Yehudi K.

    1995-03-01

    Fluorescent molecules having single-photon absorption in the blue and the UV can be excited with infra-red light via a process known as two-photon excitation. The combination of this technique with scanning techniques can be exploited for 3D microscopic imaging. The two- photon process is confined to a restricted volume in the sample determined by the laser focus, resulting in inherent confocality. Other advantages are reduced photo-bleaching of the samples and a larger penetration depth of the excitation light. The implementation of time-gated detection techniques allows fluorescent lifetime imaging. This drastically improves the selectivity and contrast of the images.

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

    NASA Technical Reports Server (NTRS)

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

    2007-01-01

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

  10. Visualising fouling of a chromatographic matrix using confocal scanning laser microscopy.

    PubMed

    Siu, Sun Chau; Boushaba, Rihab; Topoyassakul, Vithaya; Graham, Alex; Choudhury, Sorwar; Moss, Guy; Titchener-Hooker, Nigel J

    2006-11-01

    Confocal scanning laser microscopy (CSLM) was used to visualise the spatial location of foulants during the fouling of Q Sepharose FF matrix in finite batch experiments and for examining the subsequent effectiveness of clean-in-place (CIP) treatments in cleaning the heavily fouled beads. Beads were severely fouled with partially clarified E. coli homogenate by contacting the beads with the foulant for contact times of 5 min, 1 or 12 h. The use of two different fluorescent dyes, PicoGreen and Cy5.5, for labelling genomic PicoGreen-labelled dsDNA and protein respectively, allowed the direct observation of the chromatographic beads. The extent of fouling was assessed by measuring the subsequent adsorption of Cy5.5-labelled BSA to the beads. Control studies established that the labelling of BSA did not affect significantly the protein properties. In the control case of contacting the unfouled matrix with Cy5.5-labelled BSA, protein was able to penetrate the entire matrix volume. After fouling, Cy5.5-labelled BSA was unable to penetrate the bead but only to bind near the bead surface where it slowly displaced PicoGreen-conjugated dsDNA, which bound only at the exterior of the beads. Labelled host cell proteins bound throughout the bead interior but considerably less at the core; suggesting that other species might have occupied that space. The gross levels of fouling achieved drastically reduced the binding capacity and maximum Cy5.5-labelled BSA uptake rate. The capacity of the resin was reduced by 2.5-fold when incubated with foulant for up to 1 h. However, when the resin was fouled for a prolonged time of 12 h a further sixfold decrease in capacity was seen. The uptake rate of Cy5.5-labelled BSA decreased with increased fouling time of the resin. Incubating the fouled beads in 1 M NaCl dissociated PicoGreen-labelled dsDNA from the bead exterior within 15 min of incubation but proved ineffective in removing all the foulant protein. Cy5.5-labelled BSA was still unable

  11. Real-time mapping of the corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

    Guthoff, Rudolf F.; Zhivov, Andrey; Stachs, Oliver

    2010-02-01

    The aim of the study was to produce two-dimensional reconstruction maps of the living corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy in real time. CLSM source data (frame rate 30Hz, 384x384 pixel) were used to create large-scale maps of the scanned area by selecting the Automatic Real Time (ART) composite mode. The mapping algorithm is based on an affine transformation. Microscopy of the sub-basal nerve plexus was performed on normal and LASIK eyes as well as on rabbit eyes. Real-time mapping of the sub-basal nerve plexus was performed in large-scale up to a size of 3.2mm x 3.2mm. The developed method enables a real-time in vivo mapping of the sub-basal nerve plexus which is stringently necessary for statistically firmed conclusions about morphometric plexus alterations.

  12. Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms.

    PubMed

    Kania, Romain E; Lamers, Gerda E M; van de Laar, Nicole; Dijkhuizen, Marloes; Lagendijk, Ellen; Huy, Patrice Tran Ba; Herman, Philippe; Hiemstra, Pieter; Grote, Jan J; Frijns, Johan; Bloemberg, Guido V

    2010-07-01

    The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs. PMID:20473799

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

    NASA Astrophysics Data System (ADS)

    Hefti, Ryan Alf

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

  14. Cytogenetic Characterization of the TM4 Mouse Sertoli Cell Line. II. Chromosome Microdissection, FISH, Scanning Electron Microscopy, and Confocal Laser Scanning Microscopy.

    PubMed

    Schmid, Michael; Guttenbach, Martina; Steinlein, Claus; Wanner, Gerhard; Houben, Andreas

    2015-01-01

    The chromosomes and interphase cell nuclei of the permanent mouse Sertoli cell line TM4 were examined by chromosome microdissection, FISH, scanning electron microscopy, and confocal laser scanning microscopy. The already known marker chromosomes m1-m5 were confirmed, and 2 new large marker chromosomes m6 and m7 were characterized. The minute heterochromatic marker chromosomes m4 and m5 were microdissected and their DNA amplified by DOP-PCR. FISH of this DNA probe on TM4 metaphase chromosomes demonstrated that the m4 and m5 marker chromosomes have derived from the centromeric regions of normal telocentric mouse chromosomes. Ectopic pairing of the m4 and m5 marker chromosomes with the centromeric region of any of the other chromosomes (centromeric associations) was apparent in ∼60% of the metaphases. Scanning electron microscopy revealed DNA-protein bridges connecting the centromeric regions of normal chromosomes and the associated m4 and m5 marker chromosomes. Interphase cell nuclei of TM4 Sertoli cells did not exhibit the characteristic morphology of Sertoli cells in the testes of adult mice as shown by fluorescence microscopy and confocal laser scanning microscopy. PMID:26900862

  15. Investigation of metallurgical phenomena related to process and product development by means of High Temperature Confocal Scanning Laser Microscopy

    NASA Astrophysics Data System (ADS)

    Diéguez-Salgado, U.; Michelic, S.; Bernhard, C.

    2016-03-01

    An increased interest for high temperature metallurgical processes appeared during the last decades, in order to achieve the high quality requirements in steel products. A defined steel cleanness and microstructure essentially influence the final product quality. The high temperatures involved in metallurgical processes and the lack of in situ observations do not only complicate the verification of simulation model predictions but also make significant conclusions regarding the industrial processes difficult. For that reason, new tools and techniques are necessary to develop. By combining the advances of a laser, confocal optics and an infrared image furnace, the High Temperature Confocal Scanning Laser Microscopy (HTCSLM) is a strong tool which enables high temperature in situ observations of different metallurgical phenomena. Next to solidification processes and phase transformations also the behavior of inclusions at different interfaces in the system steel-slag-refractory can be observed. The present study focuses on the aspects of inclusion agglomeration in the liquid steel and the inclusion behavior at the steel/refractory interface in two different steel grades. Out of the obtained experimental data, attraction forces are calculated and compared. This information provides an important basis for a better understanding of inclusion behavior in industrial processes and the therewith related process optimization, like for example the clogging phenomenon during continuous casting.

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

    NASA Astrophysics Data System (ADS)

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

    2002-06-01

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

  17. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: LASER POWER MEASUREMENTS

    EPA Science Inventory

    Laser power abstract
    The reliability of the confocal laser-scanning microscope (CLSM) to obtain intensity measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. The laser power test appears to be one ...

  18. Penetration of tamoxifen citrate loaded ethosomes and liposomes across human skin: a comparative study with confocal laser scanning microscopy.

    PubMed

    Sarwa, Khomendra K; Suresh, Preeti K; Rudrapal, Mithun; Verma, Vinod K

    2014-01-01

    In the present study, ethosomal and liposomal formulations containing tamoxifen citrate were prepared and evaluated for their penetration properties in human cadaver skin using Franz diffusion cell and confocal laser scanning microscope (CLSM). The results clearly revealed that ethosomal vesicles showed a better drug permeation profile than that of liposomal vesicles. In addition, low fluorescence intensity in CLSM was recorded with liposomes as compared to ethosomes, indicating lower cumulative amount of drug permeation from liposomal vesicles. Furthermore, CLSM showed uniform fluorescence intensity across the entire depth of skin in ethosomal treatment, indicating high penetrability of ethosomal vesicles through human cadaver skin. In contrast, low penetrability of conventional liposomal vesicles was recorded as penetration was limited to the 7(th) section (i.e. upper epidermis layer) of skin as evident from visualization of intact liposomal vesicles in CLSM. PMID:24428443

  19. Investigation of biological cell-protein interactions using SPR sensor through laser scanning confocal imaging-surface plasmon resonance system

    NASA Astrophysics Data System (ADS)

    Zhang, Hongyan; Yang, Liquan; Zhou, Bingjiang; Wang, Xueliang; Liu, Guiying; Liu, Weimin; Wang, Pengfei

    2014-03-01

    A new method for investigating biological cell-protein interactions was developed by using a laser scanning confocal imaging-surface plasmon resonance (LSCI-SPR) system. Mouse normal IgG was modified on the SPR chip. The suspension mouse lymphocyte cancer cells (L5178Y cells) labeled by Hoechst33342 freely flowed into the surface of the SPR sensor chip. By changing the concentration of the cells, the fluorescence images and the SPR signal were synchronously recorded in real time. The red fluorescence points in the imaging region increased with increase in the concentration of the mouse lymphocyte cancer cells and fit well with the change in the SPR signal. Different suspending cells were chosen to investigate cell-protein interactions through antigen-antibody reactions on the biological cell surfaces through binding detection. This method has potential application in cell biology and pharmacology.

  20. Line-scanning, stage scanning confocal microscope

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  1. In vivo analysis of THz wave irradiation induced acute inflammatory response in skin by laser-scanning confocal microscopy.

    PubMed

    Hwang, Yoonha; Ahn, Jinhyo; Mun, Jungho; Bae, Sangyoon; Jeong, Young Uk; Vinokurov, Nikolay A; Kim, Pilhan

    2014-05-19

    The recent development of THz sources in a wide range of THz frequencies and power levels has led to greatly increased interest in potential biomedical applications such as cancer and burn wound diagnosis. However, despite its importance in realizing THz wave based applications, our knowledge of how THz wave irradiation can affect a live tissue at the cellular level is very limited. In this study, an acute inflammatory response caused by pulsed THz wave irradiation on the skin of a live mouse was analyzed at the cellular level using intravital laser-scanning confocal microscopy. Pulsed THz wave (2.7 THz, 4 μs pulsewidth, 61.4 μJ per pulse, 3Hz repetition), generated using compact FEL, was used to irradiate an anesthetized mouse's ear skin with an average power of 260 mW/cm(2) for 30 minutes using a high-precision focused THz wave irradiation setup. In contrast to in vitro analysis using cultured cells at similar power levels of CW THz wave irradiation, no temperature change at the surface of the ear skin was observed when skin was examined with an IR camera. To monitor any potential inflammatory response, resident neutrophils in the same area of ear skin were repeatedly visualized before and after THz wave irradiation using a custom-built laser-scanning confocal microscopy system optimized for in vivo visualization. While non-irradiated control skin area showed no changes in the number of resident neutrophils, a massive recruitment of newly infiltrated neutrophils was observed in the THz wave irradiated skin area after 6 hours, which suggests an induction of acute inflammatory response by the pulsed THz wave irradiation on the skin via a non-thermal process. PMID:24921268

  2. Influence of confocal scanning laser microscopy specific acquisition parameters on the detection and matching of speeded-up robust features.

    PubMed

    Stanciu, Stefan G; Hristu, Radu; Stanciu, George A

    2011-04-01

    The robustness and distinctiveness of local features to various object or scene deformations and to modifications of the acquisition parameters play key roles in the design of many computer vision applications. In this paper we present the results of our experiments on the behavior of a recently developed technique for local feature detection and description, Speeded-Up Robust Features (SURF), regarding image modifications specific to Confocal Scanning Laser Microscopy (CSLM). We analyze the repeatability of detected SURF keypoints and the precision-recall of their matching under modifications of three important CSLM parameters: pinhole aperture, photomultiplier (PMT) gain and laser beam power. During any investigation by CSLM these three parameters have to be modified, individually or together, in order to optimize the contrast and the Signal Noise Ratio (SNR), being also inherently modified when changing the microscope objective. Our experiments show that an important amount of SURF features can be detected at the same physical locations in images collected at different values of the pinhole aperture, PMT gain and laser beam power, and further on can be successfully matched based on their descriptors. In the final part, we exemplify the potential of SURF in CSLM imaging by presenting a SURF-based computer vision application that deals with the mosaicing of images collected by this technique. PMID:21349249

  3. Scanned optical fiber confocal microscope

    NASA Astrophysics Data System (ADS)

    Dickensheets, David L.; Kino, Gordon S.

    1994-04-01

    The size and weight of conventional optical microscopes often makes them inconvenient for use on the human body or for in-situ examination during materials processing. We describe a new fiber-optic scanning confocal optical microscope which could have a total outside diameter as small as 1 mm, and should lend itself to applications in endoscopy or to optical in vivo histology. The first experimental device utilizes a single-mode optical fiber for illumination and detection. The scanning element is a mechanically resonant fused silica cantilever 1.5 cm long and 0.8 mm across, with a micromachined two-phase zone plate objective mounted at one end. The cantilever is electrostatically scanned near resonance in two dimensions, generating a Lissajous pattern which is scan converted to conventional video for real time display or digitization. The objective lens has N.A. equals 0.25 at (lambda) equals 0.6328 micrometers , with a measured spot size of 1.8 micrometers FWHM.

  4. Design of an affordable fluorescence confocal laser scanning microscope for medical diagnostics

    NASA Astrophysics Data System (ADS)

    Bechtel, Christin; Knobbe, Jens; Grüger, Heinrich; Lakner, Hubert

    2012-12-01

    Confocal fluorescence microscopes are a promising imaging tool in medical diagnostics due to their capability to selectively survey cross-sections of individual layers from `thick' samples. Non-invasive depth resolved investigation of neoplastic skin disorders is one example among other applications. However these microscopes are at present uncommon in medical practice. This is due to their main application area in research. The instruments dealt with here are generally complex, stationary units and are accordingly cost-intensive. It is for this reason, that we have designed a robust and portable MEMS based confocal fluorescence microscope with a field of view of 0.6mm x 0.6mm. This has been made possible by the integration of a 2D micro scanner mirror developed at Fraunhofer IPMS. A variable acquisition depth of cross-sectional images of the fluorescence specimen is enabled by an integrated z-shifter. With the use of commercially available optics an optical demonstrator set up has been realized. To characterize and to demonstrate the ability of this system test measurements were performed. The resolution of the microscope is better than 228 lp/mm determined by 1951 USAF resolution test target. Images of various biological samples are presented and optical sectioning capabilities are shown. A comparison of the measured with the predicted system performance will be given.

  5. Optical biopsy of early gastroesophageal cancer by catheter-based reflectance-type laser-scanning confocal microscopy.

    PubMed

    Nakao, Madoka; Yoshida, Shigeto; Tanaka, Shinji; Takemura, Yoshito; Oka, Shiro; Yoshihara, Masaharu; Chayama, Kazuaki

    2008-01-01

    Magnified endoscopic observation of the gastrointestinal tract has become possible. However, such observation at the cellular level remains difficult. Laser-scanning confocal microscopy (LCM) is a novel, noninvasive optical imaging method that provides instant microscopic images of untreated tissue under endoscopy. We compare prototype catheter-based reflectance-type LCM images in vivo and histologic images of early gastroesophageal cancer to assess the usefulness of LCM in diagnosing such cancer. 20 sites in the esophagus and 40 sites in the stomach are examined by LCM under endoscopy prior to endoscopic or surgical resection. A prototype catheter LCM system, equipped with a semiconductor laser that oscillates at 685 nm and analyzes reflected light (Mauna Kea Technologies, Paris, France; Fujinon, Saitama, Japan) is used in vivo without fluorescent agent. In all normal esophageal mucosa and esophageal cancers, the nuclei are visualized. In nine of the ten normal esophageal mucosa, cell membranes are visualized, and in five of the ten esophageal cancers, cell membranes are visualized. In all normal gastric mucosa, nuclei and cell membranes are not visualized, but in ten of the 20 gastric cancers, nuclei are visualized. This novel method will aid in immediate diagnosis under endoscopy without the need for biopsy. PMID:19021423

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

    PubMed Central

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

    2016-01-01

    Purpose Optical coherence tomography angiography (OCT-A) allows noninvasive visualization of retinal vessels in vivo. OCT-A was used to characterize the vascular network of the mouse retina and was compared with fluorescein angiography (FA) and histology. Methods In the present study, OCT-A based on a Heidelberg Engineering Spectralis system was used to investigate the vascular network in mice. Data was compared with FA and confocal microscopy of flat-mount histology stained with isolectin IB4. For quantitative analysis the National Cancer Institute's AngioTool software was used. Vessel density, the number of vessel junctions, and endpoints were measured and compared between the imaging modalities. Results The configuration of the superficial capillary network was comparable with OCT-A and flat-mount histology in BALBc mice. However, vessel density and the number of vessel junctions per region of interest (P = 0.0161 and P = 0.0015, respectively) in the deep vascular network of BALBc mice measured by OCT-A was significantly higher than with flat-mount histology. In C3A.Cg-Pde6b+Prph2Rd2/J mice, where the deep capillary plexus is absent, analysis of the superficial network provided similar results for all three imaging modalities. Conclusion OCT-A is a helpful imaging tool for noninvasive, in vivo imaging of the vascular plexus in mice. It may offer advantages over FA and confocal microscopy especially for imaging the deep vascular plexus. Translational Relevance The present study shows that OCT-A can be employed for small animal imaging to assess the vascular network and offers advantages over flat-mount histology and FA. PMID:27570710

  7. Noninvasive in vivo confocal laser scanning microscopy is effective in differentiating allergic from nonallergic equivocal patch test reactions.

    PubMed

    Slodownik, D; Levi, A; Lapidoth, M; Ingber, A; Horev, L; Enk, C D

    2015-04-01

    Patch testing is the gold standard for the validation of contact dermatitis. It relies on the subjective scoring by an evaluator of the inflammatory reaction induced by an allergen applied to the skin. Equivocal reactions imply faint erythema and could represent allergic, irritant, or negative reactions. They constitute approximately 1 % of the positive reactions encountered in patch test practice. Histological evaluation of the equivocal reaction has proven helpful for the correct interpretation but is however time consuming, and its invasive nature is often unacceptable to the patient. In vivo confocal laser scanning microscopy (CLSM) is a novel, noninvasive imaging technique which permits real-time visualization of skin structures and lesions at a resolution close to that obtained by conventional histology. CLSM has been successfully applied for the differentiation between clinically clear-cut allergic and irritant patch test reactions. The objective of this study is to determine the relevance of CLSM in differentiating between allergic, irritant, and negative equivocal patch test reactions. Fifteen patients who underwent patch testing in our clinic were observed as having 20 equivocal reactions. All 20 reactions were evaluated using in vivo CLSM and compared with adjacent normal skin. In vivo CLSM evaluation revealed that 8 of the 20 equivocal reactions (40 %) showed confocal patterns consistent with the patterns encountered in positive allergic reactions. Anamnestic exposure, i.e., detailed assessment of previous related contact with these allergens, confirmed high relevance rates. In vivo CLSM is useful in differentiating between allergic, irritant, and negative equivocal patch test reactions, a differentiation that cannot be made by conventional clinical patch test reading. PMID:25604734

  8. Oral biofilm analysis of palatal expanders by fluorescence in-situ hybridization and confocal laser scanning microscopy.

    PubMed

    Klug, Barbara; Rodler, Claudia; Koller, Martin; Wimmer, Gernot; Kessler, Harald H; Grube, Martin; Santigli, Elisabeth

    2011-01-01

    Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH). We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation. FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes. Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia. General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix), Firmicutes (LGC354 A-C; hereafter LGCmix), and Bacteroidetes (Bac303). In addition, specific probes binding to Streptococcus mutans (MUT590) and Porphyromonas gingivalis (POGI) were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle. Subsequently the samples were analyzed by confocal laser scanning

  9. Flow assisted assembly of multilayer colloidal crystals studied using confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Shereda, Laura T.

    Colloidal crystals are highly ordered particle arrays with potential applications including sensors, optical switches, and photonic materials. For production on an industrially viable scale, processes must be developed to form crystals with low defect densities, good long range order, and favorable kinetics. Application of a field to a concentrated colloidal suspension accelerates crystal formation. Ackerson et al. (Ackerson, 1991) established that systems with stress-based Peclet numbers above one resulted in crystal formation. We investigate formation of colloidal crystals by studying structural changes that occur upon shearing using confocal microscopy. Charge-stabilized poly(methylmethacrylate) particles (phi = 0.35) suspended in dioctyl phthalate were used for experiments. After application of shear, assembled structures were immobilized by UV exposure. The full sample thickness was imaged using confocal microscopy. Particle centroids were located in 3D by means of image processing and local crystallinity was quantified by application of local bond order parameter criteria (tenWolde, 1996). We present microstructural analysis of structures formed by both spin coating and uniform shear flow. Spin coating produces spatiotemporal variation in the ordering of concentrated colloidal dispersions that is a universal function of the local reduced critical stress and macroscopic strain. Samples produced at Peclet numbers greater than one and macroscopic strains above two resulted in crystal formation. A plot of the cryrstalline fraction versus Peclet number yielded a sharp order to disorder transition at Peclet number of order unity. The effect of volume fraction on the Peclet number theory was studied. Results indicated that the theory applied to volume fractions within the crystalline regime. Strain requirements for crystal formation of samples undergoing step strain deformation in a parallel plate geometry were investigated by applying stains of 1--300 to samples

  10. Evaluation of the presence of Enterococcus Faecalis in root cementum: A confocal laser scanning microscope analysis

    PubMed Central

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

    2014-01-01

    Aim: The aim of this study is to address the cause of persistent infection of root cementum by Enterococcus faecalis. Materials and Methods: A sample of 60 human single-rooted teeth were divided into three groups. Group I (control group) had no access opening and one-third of the apical root cementum was sealed using varnish. Group II had no preparation of teeth samples. In group III, apical root cementum was exposed to organic acid and roughened using diamond point to mimic apical resorption. After access opening in groups II and III, all teeth samples were sterilized using gamma irradiation (25 kGy). E. faecalis broth was placed in the root canal and apical one-third of the tooth was immersed in the broth for 8 weeks with alternate day refreshment followed by biomechanical preparation, obturation and coronal seal. Apical one-third of all teeth samples were again immersed in the broth for 8 weeks with alternate day refreshment to mimic secondary infection. The samples were observed under a confocal microscope after splitting the teeth into two halves. Results: E. faecalis penetrated 160 μm deep into the root cementum in group III samples and only showed adhesion in group II samples. Conclusion: Penetration and survival of E. faecalis deep inside the cementum in extreme conditions could be the reason for persistent infection. PMID:24778505

  11. Automated Confocal Laser Scanning Microscopy and Semiautomated Image Processing for Analysis of Biofilms

    PubMed Central

    Kuehn, Martin; Hausner, Martina; Bungartz, Hans-Joachim; Wagner, Michael; Wilderer, Peter A.; Wuertz, Stefan

    1998-01-01

    The purpose of this study was to develop and apply a quantitative optical method suitable for routine measurements of biofilm structures under in situ conditions. A computer program was designed to perform automated investigations of biofilms by using image acquisition and image analysis techniques. To obtain a representative profile of a growing biofilm, a nondestructive procedure was created to study and quantify undisturbed microbial populations within the physical environment of a glass flow cell. Key components of the computer-controlled processing described in this paper are the on-line collection of confocal two-dimensional (2D) cross-sectional images from a preset 3D domain of interest followed by the off-line analysis of these 2D images. With the quantitative extraction of information contained in each image, a three-dimensional reconstruction of the principal biological events can be achieved. The program is convenient to handle and was generated to determine biovolumes and thus facilitate the examination of dynamic processes within biofilms. In the present study, Pseudomonas fluorescens or a green fluorescent protein-expressing Escherichia coli strain, EC12, was inoculated into glass flow cells and the respective monoculture biofilms were analyzed in three dimensions. In this paper we describe a method for the routine measurements of biofilms by using automated image acquisition and semiautomated image analysis. PMID:9797255

  12. Reconstruction and representation of caudal vasculature of zebrafish embryo from confocal scanning laser fluorescence microscopic images.

    PubMed

    Feng, Jun; Cheng, Shuk Han; Chan, Po K; Ip, Horace H S

    2005-12-01

    Three-dimensional (3D) reconstruction from a series of sections is an important technique in medical imaging, particularly for visualization of blood vessels from angiography. Here, we present a framework for automatic segmentation and registration of different kind of blood vessels from 2-day-old zebrafish embryos. Series of optical sections were acquired from confocal microscopy with the blood vessels labeled by fluorescent microbeads (0.02 microm) injected into blood stream of 2-day-old zebrafish embryos. Blood vessels were extracted and their morphological parameters, including length and diameter, were calculated. At the same time, individual blood vessels were registered automatically. Vasculature was represented by attributed vessel represent graph (AVRG), which contained morphological data and connectivity of every blood vessel. Using AVRG to represent a vasculature made the comparison between vasculatures of different embryos more easy. Visualization, as well as quantification, of reconstructed 3D model of AVRG was presented in an interactive interface. The framework was implemented by Visual C++ as Windows-based program. PMID:16263106

  13. Sealing ability of three root-end filling materials prepared using an erbium: Yttrium aluminium garnet laser and endosonic tip evaluated by confocal laser scanning microscopy

    PubMed Central

    Nanjappa, A Salin; Ponnappa, KC; Nanjamma, KK; Ponappa, MC; Girish, Sabari; Nitin, Anita

    2015-01-01

    Aims: (1) To compare the sealing ability of mineral trioxide aggregate (MTA), Biodentine, and Chitra-calcium phosphate cement (CPC) when used as root-end filling, evaluated under confocal laser scanning microscope using Rhodamine B dye. (2) To evaluate effect of ultrasonic retroprep tip and an erbium:yttrium aluminium garnet (Er:YAG) laser on the integrity of three different root-end filling materials. Materials and Methods: The root canals of 80 extracted teeth were instrumented and obturated with gutta-percha. The apical 3 mm of each tooth was resected and 3 mm root-end preparation was made using ultrasonic tip (n = 30) and Er:YAG laser (n = 30). MTA, Biodentine, and Chitra-CPC were used to restore 10 teeth each. The samples were coated with varnish and after drying, they were immersed in Rhodamine B dye for 24 h. The teeth were then rinsed, sectioned longitudinally, and observed under confocal laser scanning microscope. Statistical Analysis Used: Data were analyzed using one-way analysis of variance (ANOVA) and a post-hoc Tukey's test at P < 0.05 (R software version 3.1.0). Results: Comparison of microleakage showed maximum peak value of 0.45 mm for Biodentine, 0.85 mm for MTA, and 1.05 mm for Chitra-CPC. The amount of dye penetration was found to be lesser in root ends prepared using Er:YAG laser when compared with ultrasonics, the difference was found to be statistically significant (P < 0.05). Conclusions: Root-end cavities prepared with Er:YAG laser and restored with Biodentine showed superior sealing ability compared to those prepared with ultrasonics. PMID:26180420

  14. Crystallization Behavior of Perovskite in the Synthesized High-Titanium-Bearing Blast Furnace Slag Using Confocal Scanning Laser Microscope

    NASA Astrophysics Data System (ADS)

    Hu, Meilong; Liu, Lu; Lv, Xuewei; Bai, Chenguang; Zhang, Shengfu

    2013-10-01

    The isothermal phase composition of high-titanium-bearing slag (23 mass pct TiO2) under an argon atmosphere during cooling process from 1723 K (1450 °C) was calculated by FactSage.6.3 (CRCT-ThermFact Inc., Montréal, Canada). Three main phases, which were perovskite, titania spinel, and clinopyroxene, could form during the cooling process and they precipitated at 1713 K, 1603 K, and 1498 K (1440 °C, 1330 °C, and 1225 °C), respectively. The nonisothermal crystallization process of perovskite in synthesized high-titanium-bearing slag was studied in situ by a confocal scanning laser microscope (CSLM) with cooling rate of 30 K/min. The results showed that the primary phase was perovskite that precipitated at 1703 K (1430 °C). The whole precipitation and growth process of perovskite was obtained, whereas other phases formed as glass under the current experimental conditions. Perovskite grew along a specific growth track and finally appeared with snowflake morphology. The growing kinetics of perovskite formation from molten slag were also mentioned.

  15. Direct observation of the asphaltene structure in paving-grade bitumen using confocal laser-scanning microscopy.

    PubMed

    Bearsley, S; Forbes, A; Haverkamp, R G

    2004-08-01

    The structure of the asphaltene phase in the bitumen is believed to have a significant effect on its rheological properties. It has traditionally been difficult to observe the asphaltene phase in unaltered samples of bitumen. The maltenes are thought to form a continuous phase in which the asphaltenes are 'dispersed'. In this study, confocal laser-scanning microscopy (CLSM) operating in fluorescence mode was used to examine the structure of paving-grade Safaniya and San Joaquin bitumen. The asphaltene fraction fluoresces in the 515-545 nm wavelength range when irradiated with light with a wavelength of 488 nm. The major advantages of CLSM are that the bitumen sample requires little pretreatment or preparation that may affect the original dispersion of asphaltenes and the bitumen is observed at ambient temperature and pressure. This reduces the possibility of producing images that are not representative of the original material. CLSM was able to show the distribution of maltene and asphaltene components in bitumen. The asphaltene aggregates in the bitumen were observed to be 2-7 micro m in size and formed a dispersed 'sol' structure in the continuous maltene matrix rather than a network 'gel' structure. Surprisingly, the structure and fluorescence of the asphaltene phase does not appear to alter radically upon oxidative ageing. The structure of the asphaltene phase of an AR4000 San Joaquin bitumen was found to be more homogeneous than that of Safaniya bitumen, illustrating the range of structures that can be observed in bitumens by this method. PMID:15315501

  16. Tracking features in retinal images of adaptive optics confocal scanning laser ophthalmoscope using KLT-SIFT algorithm.

    PubMed

    Li, Hao; Lu, Jing; Shi, Guohua; Zhang, Yudong

    2010-01-01

    With the use of adaptive optics (AO), high-resolution microscopic imaging of living human retina in the single cell level has been achieved. In an adaptive optics confocal scanning laser ophthalmoscope (AOSLO) system, with a small field size (about 1 degree, 280 μm), the motion of the eye severely affects the stabilization of the real-time video images and results in significant distortions of the retina images. In this paper, Scale-Invariant Feature Transform (SIFT) is used to abstract stable point features from the retina images. Kanade-Lucas-Tomasi(KLT) algorithm is applied to track the features. With the tracked features, the image distortion in each frame is removed by the second-order polynomial transformation, and 10 successive frames are co-added to enhance the image quality. Features of special interest in an image can also be selected manually and tracked by KLT. A point on a cone is selected manually, and the cone is tracked from frame to frame. PMID:21258443

  17. Precision Automation of Cell Type Classification and Sub-Cellular Fluorescence Quantification from Laser Scanning Confocal Images

    PubMed Central

    Hall, Hardy C.; Fakhrzadeh, Azadeh; Luengo Hendriks, Cris L.; Fischer, Urs

    2016-01-01

    While novel whole-plant phenotyping technologies have been successfully implemented into functional genomics and breeding programs, the potential of automated phenotyping with cellular resolution is largely unexploited. Laser scanning confocal microscopy has the potential to close this gap by providing spatially highly resolved images containing anatomic as well as chemical information on a subcellular basis. However, in the absence of automated methods, the assessment of the spatial patterns and abundance of fluorescent markers with subcellular resolution is still largely qualitative and time-consuming. Recent advances in image acquisition and analysis, coupled with improvements in microprocessor performance, have brought such automated methods within reach, so that information from thousands of cells per image for hundreds of images may be derived in an experimentally convenient time-frame. Here, we present a MATLAB-based analytical pipeline to (1) segment radial plant organs into individual cells, (2) classify cells into cell type categories based upon Random Forest classification, (3) divide each cell into sub-regions, and (4) quantify fluorescence intensity to a subcellular degree of precision for a separate fluorescence channel. In this research advance, we demonstrate the precision of this analytical process for the relatively complex tissues of Arabidopsis hypocotyls at various stages of development. High speed and robustness make our approach suitable for phenotyping of large collections of stem-like material and other tissue types. PMID:26904081

  18. Precision Automation of Cell Type Classification and Sub-Cellular Fluorescence Quantification from Laser Scanning Confocal Images.

    PubMed

    Hall, Hardy C; Fakhrzadeh, Azadeh; Luengo Hendriks, Cris L; Fischer, Urs

    2016-01-01

    While novel whole-plant phenotyping technologies have been successfully implemented into functional genomics and breeding programs, the potential of automated phenotyping with cellular resolution is largely unexploited. Laser scanning confocal microscopy has the potential to close this gap by providing spatially highly resolved images containing anatomic as well as chemical information on a subcellular basis. However, in the absence of automated methods, the assessment of the spatial patterns and abundance of fluorescent markers with subcellular resolution is still largely qualitative and time-consuming. Recent advances in image acquisition and analysis, coupled with improvements in microprocessor performance, have brought such automated methods within reach, so that information from thousands of cells per image for hundreds of images may be derived in an experimentally convenient time-frame. Here, we present a MATLAB-based analytical pipeline to (1) segment radial plant organs into individual cells, (2) classify cells into cell type categories based upon Random Forest classification, (3) divide each cell into sub-regions, and (4) quantify fluorescence intensity to a subcellular degree of precision for a separate fluorescence channel. In this research advance, we demonstrate the precision of this analytical process for the relatively complex tissues of Arabidopsis hypocotyls at various stages of development. High speed and robustness make our approach suitable for phenotyping of large collections of stem-like material and other tissue types. PMID:26904081

  19. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.

    PubMed

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

  20. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

    PubMed Central

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

  1. The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images.

    PubMed

    Hidayat, Budi J; Weisskopf, Carmen; Felby, Claus; Johansen, Katja S; Thygesen, Lisbeth G

    2015-12-01

    Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations. PMID:26626331

  2. Real-time in vivo confocal laser scanning microscopy of melanin-containing cells: A promising diagnostic intervention.

    PubMed

    Xiang, Wenzhong; Song, Xiuzu; Peng, Jianzhong; Xu, Aie; Bi, Zhigang

    2015-12-01

    The use of noninvasive imaging techniques to evaluate different types of skin lesions is increasing popular. In vivo confocal laser scanning microscopy (CLSM) is a new method for high resolution non-invasive imaging of intact skin in situ and in vivo. Although many studies have investigated melanin-containing cells in lesions by in vivo CLSM, few studies have systematically characterized melanin-containing cells based on their morphology, size, arrangement, density, borders, and brightness. In this study, the characteristics of melanin-containing cells were further investigated by in vivo CLSM. A total of 130 lesions, including common nevi, giant congenital pigmented nevi, vitiligo, melasma, melanoma, and chronic eczema, were imaged by in vivo CLSM. This research helps dermatologists understand the characteristics of melanin-containing cells and facilitate the clinical application of melanin-containing cells in the investigation of dermatological disease. In summary, melanin-containing cells include keratinocytes, melanocytes, macrophages, and melanocytic skin tumor cells. Our study presents the CLSM characteristics of melanin-containing cells to potentially facilitate in vivo diagnosis based on shape, size, arrangement, density, borders, and brightness. PMID:26515646

  3. Imaging genes, chromosomes, and nuclear structures using laser-scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

    Ballard, Stephen G.

    1990-08-01

    condensed metaphase chromosomes and in interphase nuclei. The ability to image the loci of fluorescent-labelled gene probes hybridized to chromosomes and to interphase nuclei will play a major role in the mapping of the human genome. This presentation is an overview of our laboratory's efforts to use confocal imaging to address fundamental questions about the structure and organization of genes, chromosomes and cell nuclei, and to develop applications useful in clinical diagnosis of inherited diseases.

  4. Direct observation by laser scanning confocal microscopy of microstructure and phase migration of PVC gels in an applied electric field.

    PubMed

    Xia, Hong; Ueki, Takamitsu; Hirai, Toshihiro

    2011-02-01

    The fluorescent probe lucigenin was incorporated in poly(vinyl chloride) (PVC) gels, and laser scanning confocal microscopy (LSCM) was used to clarify the internal structures of the gels. From the two-dimensional and three-dimensional information by LSCM, we first observed the internal structure of the PVC gel at a wet status, where the PVC gels comprised a polymer-rich phase and a polymer-poor phase uniformly with a three-dimensional network structure. After an electric field was applied, an effect of the electric field resulted in the change of internal structure in the gels. The polymer-poor phase moved from the cathode to the anode and the polymer-rich phase formed linelike arrangement between electrodes due to the attraction force. On the other hand, the freeze-dried PVC gels with/without in-situ dc voltage casting were particularly fabricated to confirm above results by the field emission scanning electron microscopy (FE-SEM). It was found that many craters remained on the surface of the gel near the anode due to sublimation in freeze-drying. This phenomenon did not appear on the surface near the cathode. The results of in-situ dc voltage casting also suggested that a substantial amount of polymer-poor phase was moved and fixed at the anode. Thus, results of both LSCM and in-situ dc voltage casting corresponded to the effect of electric field on PVC gels and provided a convincing evidence for the interpretation of the deformation mechanism of PVC gel actuators by an applied electric field. PMID:21174424

  5. Evaluation of penetration depth of 2% chlorhexidine digluconate into root dentinal tubules using confocal laser scanning microscope

    PubMed Central

    Latha, Jothi; Velmurugan, Natanasabapathy

    2015-01-01

    Objectives This study evaluated the penetration depth of 2% chlorhexidine digluconate (CHX) into root dentinal tubules and the influence of passive ultrasonic irrigation (PUI) using a confocal laser scanning microscope (CLSM). Materials and Methods Twenty freshly extracted anterior teeth were decoronated and instrumented using Mtwo rotary files up to size 40, 4% taper. The samples were randomly divided into two groups (n = 10), that is, conventional syringe irrigation (CSI) and PUI. CHX was mixed with Rhodamine B dye and was used as the final irrigant. The teeth were sectioned at coronal, middle and apical levels and viewed under CLSM to record the penetration depth of CHX. The data were statistically analyzed using Kruskal-Wallis and Mann-Whitney U tests. Results The mean penetration depths of 2% CHX in coronal, middle and apical thirds were 138 µm, 80 µm and 44 µm in CSI group, respectively, whereas the mean penetration depths were 209 µm, 138 µm and 72 µm respectively in PUI group. Statistically significant difference was present between CSI group and PUI group at all three levels (p < 0.01 for coronal third and p < 0.001 for middle and apical thirds). On intragroup analysis, both groups showed statistically significant difference among three levels (p < 0.001). Conclusions Penetration depth of 2% CHX into root dentinal tubules is deeper in coronal third when compared to middle and apical third. PUI aided in deeper penetration of 2% CHX into dentinal tubules when compared to conventional syringe irrigation at all three levels. PMID:25984477

  6. Mapping microclimate pH distribution inside protein-encapsulated PLGA microspheres using confocal laser scanning microscopy

    PubMed Central

    Liu, Yajun; Schwendeman, Steven P.

    2012-01-01

    The pH in the aqueous pores of poly(lactide-co-glycolide) (PLGA) matrix, also referred to microclimate pH (μpH), is often uncontrolled ranging from highly acidic to neutral pH range. The μpH distribution inside protein-encapsulated PLGA microspheres was quantitatively evaluated using confocal laser scanning microscopy. The fluorescent response of Lysosensor yellow/blue® dextran used to map μpH in PLGA was influenced by the presence of encapsulated protein. The nonprotonated form of pyridyl group on the fluorescence probe at neutral pH was responsible for the interference, which was dependent on the type and concentration of protein. A method for correction of this interference based on estimating protein concentration inside the microspheres was established and validated. After correction of the influence, the μpH distribution kinetics inside microspheres was evaluated for different PLGA 50/50 microsphere formulations under physiological conditions for 4 weeks. Generally, the μpH acidity increased with the progression of incubation time. The co-incorporation of poorly soluble base, magnesium carbonate, in the microspheres prolonged the appearance of detectable acidity for up to 3 weeks. Co-addition of an acetate buffer was able to control the μpH over a slightly acidic range (around pH 4.7) after two weeks incubation. Microspheres prepared from a lower polymer concentration exhibited a higher μpH, likely owing to reduced diffusional resistance to acidic degradation products. The stability of protein was enhanced by addition of MgCO3, acetate buffer, or by reduced polymer concentration in the preparation, as evidenced by more soluble protein recovered after incubation. Hence, the μpH imaging technique developed can be employed in the future for optimization of formulation strategies for controlling μpH and stabilizing encapsulated proteins. PMID:22428586

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

    PubMed Central

    Mueller, Lukas N; de Brouwer, Jody FC; Almeida, Jonas S; Stal, Lucas J; Xavier, João B

    2006-01-01

    Background Confocal laser scanning microscopy (CLSM) is the method of choice to study interfacial biofilms and acquires time-resolved three-dimensional data of the biofilm structure. CLSM can be used in a multi-channel modus where the different channels map individual biofilm components. This communication presents a novel image quantification tool, PHLIP, for the quantitative analysis of large amounts of multichannel CLSM data in an automated way. PHLIP can be freely downloaded from Results PHLIP is an open source public license Matlab toolbox that includes functions for CLSM imaging data handling and ten image analysis operations describing various aspects of biofilm morphology. The use of PHLIP is here demonstrated by a study of the development of a natural marine phototrophic biofilm. It is shown how the examination of the individual biofilm components using the multi-channel capability of PHLIP allowed the description of the dynamic spatial and temporal separation of diatoms, bacteria and organic and inorganic matter during the shift from a bacteria-dominated to a diatom-dominated phototrophic biofilm. Reflection images and weight measurements complementing the PHLIP analyses suggest that a large part of the biofilm mass consisted of inorganic mineral material. Conclusion The presented case study reveals new insight into the temporal development of a phototrophic biofilm where multi-channel imaging allowed to parallel monitor the dynamics of the individual biofilm components over time. This application of PHLIP presents the power of biofilm image analysis by multi-channel CLSM software and demonstrates the importance of PHLIP for the scientific community as a flexible and extendable image analysis platform for automated image processing. PMID:16412253

  8. Adhesion of rice flour-based batter to chicken drumsticks evaluated by laser scanning confocal microscopy and texture analysis.

    PubMed

    Mukprasirt, A; Herald, T J; Boyle, D L; Rausch, K D

    2000-09-01

    The convenience and appeal of battered or breaded products have resulted in a sales increase of 100% since 1980. Because of the rapid growth of the Asian-American population and increasing consumption of rice and rice products, rice flour is a logical alternative for wheat flour in traditional batter formulation. The effects of ingredients used in rice flour-based batters on adhesion characteristic for deep-fat fried chicken drumsticks were studied by laser scanning confocal microscopy (LSCM) and texture analysis. Raw chicken drumsticks were predusted with egg albumin powder before dipping into batters prepared from combinations of rice flour, yellow corn flour, oxidized cornstarch, methylcellulose, or xanthan gum. The drumsticks were fried at 175+/-5 C until the internal temperature reached at least 71 C. For LSCM, samples were fixed overnight and were sectioned by vibratome (200 microm) before viewing. Batter adhesion was determined using an attachment specifically designed for chicken drumsticks. Microstructural analysis showed that batter formulated with a 50:50 mixture of rice and corn flours adhered better to drumsticks than batter with other rice flour ratios. Xanthan gum (0.2%) or methylcellulose (0.3%) alone had poor adhesion to chicken skin. However, when combined with other ingredients, xanthan gum increased the amount of batter pick-up before frying by increasing viscosity. Egg albumin significantly facilitated batter adhesion. The results from texture analysis supported the microstructural studies. As rice flour ratio increased from 50 to 70%, the binding force decreased. Rice flour showed potential as an alternative to wheat flour for batter formulas when the appropriate levels of oxidized starch, xanthan gum, and methylcellulose were included in the formulation. PMID:11020085

  9. Characterization and quantification of wound-induced hair follicle neogenesis using in vivo confocal scanning laser microscopy

    PubMed Central

    Fan, Chengxiang; Luedtke, Michael A.; Prouty, Stephen M.; Burrows, Michelle; Kollias, Nikiforos

    2011-01-01

    Background In vivo confocal scanning laser microscopy (CSLM) is a recently-developed non-invasive technique for visualizing microscopic structures with the skin. CSLM has been used to characterize proliferative and inflammatory skin diseases, neoplastic skin lesions and pigmented lesions. Objective Here, we assessed the ability of CSLM to evaluate the formation of neogenic hair follicles after a full thickness wound in mice. Methods Full-thickness wounds were made on the dorsal skin of 3-week old mice. After scab detachment (SD), the number, width, length, space and volume of neogenic hair follicles were analyzed using CSLM. The results were compared with those from conventional methods, including staining for alkaline phosphatase (AP) and keratin 17 (K17) as well as histology. Results Quantification of neogenic hair follicles using CSLM compared favorably with results from direct measurements on isolated epidermal tissue after immunostaining for K17, a marker for the epithelial portion of new hair follicles. CSLM detected 89% of K17-stained follicles. CSLM more accurately quantitated the number of new follicles compared to AP staining, which detects the dermal portion of the new follicle. The width and length measurement from CSLM and histology were very close and correlated with each other. The minimum length of a neogenic hair follicle that could be detected by CSLM was 21 μm. The space between neogenic hair follicles was decreased in histological sections compared to CSLM. Conclusions CSLM is an accurate and valuable method for counting and measuring neogenic hair follicles non-invasively. CSLM produces images similar to histology in mice. Measurements of microstructures using CSLM more accurately reflect actual sizes since this technique avoids fixation artifact. In vivo visualization of developing follicles with CSLM permits detection of serial changes in hair follicle formation, thus conserving numbers of mice required for studies and improving detection of

  10. Mapping microclimate pH distribution inside protein-encapsulated PLGA microspheres using confocal laser scanning microscopy.

    PubMed

    Liu, Yajun; Schwendeman, Steven P

    2012-05-01

    The pH in the aqueous pores of poly(lactide-co-glycolide) (PLGA) matrix, also referred to as microclimate pH (μpH), is often uncontrolled, ranging from highly acidic to neutral pH range. The μpH distribution inside protein-encapsulated PLGA microspheres was quantitatively evaluated using confocal laser scanning microscopy. The fluorescent response of Lysosensor yellow/blue dextran used to map μpH in PLGA was influenced by the presence of encapsulated protein. The nonprotonated form of pyridyl group on the fluorescence probe at neutral pH was responsible for the interference, which was dependent on the type and concentration of protein. A method for correction of this interference based on estimating protein concentration inside the microspheres was established and validated. After correction of the influence, the μpH distribution kinetics inside microspheres was evaluated for different PLGA 50/50 microsphere formulations under physiological conditions for 4 weeks. Generally, the μpH acidity increased with the progression of incubation time. The coincorporation of poorly soluble base, magnesium carbonate, in the microspheres prolonged the appearance of detectable acidity for up to 3 weeks. Co-addition of an acetate buffer was able to control the μpH over a slightly acidic range (around pH 4.7) after two week incubation. Microspheres prepared from a lower polymer concentration exhibited a higher μpH, likely owing to reduced diffusional resistance to acidic degradation products. The stability of protein was enhanced by addition of MgCO(3), acetate buffer, or by reduced polymer concentration in the preparation, as evidenced by more soluble protein recovered after incubation. Hence, the μpH imaging technique developed can be employed in the future for optimization of formulation strategies for controlling μpH and stabilizing encapsulated proteins. PMID:22428586

  11. Scanning electron and confocal scanning laser microscopy imaging of the ultrastructure and viability of vaginal Candida albicans and non- albicans species adhered to an intrauterine contraceptive device.

    PubMed

    Paiva, Luciene C Farias; Donatti, Lucélia; Patussi, Eliana V; Svizdinski, Terezinha I E; Lopes-Consolaro, Márcia E

    2010-10-01

    Although bacterial biofilms have been studied in detail, adhesion of Candida albicans and non-albicans species to an intrauterine contraceptive device (IUD) is not clear. The objective of this study was to evaluate aspects of imaging of the ultrastructure and viability of vaginal yeasts adhered to different parts of an IUD, through scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). We studied yeasts isolated from different patients with vulvovaginal candidiasis: C. albicans, C. glabrata, C. guillermondii, C. parapsilosis, C. tropicalis, and Saccharomyces cerevisiae. A suspension of the each yeast was prepared and incubated with IUD parts (tail, without copper, and copper-covered). SEM and CSLM showed that all the vaginal yeasts adhered to all the parts of the IUD and demonstrated viability, including 30 days after contact for C. albicans. Possibly irregularities of IUD surface contribute to the adherence process. Although all of the IUD parts contribute to retention of yeasts in the genital tract, high concentration of yeast cells on the tail may indicate the importance of this segment in maintaining the colonization by yeast cells because the tail forms a bridge between the external environment, the vagina that is colonized by yeast cells, and the upper genital tract where there is no colonization. PMID:20804637

  12. 3D digital image processing for biofilm quantification from confocal laser scanning microscopy: Multidimensional statistical analysis of biofilm modeling

    NASA Astrophysics Data System (ADS)

    Zielinski, Jerzy S.

    The dramatic increase in number and volume of digital images produced in medical diagnostics, and the escalating demand for rapid access to these relevant medical data, along with the need for interpretation and retrieval has become of paramount importance to a modern healthcare system. Therefore, there is an ever growing need for processed, interpreted and saved images of various types. Due to the high cost and unreliability of human-dependent image analysis, it is necessary to develop an automated method for feature extraction, using sophisticated mathematical algorithms and reasoning. This work is focused on digital image signal processing of biological and biomedical data in one- two- and three-dimensional space. Methods and algorithms presented in this work were used to acquire data from genomic sequences, breast cancer, and biofilm images. One-dimensional analysis was applied to DNA sequences which were presented as a non-stationary sequence and modeled by a time-dependent autoregressive moving average (TD-ARMA) model. Two-dimensional analyses used 2D-ARMA model and applied it to detect breast cancer from x-ray mammograms or ultrasound images. Three-dimensional detection and classification techniques were applied to biofilm images acquired using confocal laser scanning microscopy. Modern medical images are geometrically arranged arrays of data. The broadening scope of imaging as a way to organize our observations of the biophysical world has led to a dramatic increase in our ability to apply new processing techniques and to combine multiple channels of data into sophisticated and complex mathematical models of physiological function and dysfunction. With explosion of the amount of data produced in a field of biomedicine, it is crucial to be able to construct accurate mathematical models of the data at hand. Two main purposes of signal modeling are: data size conservation and parameter extraction. Specifically, in biomedical imaging we have four key problems

  13. Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

    PubMed Central

    Kumar, Deepak; Sahoo, Dipak K.; Maiti, Indu B.; Dey, Nrisingha

    2011-01-01

    Background Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. Methodology/Principal Findings We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, −271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. Conclusion and Significance We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety

  14. Spiral ganglion neuron quantification in the guinea pig cochlea using Confocal Laser Scanning Microscopy compared to embedding methods.

    PubMed

    Wrzeszcz, Antonina; Reuter, Günter; Nolte, Ingo; Lenarz, Thomas; Scheper, Verena

    2013-12-01

    Neuron counting in the cochlea is a crucial but time-consuming operation for which various methods have been developed. To improve simplicity and efficiency, we tested an imaging method of the cochlea, and based on Confocal Laser Scanning Microscopy (CLSM), we visualised Rosenthal's Canal and quantified the spiral ganglion neurons (SGN) within. Cochleae of 8 normal hearing guinea pigs and one implanted with a silicone filament were fixed in paraformaldehyde (PFA), decalcified, dehydrated and cleared in Spalteholz solution. Using the tissue's autofluorescence, CLSM was performed at 100 fold magnification generating z-series stacks of about 20 slices of the modiolus. In 5 midmodiolar slices per cochlea the perimeters of the Rosenthal's Canal were surveyed, representative neuron diameters were measured and the neurons first counted manually and then software-assisted. For comparison, 8 normal hearing guinea pig cochleae were embedded in paraffin and examined similarly. The CLSM method has the advantage that the cochleae remain intact as an organ and keep their geometrical structure. Z-stack creation is nearly fully-automatic and frequently repeatable with various objectives and step sizes and without visible bleaching. The tissue shows minimal or no shrinking artefacts and damage typical of embedding and sectioning. As a result, the cells in the cleared cochleae reach an average diameter of 21 μm and a density of about 18 cells/10,000 μm(2) with no significant difference between the manual and the automatical counts. Subsequently we compared the CLSM data with those generated using the established method of paraffin slides, where the SGN reached a mean density of 9.5 cells/10,000 μm(2) and a mean soma diameter of 13.6 μm. We were able to prove that the semi-automatic CLSM method is a simple and effective technique for auditory neuron count. It provides a high grade of tissue preservation and the automatic stack-generation as well as the counter software reduces

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

    NASA Astrophysics Data System (ADS)

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

    2008-10-01

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

  16. Rhodamine 123 phototoxicity in laser-irradiated MGH-U1 human carcinoma cells studied in vitro by electron microscopy and confocal laser scanning microscopy

    SciTech Connect

    Shea, C.R.; Sherwood, M.E.; Flotte, T.J.; Chen, N.; Scholz, M.; Hasan, T. )

    1990-07-01

    Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that localizes preferentially within mitochondria of living carcinoma cells. MGH-U1 human bladder carcinoma cells incubated in vitro with 10 microM R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser underwent selective, phototoxic injury to mitochondria. Ultrastructurally, treatment with R123 plus irradiation with 10 J/cm2 caused selective, progressive mitochondrial alterations consisting of disruption of cristae, vacuolization, swelling, increasing numbers of ring-shaped and angulated mitochondria at 4 to 8 h after irradiation, and obliteration of many mitochondria at 24 to 48 h. Confocal laser scanning microscopy after treatment with R123 plus irradiation with 10 to 30 J/cm2 demonstrated altered uptake and localization of subsequently administered R123, accompanied by striking mitochondrial fragmentation. Irradiation caused a dose-dependent depletion of extractable R123, due to a photosensitized efflux that began immediately and progressed by 4 h after irradiation with 10 to 30 J/cm2; further uptake after reincubation in the presence of R123 was also quantitatively impaired in cells previously irradiated with 30 J/cm2.

  17. Confocal Laser Induced Fluorescence of Argon Plasmas

    NASA Astrophysics Data System (ADS)

    Scime, Earl; Soderholm, Mark

    2015-11-01

    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 pair of axicon lenses create an annular beam path for the emission collection and the pump laser light is confined inside the annulus of the collection beam. The measurement location is scanned radially by manually adjusting the final focusing lens position. Here we present optical modeling of and initial results from the axicon based confocal optical system. The confocal measurements are compared to traditional, two-port, LIF measurements over the same radial range. This work is supported by US National Science Foundation grant number PHY-1360278.

  18. Laser differential confocal radius measurement.

    PubMed

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

    2010-02-01

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

  19. Biofilm forming capacity of Enterococcus faecalis on Gutta-percha points treated with four disinfectants using confocal scanning laser microscope: An in vitro study

    PubMed Central

    Ravi Chandra, Polavarapu Venkata; Kumar, Vemisetty Hari; Reddy, Surakanti Jayaprada; Kiran, Dandolu Ram; Krishna, Muppala Nagendra; Kumar, Golla Vinay

    2015-01-01

    Background: The aim of this study was to evaluate and compare the in vitro biofilm forming capacity of Enterococcus faecalis on Gutta-percha points disinfected with four disinfectants. Materials and Methods: A total of 50 Gutta-percha points used in this study were divided into four test groups based on disinfectant (5.25% sodium hypochlorite, 2% chlorhexidine gluconate, 20% neem, 13% benzalkonium chloride [BAK]), and one control group. The Gutta-percha points were initially treated with corresponding disinfectants followed by anaerobic incubation in Brain Heart Infusion broth suspended with human serum and E. faecalis strain for 14 days. After incubation, these Gutta-percha points were stained with Acridine Orange (Sigma – Aldrich Co., St. Louis, MO, USA) and 0.5 mm thick cross section samples were prepared. The biofilm thickness of E. faecalis was analyzed quantitatively using a confocal scanning laser microscope. Results statistically analyzed using analysis of variance. P < 0.05 was considered to be significant. Results: Confocal scanning laser microscope showed reduced amount of E. faecalis biofilm on Gutta-percha points treated with BAK and sodium hypochlorite. Post-hoc (least square differences) test revealed that there is no statistically significant difference between BAK and sodium hypochlorite groups (P > 0.05). Conclusion: This study illustrates that the Gutta-percha points disinfected with sodium hypochlorite and BAK showed minimal biofilm growth on its surface. PMID:26288622

  20. Confocal scanning laser microscopy with complementary 3D image analysis allows quantitative studies of functional state of ionoregulatory cells in the Nile tilapia (Oreochromis niloticus) following salinity challenge.

    PubMed

    Fridman, Sophie; Rana, Krishen J; Bron, James E

    2013-04-01

    The development of a novel three-dimensional image analysis technique of stacks generated by confocal laser scanning microscopy is described allowing visualization of mitochondria-rich cells (MRCs) in the seawater-adapted Nile tilapia in relation to their spatial location. This method permits the assessment and classification of both active and nonactive MRCs based on the distance of the top of the immunopositive cell from the epithelial surface. In addition, this technique offers the potential for informative and quantitative studies, for example, densitometric and morphometric measurements based on MRC functional state. Confocal scanning laser microscopy used with triple staining whole-mount immunohistochemistry was used to detect integumental MRCs in the yolk-sac larvae tail of the Nile tilapia following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt. Mean active MRC volume was always significantly larger and displayed a greater staining intensity (GLM; P<0.05) than nonactive MRCs. Following transfer, the percentage of active MRCs was seen to increase as did MRC volume (GLM; P<0.05). PMID:23390074

  1. High-temperature laser-scanning confocal microscopy as a tool to study the interface instability during unsteady-state solidification of low-carbon steel.

    PubMed

    Niknafs, S; Phelan, D; Dippenaar, R

    2013-01-01

    Solidification microstructure is a defining link between production techniques and the mechanical properties of metals and in particular steel. Due to the difficulty of conducting solidification studies at high temperature, knowledge of the development of solidification microstructure in steel is scarce. In this study, a laser-scanning confocal microscopy (LSCM) has been used to observe in situ and in real-time the planar to cellular to dendritic transition of the progressing solid/liquid interface in low carbon steel. Because the in situ observations in the laser-scanning confocal microscopy are restricted to the surface, the effect of sample thickness on surface observations was determined. Moreover, the effect of cooling rate and alloy composition on the planar to cellular interface transition was investigated. In the low-alloyed, low-carbon steel studied, the cooling rate does not seem to have an effect on the spacing of the cellular microstructure. However, in the presence of copper and manganese, the cell spacing decreased at higher cooling rates. Higher concentrations of copper in steel resulted on an increased cell spacing at the same cooling rates. PMID:23170969

  2. A novel confocal line scanning sensor

    NASA Astrophysics Data System (ADS)

    Chanbai, Sirichanok; Wiora, Georg; Weber, Mark; Roth, Hubert

    2009-05-01

    Optical methods, including confocal microscopes, are widely used for measurements of surface topography. The knowledge of surface morphology and roughness parameters is crucial for many applications, i.e. in industrial and automotive environment, in tribology, wear and functionality prediction. However, confocal microscopy has a limited field of view. A time consuming stitching process is required for extending to long profile lines measurement. Therefore, in this paper we present a novel concept of a Confocal Line Scanning Sensor (CLSS) to cover theoretically infinite profile lengths. The new technique is proposed with no moving parts required for axial scanning, and it has a simpler setup than those of Chromatic Confocal Sensor (CCS). The idea is to produce a stack of focal points on an inclined plane covering a certain axial measurement range. Therefore, by scanning the stack of focal points in lateral direction we can realize a long profile line. By doing that we expect to achieve shorter scanning time, while providing high lateral and axial resolution by using a true confocal principle. A long profile line of a few ten millimeters with a lateral resolution in sub-micrometer range and an axial resolution in tens of nanometers can be expected. Moreover, this concept is easily extensible to an areal measurement. Among other key components, a new design of the pinhole mask has been developed. We design it to produce an inclined focal line with optimum optical parameters. Optimization of the pinhole design fulfills two objectives; minimizing its size by allowing optimal reflected-light intensity, and minimizing crosstalk between nearby pinholes. Further detail of the pinhole design is beyond a scope of this paper. In this paper an overview of the new concept is presented, accompanied by validation of first experimental results.

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

  4. Atomic force microscopy and laser confocal scanning microscopy analysis of callose fibers developed from protoplasts of embryogenic cells of a conifer.

    PubMed

    Fukumoto, Takeshi; Hayashi, Noriko; Sasamoto, Hamako

    2005-12-01

    Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 mum, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for beta-1,4-glucan and beta-1,3-glucan. PMID:16034590

  5. Time-variant analysis of organelle and vesicle movement during phagocytosis in Paramecium primaurelia by means of fluorescence confocal laser scanning microscopy.

    PubMed

    Ramoino, P; Beltrame, F; Diaspro, A; Fato, M

    1996-12-01

    Vital fluorescent dyes (FITC-albumin, Texas Red-albumin, and acridine orange) were used together with a confocal laser scanning optical microscope (CLSM) to display and analyze formation, movement, and fusion of vesicles during the phagocytosis of Paramecium primaurelia, in the x-y-z-t space. By immobilizing living cells pulsed with a food vacuole marker at successive times after chasing in unlabeled medium, the intracellular movement of food vacuoles from their formation at the cytostome to their egestion at the cytoproct was visualized, and food vacuoles were selected in a specific digestion stage. Small pinocytic vesicles are shown to evaginate from the vacuoles and move in the cytoplasm. These vesicles are transported toward the cytopharynx where they enlarge the membrane of the nascent food vacuoles or fuse with stage II food vacuoles, when the vacuoles of stage II increase their size, changing from an acidic to an alkaline status. A multimodal analysis of confocal fluorescence images and the false-color technique were used to visualize vesicle movement vs. time. Starting from three images of the same cell at succeeding time points, a composite image was generated by associating with each originally acquired image a different color corresponding to each sampling point in time. The composite image shows that vesicles move away from the food vacuole in a scattered manner exhibiting changes in direction. PMID:8989767

  6. Video-rate Scanning Confocal Microscopy and Microendoscopy

    PubMed Central

    Nichols, Alexander J.; Evans, Conor L.

    2011-01-01

    Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9. Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples. Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will

  7. Video-rate scanning confocal microscopy and microendoscopy.

    PubMed

    Nichols, Alexander J; Evans, Conor L

    2011-01-01

    Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets, monitor dynamics in living cells, and visualize the three dimensional evolution of entire organisms. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo and are currently being applied to disease imaging and diagnosis in clinical settings. Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples. Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not

  8. In vivo molecular imaging of somatostatin receptors in pancreatic islet cells and neuroendocrine tumors by miniaturized confocal laser-scanning fluorescence microscopy.

    PubMed

    Fottner, C; Mettler, E; Goetz, M; Schirrmacher, E; Anlauf, M; Strand, D; Schirrmacher, R; Klöppel, G; Delaney, P; Schreckenberger, M; Galle, P R; Neurath, M F; Kiesslich, R; Weber, M M

    2010-05-01

    The aim of the study was to evaluate real time in vivo molecular imaging of somatostatin receptors (sstrs) using a handheld miniaturized confocal laser scan microscope (CLM) in conjunction with fluorescein-labeled octreotate (OcF) in healthy mice and murine models of neuroendocrine tumors. For CLM a small rigid probe (diameter 7 mm) with an integrated single line laser (488 nm) was used (optical slice thickness 7 mum; lateral resolution 0.7 mum). OcF was synthesized via Fmoc solid-phase peptide synthesis and purified by HPLC showing high-affinity binding to the sstr2 (IC(50) 6.2 nmol). For in vitro evaluation, rat and human pancreatic cancer cells were used and characterized with respect to its sstr subtype expression and functional properties. For in vivo confocal imaging, healthy mouse pancreatic islet and renal tubular cells as well as immunoincompetent nude mice harboring sstr-expressing tumors were evaluated. Incubation of sstr-positive cells with OcF showed a specific time- and dose-dependent staining of sstr-positive cells. CLM showed rapid internalization and homogenous cytoplasmatic distribution. After systemic application to mice (n = 8), specific time-dependent internalization and cytoplasmatic distribution into pancreatic islet cells and tubular cells of the renal cortex was recorded. After injection in tumor-harboring nude mice (n = 8), sstr-positive cells selectively displayed a cell surface and cytoplasmatic staining. CLM-targeted biopsies detected sstr-positive tumor cells with a sensitivity of 87.5% and a specificity of 100% as correlated with ex vivo immunohistochemistry. CLM with OcF permits real-time molecular, functional, and morphological imaging of sstr-expressing cell structures, allowing the specific visualization of pancreatic islet cells and neuroendocrine tumors in vivo. PMID:20233796

  9. The Superficial Stromal Scar Formation Mechanism in Keratoconus: A Study Using Laser Scanning In Vivo Confocal Microscopy

    PubMed Central

    Song, Peng; Wang, Shuting; Zhang, Peicheng; Sui, Wenjie; Zhang, Yangyang; Liu, Ting; Gao, Hua

    2016-01-01

    To investigate the mechanism of superficial stromal scarring in advanced keratoconus using confocal microscopy, the keratocyte density, distribution, micromorphology of corneal stroma, and SNP in three groups were observed. Eight corneal buttons of advanced keratoconus were examined by immunohistochemistry. The keratocyte densities in the sub-Bowman's stroma, anterior stroma, and posterior stroma and the mean SNP density were significantly different among the three groups. In the mild-to-moderate keratoconus group, activated keratocyte nuclei and comparatively highly reflective ECM were seen in the sub-Bowman's stroma, while fibrotic structures with comparatively high reflection were visible in the anterior stroma in advanced keratoconus. The alternating dark and light bands in the anterior stroma of the mild-to-moderate keratoconus group showed great variability in width and direction. The wide bands were localized mostly in the posterior stroma that corresponded to the Vogt striae in keratoconus and involved the anterior stroma only in advanced keratoconus. Histopathologically, high immunogenicity of α-SMA, vimentin, and FAP was expressed in the region of superficial stromal scarring. In vivo confocal microscopy revealed microstructural changes in the keratoconic cone. The activation of superficial keratocytes and abnormal remodeling of ECM may both play a key role in the superficial stromal scar formation in advanced keratoconus. PMID:26885515

  10. Structural characterization by confocal laser scanning microscopy and electrochemical study of multi-walled carbon nanotube tyrosinase matrix for phenol detection.

    PubMed

    Guix, Maria; Pérez-López, Briza; Sahin, Melike; Roldán, Mònica; Ambrosi, Adriano; Merkoçi, Arben

    2010-08-01

    A novel visualization methodology based on the use of immunofluorescence and Confocal Laser Scanning Microscopy (CLSM) was used to quantify and visualize tyrosinase enzyme within a MWCNTs matrix immobilized onto carbon based screen-printed electrodes. CLSM was shown to be an extremely powerful technique which allowed a clear visualization of the distribution of the enzyme within both the MWCNTs and carbon based layers and provided additional and useful morphological data for a better understanding of the interaction between biomolecules and electrode materials. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) were also employed to fully characterize the system components. The proposed MWCNT/Tyrosinase matrix was applied to the detection of phenol, as an alternative biosensor material. Electrochemical analytical performances of the biosensor were investigated in order to determine the optimal fabrication design along with the enzyme stability. The biosensor based on the developed biomaterial matrix proved promising results in terms of cost, simplicity and analytical performance. A detection limit of 1.35 microM and a sensitivity of 47.4 microA mM(-1) within a linear response range of 2.5 to 75 microM phenol were obtained. The biosensor performed well as a disposable device and could be stored in a refrigerator (-18 degrees C) without loss of activity for up to 2 months. PMID:20532304

  11. Classification of nanoparticle diffusion processes in vital cells by a multifeature random forests approach: application to simulated data, darkfield, and confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Wagner, Thorsten; Kroll, Alexandra; Wiemann, Martin; Lipinski, Hans-Gerd

    2016-04-01

    Darkfield and confocal laser scanning microscopy both allow for a simultaneous observation of live cells and single nanoparticles. Accordingly, a characterization of nanoparticle uptake and intracellular mobility appears possible within living cells. Single particle tracking makes it possible to characterize the particle and the surrounding cell. In case of free diffusion, the mean squared displacement for each trajectory of a nanoparticle can be measured which allows computing the corresponding diffusion coefficient and, if desired, converting it into the hydrodynamic diameter using the Stokes-Einstein equation and the viscosity of the fluid. However, within the more complex system of a cell's cytoplasm unrestrained diffusion is scarce and several other types of movements may occur. Thus, confined or anomalous diffusion (e.g. diffusion in porous media), active transport, and combinations thereof were described by several authors. To distinguish between these types of particle movement we developed an appropriate classification method, and simulated three types of particle motion in a 2D plane using a Monte Carlo approach: (1) normal diffusion, using random direction and step-length, (2) subdiffusion, using confinements like a reflective boundary with defined radius or reflective objects in the closer vicinity, and (3) superdiffusion, using a directed flow added to the normal diffusion. To simulate subdiffusion we devised a new method based on tracks of different length combined with equally probable obstacle interaction. Next we estimated the fractal dimension, elongation and the ratio of long-time / short-time diffusion coefficients. These features were used to train a random forests classification algorithm. The accuracy for simulated trajectories with 180 steps was 97% (95%-CI: 0.9481-0.9884). The balanced accuracy was 94%, 99% and 98% for normal-, sub- and superdiffusion, respectively. Nanoparticle tracking analysis was used with 100 nm polystyrene particles

  12. The determination of firing distance applying a microscopic quantitative method and confocal laser scanning microscopy for detection of gunshot residue particles.

    PubMed

    Neri, Margherita; Turillazzi, Emanuela; Riezzo, Irene; Fineschi, Vittorio

    2007-07-01

    In this study, we applied a microscopic quantitative method based on the use of sodium rhodizonate to verify the presence of residues and their distribution on the cutis of gunshot wounds. A total of 250 skin samples were selected from cases in which the manner of death (accidental, suicide, and homicide) and the shooting distance could be reliably determined. The samples were examined under a light microscope, in transmitted bright field illumination and phase contrast mode, and with confocal laser scanning microscopy. In all skin specimens the area of each histological section was directly measured by an image analysis system. Both the number and the size of powder particles were measured. The distribution of gunshot residues (GSR) in the epidermal and subepidermal layers was also analyzed. The evaluation of the microscopic entrance wounds demonstrated different findings related to the range of fire. The data derived from the evaluation of both macroscopic and microscopic features demonstrated that the amount and the spatial distribution of GSR deposits in the skin surrounding entrance wounds strictly correlate with shooting distance. PMID:16862444

  13. Measurement of the retinal arteriolar response to a hyperoxic provocation in nonsmokers and smokers, using a high-resolution confocal scanning laser ophthalmoscope

    NASA Astrophysics Data System (ADS)

    O' Halloran, Margaret; O'Donoghue, Eamonn; Dainty, Chris

    2014-07-01

    We used a high-resolution confocal scanning laser ophthalmoscope to measure the magnitude of change in retinal arteriolar diameters in response to oxygen breathing in young, healthy nonsmokers and smokers. Image sequences were obtained before and during oxygen breathing. Image sequences were desinusoided, registered, and averaged, before vessel diameters were measured using a sliding linear regression filter. Arteriole diameters were observed to constrict during the first 5 min. of oxygen breathing, plateau, and remain stable while hyperoxia was maintained, returning to baseline at the end of the hyperoxic period. Blood flow to the temporal retina was found to be higher than to the nasal retina (p=0.008). The percentage constriction of vessels did not vary across retinal quadrants (p=0.372, analysis of variance) and did not depend on vessel size (p=0.538). Baseline diameters were unaffected by acute cigarette smoking. The magnitude of vasoconstriction was diminished in smokers compared to nonsmokers (p=0.017), while acute smoking did not influence the percentage constriction attained by the vessels (p=0.621). Using a high-resolution imaging technique allowed us to measure reactivity to a high degree of accuracy and to assess it in vessels of smaller caliber than were previously studied.

  14. Attachment of Escherichia coli O157:H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demonstrated by using confocal scanning laser microscopy.

    PubMed

    Seo, K H; Frank, J F

    1999-01-01

    Confocal scanning laser microscopy was used to observe the location of Escherichia coli O157:H7 on and within lettuce leaves. Sections of leaves (ca. 0.5 by 0.5 cm) were inoculated by submersion in a suspension of E. coli O157:H7 (ca. 10(7) to 10(8) CFU/ml) overnight at 7 degrees C. Fluorescein isothiocyanate-labeled antibody was used to visualize the attached bacteria. E. coli O157:H7 was found attached to the surface, trichomes, stomata, and cut edges. Three-dimensional volume reconstruction of interior portions of leaves showed that E. coli O157:H7 was entrapped 20 to 100 microm below the surface in stomata and cut edges. Agar plate culturing and microscopic observation indicated that E. coli O157:H7 preferentially attached to cut edges, as opposed to the intact leaf surface. Dual staining with fluorescein isothiocyanate-labeled antibody and propidium iodide was used to determine viability of cells on artificially contaminated lettuce leaves after treatment with 20 mg/liter chlorine solution for 5 min. Many live cells were found in stomata and on cut edges following chlorine treatment. E. coli O157:H7 did not preferentially adhere to biofilm produced by Pseudomonas fluorescens on the leaf surface. In contrast to E. coli O157:H7, Pseudomonas adhered to and grew mainly on the intact leaf surface rather than on the cut edges. PMID:9921820

  15. Benford's Law based detection of latent fingerprint forgeries on the example of artificial sweat printed fingerprints captured by confocal laser scanning microscopes

    NASA Astrophysics Data System (ADS)

    Hildebrandt, Mario; Dittmann, Jana

    2015-03-01

    The possibility of forging latent fingerprints at crime scenes is known for a long time. Ever since it has been stated that an expert is capable of recognizing the presence of multiple identical latent prints as an indicator towards forgeries. With the possibility of printing fingerprint patterns to arbitrary surfaces using affordable ink- jet printers equipped with artificial sweat, it is rather simple to create a multitude of fingerprints with slight variations to avoid raising any suspicion. Such artificially printed fingerprints are often hard to detect during the analysis procedure. Moreover, the visibility of particular detection properties might be decreased depending on the utilized enhancement and acquisition technique. In previous work primarily such detection properties are used in combination with non-destructive high resolution sensory and pattern recognition techniques to detect fingerprint forgeries. In this paper we apply Benford's Law in the spatial domain to differentiate between real latent fingerprints and printed fingerprints. This technique has been successfully applied in media forensics to detect image manipulations. We use the differences between Benford's Law and the distribution of the most significant digit of the intensity and topography data from a confocal laser scanning microscope as features for a pattern recognition based detection of printed fingerprints. Our evaluation based on 3000 printed and 3000 latent print samples shows a very good detection performance of up to 98.85% using WEKA's Bagging classifier in a 10-fold stratified cross-validation.

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

  17. Novel Application of Confocal Laser Scanning Microscopy and 3D Volume Rendering toward Improving the Resolution of the Fossil Record of Charcoal

    PubMed Central

    Belcher, Claire M.; Punyasena, Surangi W.; Sivaguru, Mayandi

    2013-01-01

    Variations in the abundance of fossil charcoals between rocks and sediments are assumed to reflect changes in fire activity in Earth’s past. These variations in fire activity are often considered to be in response to environmental, ecological or climatic changes. The role that fire plays in feedbacks to such changes is becoming increasingly important to understand and highlights the need to create robust estimates of variations in fossil charcoal abundance. The majority of charcoal based fire reconstructions quantify the abundance of charcoal particles and do not consider the changes in the morphology of the individual particles that may have occurred due to fragmentation as part of their transport history. We have developed a novel application of confocal laser scanning microscopy coupled to image processing that enables the 3-dimensional reconstruction of individual charcoal particles. This method is able to measure the volume of both microfossil and mesofossil charcoal particles and allows the abundance of charcoal in a sample to be expressed as total volume of charcoal. The method further measures particle surface area and shape allowing both relationships between different size and shape metrics to be analysed and full consideration of variations in particle size and size sorting between different samples to be studied. We believe application of this new imaging approach could allow significant improvement in our ability to estimate variations in past fire activity using fossil charcoals. PMID:23977267

  18. In situ detection of the Zn(2+) release process of ZnO NPs in tumour cells by confocal laser scanning fluorescence microscopy.

    PubMed

    Song, Wenshuang; Tang, Xiaoling; Li, Yong; Sun, Yang; Kong, Jilie; Qingguang, Ren

    2016-08-01

    The use of zinc oxide (ZnO) nanoparticles (NPs) for cancer is not yet clear for human clinical applications, which is primarily due to the lack of a better understanding of the action mechanisms and cellular consequences of the direct exposure of cells to these NPs. In this work, the authors have selected zinquin ethyl ester, a Zn(2+)-specific fluorescent molecular probe, to efficiently differentiate ZnO NPs and Zn(2+), and combined with confocal laser scanning microscopy (CLSM) to in situ study the Zn(2+) release process of ZnO NPs in cancer cell system through detecting the change of Zn(2+) level over time. During the experiments, the authors have designed the test group ZnO-2 in addition to assess the influence of a long-term storage on the characteristics of ZnO NPs in aqueous solution, and the Zn(2+) release process of ZnO NPs in cancer cell system. After three-month storage at room temperature, the release process became earlier and faster, which was consistent with previous results of transmission electron microscope, UV-Vis and PL spectra. It is a good detection method that combination of Zn(2+)-specific fluorescent molecular probe and CLSM, which will be helpful for ZnO NPs using in clinical research. PMID:27463786

  19. Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part II. Impact on chromatographic separations.

    PubMed

    Hubbuch, Jürgen; Linden, Thomas; Knieps, Esther; Thömmes, Jörg; Kula, Maria-Regina

    2003-12-22

    The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent. PMID:14735979

  20. Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions.

    PubMed

    Valle, Edith R; Henderson, Gemma; Janssen, Peter H; Cox, Faith; Alexander, Trevor W; McAllister, Tim A

    2015-06-01

    In this study, methanogen-specific coenzyme F420 autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis. PMID:25924182

  1. Differentiation of Methanosaeta concilii and Methanocarcina barkeri in anaerobic mesophilic granular sludge by fluorescent in situ hybridization and confocal scanning laser microscopy

    SciTech Connect

    Rocheleau, S.; Greer, C.W.; Cantin, C.; Laramee, L.; Guiot, S.R.; Lawrence, J.R.

    1999-05-01

    Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of al mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.

  2. Serotonin-immunoreactive neurones in the visual system of the praying mantis: an immunohistochemical, confocal laser scanning and electron microscopic study.

    PubMed

    Leitinger, G; Pabst, M A; Kral, K

    1999-03-27

    The distribution, number, and morphology of serotonin-immunoreactive (5-HTi) neurones in the optic lobe of the praying mantis Tenodera sinensis were studied using conventional microscopy and confocal laser scanning microscopy. Five or six 5-HTi neurones connect the lobula complex with the medulla, and at least 50 5-HTi neurones appear to be confined to the medulla. In addition, a few large 5-HTi processes from the protocerebrum supply the lobula complex, and two large 5-HTi processes from the protocerebrum ramify in the medulla and lamina, where they show wide field arborisations. In order to provide a basis for understanding the action of serotonin in the lamina, the ultrastructure of its 5-HTi terminals was examined by conventional and immunohistochemical electron microscopy. The 5-HTi profiles were filled with dense core vesicles and made synapses. Output synapses from 5-HTi profiles outnumbered inputs by about 3 to 1. The terminals of the 5-HTi neurones were in close contact with cells of various types, including large monopolar cells, but close apposition to photoreceptor terminals was rare, and no synapses were found between 5-HTi terminals and photoreceptor terminals. PMID:10095007

  3. Comparison of fungiform taste-bud distribution among age groups using confocal laser scanning microscopy in vivo in combination with gustatory function.

    PubMed

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

    2016-04-01

    The aim of this study was to compare the distribution of taste buds in fungiform papillae (FP) and gustatory function between young and elderly age groups. Confocal laser scanning microscopy was used because it allows many FP to be observed non-invasively in a short period of time. The age of participants (n = 211) varied from 20 to 83 yr. The tip and midlateral region of the tongue were observed. Taste buds in an average of 10 FP in each area were counted. A total of 2,350 FP at the tongue tip and 2,592 FP in the midlateral region could be observed. The average number of taste buds was similar among all age groups both at the tongue tip and in the midlateral region. The taste function, measured by electrogustometry, among participants 20-29 yr of age was significantly lower than that in the other age groups; however, there was no difference among any other age groups in taste function. These results indicate that the peripheral gustatory system is well maintained anatomically and functionally in elderly people. PMID:26917278

  4. Evaluation of the extracellular polymeric substances by confocal laser scanning microscopy in conventional activated sludge and advanced membrane bioreactors treating hospital wastewater.

    PubMed

    Alrhmoun, Mousaab; Carrion, Claire; Casellas, Magali; Dagot, Christophe

    2014-01-01

    Confocal laser scanning microscopy (CLSM) combined with fluorescent viability indicators, was used in this study to investigate the impact of hospital wastewaters on floc structure and composition. In this work, three pilot-scale projects, two membrane bioreactors (MBRs) with a submerged or external membrane bioreactor and a conventional activated sludge, were installed and operated for 65 days. They were fed with an influent sampled directly from the hospital drainage system, which contained micropollutant concentrations ranging from ng/L to mg/L. Samples of flocs were observed using CLSM to characterize the extracellular polymeric substances (EPS) stained with concanavalin A-tetra methylrhodamine and fluorescein isothiocyanate solution and combined with a fluorescent viability indicator (Baclight(®) Bacterial Viability Kit, Molecular Probes), allowing visualization of isolated stained cells in the three-dimensional structure of flocs (damaged or not). The results of CLSM of the sludge composition were compared with classical biochemical analysis of EPS made through a thermal extraction method. The results showed a good relation between these analyses and the statistical treatment of microscopic pictures. PMID:24901624

  5. Characterization of Nanoscale Transformations in Polyelectrolyte Multilayers Fabricated from Plasmid DNA Using Laser Scanning Confocal Microscopy in Combination with Atomic Force Microscopy

    PubMed Central

    Fredin, Nathaniel J.; Flessner, Ryan M.; Jewell, Christopher M.; Bechler, Shane L.; Buck, Maren E.; Lynn, David M.

    2010-01-01

    Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) were used to characterize changes in nanoscale structure that occur when ultrathin polyelectrolyte multilayers (PEMs) are incubated in aqueous media. The PEMs investigated here were fabricated by the deposition of alternating layers of plasmid DNA and a hydrolytically degradable polyamine onto a precursor film composed of alternating layers of linear poly(ethylene imine) (LPEI) and sodium poly(styrene sulfonate) (SPS). Past studies of these materials in the context of gene delivery revealed transformations from a morphology that is smooth and uniform to one characterized by the formation of nanometer-scale particulate structures. We demonstrate that in-plane registration of LSCM and AFM images acquired from the same locations of films fabricated using fluorescently labeled polyelectrolytes allows the spatial distribution of individual polyelectrolyte species to be determined relative to the locations of topographic features that form during this transformation. Our results suggest that this physical transformation leads to a morphology consisting of a relatively less disturbed portion of film composed of polyamine and DNA juxtaposed over an array of particulate structures composed predominantly of LPEI and SPS. Characterization by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) microanalysis provides additional support for this interpretation. The combination of these different microscopy techniques provides insight into the structures and dynamics of these multicomponent thin films that cannot be achieved using any one method alone, and that could prove useful for the further development of these assemblies as platforms for the surface-mediated delivery of DNA. PMID:20155860

  6. Confocal unstable-resonator semiconductor laser

    NASA Technical Reports Server (NTRS)

    Salzman, J.; Lang, R.; Yariv, A.; Larson, A.

    1986-01-01

    GaAs/GaAlAs heterostructure lasers with a monolithic confocal unstable resonator were demonstrated. The curved mirrors satisfying the confocal condition were fabricated by etching. Close to threshold, the lasers operate in a single lateral mode with a nearly collimated output beam. A single-lobe far-field intensity distribution as narrow as 1.9-deg full width at half maximum was measured.

  7. A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy.

    PubMed

    Dubert-Ferrandon, Alix; Niranjan, Keshaven; Grandison, Alistair S

    2006-11-01

    The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g glucono-delta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems. PMID:16834815

  8. Influence of various herbal irrigants as a final rinse on the adherence of Enterococcus faecalis by fluorescence confocal laser scanning microscope

    PubMed Central

    Rosaline, Hannah; Kandaswamy, D; Gogulnath, D; Rubin, MI

    2013-01-01

    Aim: The aim of this study was to assess the antibacterial efficacy of three different herbal irrigants against Enterococcus faecalis. Materials and Methods: Single rooted teeth were extracted due to orthodontic and periodontal reasons. The teeth were then inoculated with E. faecalis. The teeth were randomly divided into three experimental groups and two control groups of six samples each. Group 1 specimens were treated with 5.2% sodium hypochlorite (NaOCL) for 30 min followed by 5 mmol/L Ethylenediaminetetraacetic acid (EDTA) for 5 min and saline as final irrigant. Group 2 specimens were treated with and 5.2% NaOCl for 30 min as final irrigant. Group 3 were treated with Morinda citrifolia (MC) for 30 min as final irrigant. Group 4 were treated with Azadiracta indica (AI) as final irrigant. Group 5 were treated with green tea (GT) for 30 min as final irrigant. The dentin specimens were carefully spread onto a microscope slide and stained with BacLight and examined in a confocal laser scanning microscope set to monitor fluorescein isothiocyanate and propidium iodide. A total of nine fields were examined for each treatment and the bacteria presented were counted. Statistical Analysis: Using the one-way ANOVA with multiple comparison, significantly less bacteria were found adhering to the samples treated with Neem followed by NaOCL, GT, MC, Saline. Results: AI treatment produced the maximum reduction in adherence of E. faecalis to dentin (9.30%) followed by NaOCl (12.50%), GT (27.30%), MC (44.20%) and saline (86.70%). Conclusion: Neem is effective in preventing adhesion of E. faecalis to dentin. PMID:23956540

  9. Organic pollutant clustered in the plant cuticular membranes: visualizing the distribution of phenanthrene in leaf cuticle using two-photon confocal scanning laser microscopy.

    PubMed

    Li, Qingqing; Chen, Baoliang

    2014-05-01

    Plants play a key role in the transport and fate of organic pollutants. Cuticles on plant surfaces represent the first resistance for the uptake of airborne toxicants. In this study, a confocal scanning microscope enhanced with a two-photon laser was applied as a direct and noninvasive probe to explore the in situ uptake of a model pollutant, phenanthrene (PHE), into the cuticular membrane of a hypostomatic plant, Photinia serrulata. On the leaf cuticle surfaces, PHE forms clusters instead of being evenly distributed. The PHE distribution was quantified by the PHE fluorescence intensity. When PHE concentrations in water varying over 5 orders of magnitude were applied to the isolated cuticle, the accumulated PHE level by the cuticle was not vastly different, whether PHE was applied to the outer or inner side of the cuticle. Notably, PHE was found to diffuse via a channel-like pathway into the middle layer of the cuticle matrix, where it was identified to be composed of polymeric lipids. The strong affinity of PHE for polymeric lipids is a major contributor of the fugacity gradient driving the diffusive uptake of PHE in the cuticular membrane. Membrane lipids constitute important domains for hydrophobic interaction with pollutants, determining significant differentials of fugacities within the membrane microsystem. These, under unsteady conditions, contribute to enhance net transport and clustering along the z dimension. Moreover, the liquid-like state of polymeric lipids may promote mobility by enhancing the diffusion rate. The proposed "diffusive uptake and storage" function of polymeric lipids within the membrane characterizes the modality of accumulation of the hydrophobic contaminant at the interface between the plant and the environment. Assessing the capacity of fugacity of these constituents in detail will bring about knowledge of contaminant fate in superior plants with a higher level of accuracy. PMID:24678956

  10. The Effect of Addition of an EPS Degrading Enzyme with and without Detergent to 2% Chlorhexidine on Disruption of Enterococcus faecalis Biofilm: A Confocal Laser Scanning Microscopic Study

    PubMed Central

    Nagendrababu, Venkateshbabu; John, Aby; Deivanayagam, Kandaswamy

    2015-01-01

    Background Enterococcus faecalis is one of the most commonly occurring organisms retrieved from root canal treated teeth that show refractory apical periodontitis. Though it is well known that the ability of E. faecalis to form a matrix-encased biofilm contributes to its pathogenicity, the role of extracellular dextran and DNA in biofilm formation and its effect on the susceptibility of the biofilm to chlorhexidine remains poorly understood. It was hypothesized that the addition of an Extracellular Polymeric Substance (EPS) degrading enzyme along with a detergent to chlorhexidine may increase the susceptibility of the E. faecalis biofilm. Aim To evaluate the sensitivity of Enterococcus faecalis biofilms treated with DNase enzyme and their susceptibility to 2% chlorhexidine used alone or in conjunction with a detergent in a dentin disinfection model and examine under confocal laser scanning microscopy (CLSM). Materials and Methods Semi cylindrical shaped dentin specimens were infected with E. faecalis and incubated for 24 hours. Following incubation, the infected dentin specimens were exposed for 3 minutes to the four disinfecting solutions and grouped accordingly. {Group I- Sterile saline, Group II- 2% Chlorhexidine (CHX), Group III– Dnase1 Enzyme + 2% CHX, Group IV- DNase1 Enzyme + 2% CHX & Tween 80. Bacterial viability was then assessed by staining the specimens and examining under CLSM to analyse the proportion of dead and live bacteria within the dentinal tubules. Results The Groups II, III and IV showed statistically significant (p<0.05) percentage of dead bacteria compared to the control (Group I). However there was no significant difference in the killing effectiveness within the experimental groups (II-IV) at (p<0.05). Conclusion EPS degrading enzyme (DNase I) disrupts the biofilm and increases the susceptibility of E.faecalis when exposed to 2% Chlorhexidine and the use of a surfactant with this combination significantly contributes to improving the

  11. Fully Automatic Determination of Soil Bacterium Numbers, Cell Volumes, and Frequencies of Dividing Cells by Confocal Laser Scanning Microscopy and Image Analysis

    PubMed Central

    Bloem, J.; Veninga, M.; Shepherd, J.

    1995-01-01

    We describe a fully automatic image analysis system capable of measuring cell numbers, volumes, lengths, and widths of bacteria in soil smears. The system also determines the number of cells in agglomerates and thus provides the frequency of dividing cells (FDC). Images are acquired from a confocal laser scanning microscope. The grey images are smoothed by convolution and by morphological erosion and dilation to remove noise. The background is equalized by flooding holes in the image and is then subtracted by two top hat transforms. Finally, the grey image is sharpened by delineation, and all particles above a fixed threshold are detected. The number of cells in each detected particle is determined by counting the number of local grey-level maxima in the particle. Thus, up to 1,500 cells in 10 fields of view in a soil smear are analyzed in 30 min without human intervention. Automatic counts of cell numbers and FDC were similar to visual counts in field samples. In microcosms, automatic measurements showed significant increases in cell numbers, FDC, mean cell volume, and length-to-width ratio after amendment of the soil. Volumes of fluorescent microspheres were measured with good approximation, but the absolute values obtained were strongly affected by the settings of the detector sensitivity. Independent measurements of bacterial cell numbers and volumes by image analysis and of cell carbon by a total organic carbon analyzer yielded an average specific carbon content of 200 fg of C (mu)m(sup-3), which indicates that our volume estimates are reasonable. PMID:16534976

  12. Mapping of Heavy Metal Ion Sorption to Cell-Extracellular Polymeric Substance-Mineral Aggregates by Using Metal-Selective Fluorescent Probes and Confocal Laser Scanning Microscopy

    PubMed Central

    Li, Jianli; Kappler, Andreas; Obst, Martin

    2013-01-01

    Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro- and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe3+, Cu2+, Zn2+, and Hg2+, illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems. PMID:23974141

  13. A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy

    PubMed Central

    de ANDRADE, Flaviana Bombarda; ARIAS, Marcela Paola Castro; MALIZA, Amanda Garcia Alves; DUARTE, Marco Antonio Hungaro; GRAEFF, Márcia Sirlene Zardin; AMOROSO-SILVA, Pablo Andrés; MIDENA, Raquel Zanin; de MORAES, Ivaldo Gomes

    2015-01-01

    Objectives To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM). Material and Methods Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalis during the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD® for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn’s tests were used to evaluate the live and dead cells rates, and the scores obtained. Results The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05). Conclusion The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods. PMID:26200524

  14. Development of a surface-micromachined confocal scanning optical microscope

    NASA Astrophysics Data System (ADS)

    Dagel, Daryl James

    2001-09-01

    This dissertation describes the development of a confocal scanning optical microscope based on micro-electro- mechanical mirrors. The relatively new field of MicroElectroMechanical Systems (MEMS) has sparked unparalleled synergy between hitherto unrelated fields such as biology and microelectronics. Recently, its outgrowth into optics has resulted in several commercial devices, including switches for telecommunications networks. In essence, MEMS is a new manufacturing technology that allows for increased functionality, reliability, and sophistication of existing systems as well as for development of new, previously impossible or impractical devices. In this dissertation, the design, fabrication, and characterization of a MEMS-based confocal microscope have been accomplished. Following the introductory material on micromachining and confocal microscopy given in Chapter 1, an overview of the current microscope and a survey of previous work in the field, including that of the author, is presented in Chapter 2. Chapter 3 discusses the design and operation of the microscope components, including the laser diode, optical fiber, scanning mirrors, objective lens, and photodetector. Emphasis is placed on modeling the amplitude and frequency response of the scanning mirrors. Chapter 4 summarizes the experiments completed to characterize the response and performance of the microscope. Here, resolution, field-of-view, frequency and amplitude response, and image interpretation are the main focus. Conclusions and directions for future work appear in Chapter 5. A scanning confocal microscope capable of real-time imaging in two dimensions has been demonstrated. The overall size of the imaging head with the 2D scanner is ~2 x 2 mm2, making it suitable for endoscopy. Although not diffraction-limited, the microscope resolution approaches its theoretical limit. In the non-optimized configuration studied here, the transverse resolution is 1.5 μm, and the axial resolution is 7.0

  15. Reflectance confocal endomicroscope with optical axial scanning for in vivo imaging of the oral mucosa

    PubMed Central

    Jabbour, Joey M.; Bentley, Julie L.; Malik, Bilal H.; Nemechek, John; Warda, John; Cuenca, Rodrigo; Cheng, Shuna; Jo, Javier A.; Maitland, Kristen C.

    2014-01-01

    This paper presents the design and evaluation of a reflectance confocal laser endomicroscope using a miniature objective lens within a rigid probe in conjunction with an electrically tunable lens for axial scanning. The miniature lens was characterized alone as well as in the endoscope across a 200 µm axial scan range using the tunable lens. The ability of the confocal endoscope to probe the human oral cavity is demonstrated by imaging of the oral mucosa in vivo. The results indicate that reflectance confocal endomicroscopy has the potential to be used in a clinical setting and guide diagnostic evaluation of biological tissue. PMID:25426310

  16. Reflectance confocal endomicroscope with optical axial scanning for in vivo imaging of the oral mucosa.

    PubMed

    Jabbour, Joey M; Bentley, Julie L; Malik, Bilal H; Nemechek, John; Warda, John; Cuenca, Rodrigo; Cheng, Shuna; Jo, Javier A; Maitland, Kristen C

    2014-11-01

    This paper presents the design and evaluation of a reflectance confocal laser endomicroscope using a miniature objective lens within a rigid probe in conjunction with an electrically tunable lens for axial scanning. The miniature lens was characterized alone as well as in the endoscope across a 200 µm axial scan range using the tunable lens. The ability of the confocal endoscope to probe the human oral cavity is demonstrated by imaging of the oral mucosa in vivo. The results indicate that reflectance confocal endomicroscopy has the potential to be used in a clinical setting and guide diagnostic evaluation of biological tissue. PMID:25426310

  17. Fiber-matrix interface studies on bioabsorbable composite materials for internal fixation of bone fractures. II. A new method using laser scanning confocal microscopy.

    PubMed

    Slivka, M A; Chu, C C

    1997-12-01

    In this study, a new visual characterization method was developed using laser scanning confocal microscopy (LSCM) to study morphologic properties, particularly at the fiber-matrix interface, by optical sectioning of bioabsorbable single-fiber composites. The interface gap width (IGW) between the fiber and matrix, and the changes in IGW after in vitro hydrolysis, named the gap rate (Rg), were measured from images obtained using the LSCM. Higher values for IGW and Rg showed faster degradation of the fiber-matrix interface. These parameters were used to investigate the effects of strain, wicking, different reinforcing fibers, and gamma-irradiation on the fiber-matrix interface morphology. The component materials used were nonbioabsorbable AS4 carbon (C) fibers, bioabsorbable calcium phosphate (CaP), poly(glycolic acid) (PGA), and chitin fibers, and bioabsorbable poly(L-lactic acid) (PLLA) matrix. The application of strain on CaP/PLLA composites increased the IGW up to about 15%, after which there was no change up to 25%. The Rg for CaP/PLLA composites with the fiber ends exposed in vitro (permitting wicking) was greater than for CaP/PLLA with the fiber ends embedded completely within the matrix (preventing wicking). Open-end C/PLLA composites had the slowest rate of interface degradation in vitro, followed by chitin/PLLA, PGA/PLLA, and CaP/PLLA. The exposure of closed-end CaP/PLLA composites to 4 Mrad of gamma-irradiation, in air at room temperature or in vaccuum at 77K, accelerated the rate of interface degradation in vitro. In conclusion, an effective new visual characterization method was developed using LSCM, and it was used to show that (a) moderate strain could accelerate the degradation of the interface, (b) fiber-matrix interface wicking could accelerate the rate of degradation of the interface, (c) the rate of interface degradation depends on the type of fiber used, and (d) gamma-irradiation could accelerate the rate of interface degradation. Furthermore, the

  18. Attachment of Escherichia coli O157:H7 to the Surfaces and Internal Structures of Apples as Detected by Confocal Scanning Laser Microscopy

    PubMed Central

    Burnett, Scott L.; Chen, Jinru; Beuchat, Larry R.

    2000-01-01

    Confocal scanning laser microscopy (CSLM) was used to demonstrate the attachment of Escherichia coli O157:H7 transformed with a plasmid encoding for green fluorescent protein (GFP) to the surface and within the internal structures of nonwaxed Red Delicious cv. apples. Apples at 2 or 25°C were inoculated with an E. coli O157:H7 cell suspension at 2 or 25°C. The effect of a negative temperature differential (cold inoculum, warm apple), a positive differential (warm inoculum, cold apple), and no differential (warm inoculum, warm apple), in combination with a pressure differential (atmospheric versus 10,130 Pa), on the attachment and infiltration of cells was determined. CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachment to discontinuities in the waxy cuticle on the surface and to damaged tissue surrounding puncture wounds, where the pathogen was observed at depths up to 70 μm below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 μm. Infiltration through the floral tube and attachment to seeds, cartilaginous pericarp, and internal trichomes were observed in all apples examined, regardless of temperature differential during inoculation. The pressure differential had no effect on infiltration or attachment of E. coli O157:H7. Image analysis to count cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the effects of the temperature differential. Puncture wounds harbored greater numbers of the pathogen at greater depths than did other sites examined. Attachment or infiltration of cells was greater on the intact skin and in lenticels, russet areas, and the floral tube of apples inoculated under a negative temperature differential compared to those inoculated under no temperature differential. The results suggest that E. coli O157:H7

  19. Changes in F-actin organization induced by hard metal particle exposure in rat pulmonary epithelial cells using laser scanning confocal microscopy.

    PubMed

    Antonini, J M; Starks, K; Roberts, J R; Millecchia, L; Yang, H M; Rao, K M

    2000-01-01

    Chronic inhalation of hard metal (WC-Co) particles causes alveolitis and the eventual development of pulmonary fibrosis. The initial inflammatory response includes a change in the alveolar epithelial cell-capillary barrier, which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network. The objective of this study was to evaluate the effect WC-Co particles have on F-actin organization of lung epithelial cells in an in vitro culture system. Rat lung epithelial (L2) cells were exposed to 5, 25, and 100 microg/mL of WC-Co particles, as well as the individual components (Co and WC) of the hard metal mixture particles for 24 h. The effect on F-actin organization was visualized by laser scanning confocal microscopy (LSCM) following Bodipy-Phallacidin staining. Minimal changes in the F-actin microfilaments of L2 cells were observed by LSCM after exposure to WC and WC-Co at 5 and 25 microg/mL, while at 100 microg/mL, there was a noticeable disruption in the uniform distribution of L2 cell F-actin microfilaments. After exposure to Co, a dose-dependent change in the F-actin organization of the L2 cells was observed. Little change in F-actin assembly was observed after treatment with 5 microg/mL of Co (the concentration equivalent to the 5% amount of Co commonly present in 100 microg/mL of the WC-Co sample mixture). However, at 100 microg/mL of Co, the microfilaments aggregated into homogeneous masses within the cells, and a significant loss in the organization of L2 F-actin was observed. These dramatic alterations in F-actin organization seen after exposure to the higher doses of Co were attributed to an increase in L2 cell injury as measured by lactate dehydrogenase and trypan blue exclusion. We conclude the pulmonary response evoked in the lung by inhalation of high levels of WC-Co particles is unlikely due to alterations in the F-actin microfilaments of lung-epithelial cells. PMID:10900403

  20. Pupil engineering for a confocal reflectance line-scanning microscope

    NASA Astrophysics Data System (ADS)

    Patel, Yogesh G.; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2011-03-01

    Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current confocal point-scanning systems are large, complex, and expensive. A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner. We have developed a confocal line-scanner that combines both divided-pupil and full-pupil configurations. This combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast, signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions. Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging performance. Both results will be compared to theoretical calculations using our previously reported Fourier analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of confocal reflectance scanning microscopes for clinical and surgical dermatology.

  1. Maximum permissible exposure of the retina in the human eye in optical coherence tomography systems using a confocal scanning laser ophthalmoscopy platform

    NASA Astrophysics Data System (ADS)

    Rees, Sian; Dobre, George

    2014-01-01

    When using scanning laser ophthalmoscopy to produce images of the eye fundus, maximum permissible exposure (MPE) limits must be considered. These limits are set out in international standards such as the National Standards Institute ANSI Z136.1 Safe Use of Lasers (USA) and BS EN 60825-1: 1994 (UK) and corresponding Euro norms but these documents do not explicitly consider the case of scanned beams. Our study aims to show how MPE values can be calculated for the specific case of retinal scanning by taking into account an array of parameters, such as wavelength, exposure duration, type of scanning, line rate and field size, and how each set of initial parameters results in MPE values that correspond to thermal or photochemical damage to the retina.

  2. Laser-excited confocal-fluorescence gel scanner

    SciTech Connect

    Mathies, R.A.; Scherer, J.R.; Quesada, M.A. ); Rye, H.S.; Glazer, A.N. )

    1994-04-01

    A high-sensitivity, laser-excited, confocal-fluorescence scanner has been developed for the detection of fluorescently labeled nucleic acids separated on slab gels. The gel is placed on a motor-driven, two-dimensional scan stage and raster scanned past the optical detection system. The 488-nm argon ion laser beam is introduced into the confocal optical system at a long-pass dichroic beam splitter and focused within the gel to an [similar to]2 [mu]m diameter spot by a high-numerical aperture microscope objective. The resulting fluorescence is gathered by the objective, passed back through the first long-pass beam splitter, and relayed to a second dichroic beam splitter that separates the red and green emissions. The fluorescence is then focused on confocal spatial filters to reduce stray and scattered light, passed through spectral filters, and detected with photomultipliers. The resulting signals are amplified, filtered, and digitized for display on a computer. This system can detect as little as 5[times]10[sup [minus]12] M fluorescein, the resolution as operated is 160 [mu]m, and it can scan a 6 cm[times]6 cm gel using a scan rate of 4 cm/s in 12 min. The detection of DNA on slab gels, two-color DNA fragment sizing, and microtiter plate scanning are presented to illustrate some of the possible applications of this apparatus.

  3. Influence of erbium, chromium-doped: Yttrium scandium-gallium-garnet laser etching and traditional etching systems on depth of resin penetration in enamel: A confocal laser scanning electron microscope study

    PubMed Central

    Vijayan, Vishal; Rajasigamani, K.; Karthik, K.; Maroli, Sasidharan; Chakkarayan, Jitesh; Haris, Mohamed

    2015-01-01

    Objective: This study was performed to assess the resin tag length penetration in enamel surface after bonding of brackets to identify which system was most efficient. Methodology: Our study was based on a more robust confocal microscopy for visualizing the resin tags in enamel. Totally, 100 extracted human first and second premolars have been selected for this study and were randomly divided into ten groups of 10 teeth each. In Group 1, the buccal enamel surface was etched with 37% phosphoric acid (3M ESPE), Group 2 with 37% phosphoric (Ultradent). In Groups 5, 6, and 7, erbium, chromium-doped: Yttrium scandium-gallium-garnet (Er, Cr: YSGG) laser (Biolase) was used for etching the using following specifications: Group 5 (1.5 W/20 Hz, 15 s), Group 6 (2 W/10 Hz, 15 s), and Group 7 (2 W/20 Hz, 15 s). In Groups 8, 9, and 10, Er, Cr: YSGG laser (Biolase) using same specifications and additional to this step, conventional etching on the buccal enamel surface was etched with 37% (3M ESPE) after laser etching. In Groups 1, 5, 6, 7, 8, 9, and 10 3M Unitek Transbond XT primer was mixed with Rhodamine B dye (Sigma-Aldrich, Germany) to etched surface and then cured for 20 s. In Group 2, Ultradents bonding agent was mixed with Rhodamine B. In Group 3, 3M Unitek Transbond PLUS, Monrovia, USA, which was mixed with Rhodamine B dye (Sigma-Aldrich, Germany). Group 4, with self-etching primer (Ultradent-Peak SE, USA) was mixed with Rhodamine B dye (Sigma-Aldrich, Germany). Later (3M Unitek, Transbond XT, Monrovia USA) [Figure 1] was used to bond the modified Begg brackets (T. P. Orthodontics) in Groups 1, 3, 5, 6, 7, 8, 9, and 10. In Groups 2, 4 Ultradent-Peak LC Bond was used to bond the modified brackets. After curing brackets were debonded, and enamel depth penetration was assessed using confocal laser scanning microscope. Results: Group J had a mean maximum depth of penetration of 100.876 μm, and Group D was the least having a maximum value of 44.254 μm. Conclusions: Laser

  4. Digital adaptive optics line-scanning confocal imaging system.

    PubMed

    Liu, Changgeng; Kim, Myung K

    2015-01-01

    A digital adaptive optics line-scanning confocal imaging (DAOLCI) system is proposed by applying digital holographic adaptive optics to a digital form of line-scanning confocal imaging system. In DAOLCI, each line scan is recorded by a digital hologram, which allows access to the complex optical field from one slice of the sample through digital holography. This complex optical field contains both the information of one slice of the sample and the optical aberration of the system, thus allowing us to compensate for the effect of the optical aberration, which can be sensed by a complex guide star hologram. After numerical aberration compensation, the corrected optical fields of a sequence of line scans are stitched into the final corrected confocal image. In DAOLCI, a numerical slit is applied to realize the confocality at the sensor end. The width of this slit can be adjusted to control the image contrast and speckle noise for scattering samples. DAOLCI dispenses with the hardware pieces, such as Shack–Hartmann wavefront sensor and deformable mirror, and the closed-loop feedbacks adopted in the conventional adaptive optics confocal imaging system, thus reducing the optomechanical complexity and cost. Numerical simulations and proof-of-principle experiments are presented that demonstrate the feasibility of this idea. PMID:26140334

  5. Digital adaptive optics line-scanning confocal imaging system

    NASA Astrophysics Data System (ADS)

    Liu, Changgeng; Kim, Myung K.

    2015-11-01

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

  6. Sealing ability of mineral trioxide aggregate, calcium phosphate and polymethylmethacrylate bone cements on root ends prepared using an Erbium: Yttriumaluminium garnet laser and ultrasonics evaluated by confocal laser scanning microscopy

    PubMed Central

    Girish, C Sabari; Ponnappa, KC; Girish, TN; Ponappa, MC

    2013-01-01

    Background: Surgical endodontic therapy comprises of exposure of the involved root apex, resection of the apical end of the root, preparation of a class I cavity, and insertion of a root end filling material. Mineral trioxide aggregate (MTA) is now the gold standard among all root end filling materials. MTA is however difficult to handle, expensive and has a very slow setting reaction. Aim: (1) To compare the sealing ability of MTA, polymethylmethacrylate (PMMA) bone cement and CHITRA Calcium phosphate cement (CPC) when used as root end filling material using Rhodamine B dye evaluated under a confocal laser scanning microscope. (2) To compare the seal of root ends prepared using an ultrasonic retroprep tip and an Er: YAG laser using three different root end filling materials. Statistical Analysis: Statistical analysis was performed using a one-way ANOVA and a two-way ANOVA, independent samples t-test and Scheffe's post hoc test using SPSS Version 16 for Windows. Results: All the three materials, namely MTA, PMMA BONE CEMENT and CHITRA CPC, showed microleakage. Comparison of microleakage showed maximum peak value of 0.86 mm for MTA, 0.24 mm for PMMA bone cement and 1.37 mm for CHITRA CPC. The amount of dye penetration was found to be lesser in root ends prepared using Er: YAG laser when compared with ultrasonics, but the difference was found to be not statistically significant. Conclusion: PMMA bone cement is a better material as root end filling material to prevent apical microleakage. MTA still continues to be a gold standard root end filling material showing minimum microleakage. Er: YAG laser is a better alternative to ultrasonics for root end preparations. PMID:23956530

  7. Morphological and ultrastructural characterization of ionoregulatory cells in the teleost Oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique.

    PubMed

    Fridman, Sophie; Rana, Krishen J; Bron, James E

    2013-10-01

    Aspects of ionoregulatory or mitochondria-rich cell (MRC) differentiation and adaptation in Nile tilapia yolk-sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole-mount Nile tilapia larvae using anti- Na⁺/K⁺-ATPase as a primary antibody and Fluoronanogold™ (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk-sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺-ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level. PMID:23873584

  8. Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope.

    PubMed

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

    2011-01-01

    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. PMID:21809349

  9. Double-label confocal laser-scanning microscopy, image restoration, and real-time three-dimensional reconstruction to study axons in the central nervous system and their contacts with target neurons.

    PubMed

    Wouterlood, Floris G; van Haeften, Theo; Blijleven, Nico; Pérez-Templado, Pepa; Pérez-Templado, Helena

    2002-03-01

    The current double tracing-double confocal laser-scanning method was developed to reconstruct identified nerve fibers and their contacts with identified target neurons in the rat brain in three dimensions. It intends to fill the gap between conventional light microscopic and electron microscopic neuroanatomic tracing. The steps involved are as follows: (1) injection of two neuroanatomic tracers--Phaseolus vulgaris leucoagglutinin (PHA-L) to label fibers innervating a particular brain area and Neurobiotin to label prospective target neurons in that area; (2) immunofluorescence detection of the labeled fibers (fluorophore Cy5, infrared emission), together with fluorochromated avidin detection of the taken-up Neurobiotin (Cy2 or Alexa 488; green emission); (3) acquisition of Z-series of confocal images at high magnification with a laser-scanning microscope using the laser lines 488 nm and 647 nm; and (4) computer-processing and three-dimensional reconstruction of the labeled fibers and the presumed target dendrites. Rotation on the computer of the three-dimensional reconstructed fibers and dendrites along all three spatial axes enabled the authors to determine whether "true" or "false" contacts occur. In a true contact no space was present between the apposing structures, whereas a false contact consisted of two differently stained structures close to each other but separated by a narrow, optically empty space. One important phenomenon in the three-dimensional reconstruction of double-stained structures that needed correction was "twin image mismatch"--i.e., the observation that a three-dimensional reconstruction of a small test object (double-stained on purpose) produced two slightly shifted objects, each associated with its particular fluorochrome. To measure the actual twin image mismatch of the confocal instrument and to obtain accurate correction factors the authors took in each session in which they obtained image series of the real experiments, with both laser

  10. Clinical applications of a real-time scanning-slit confocal microscope designed for real-time observations of the in-vivo human cornea

    NASA Astrophysics Data System (ADS)

    Masters, Barry R.

    1995-05-01

    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.

  11. Imaging System With Confocally Self-Detecting Laser.

    DOEpatents

    Webb, Robert H.; Rogomentich, Fran J.

    1996-10-08

    The invention relates to a confocal laser imaging system and method. The system includes a laser source, a beam splitter, focusing elements, and a photosensitive detector. The laser source projects a laser beam along a first optical path at an object to be imaged, and modulates the intensity of the projected laser beam in response to light reflected from the object. A beam splitter directs a portion of the projected laser beam onto a photodetector. The photodetector monitors the intensity of laser output. The laser source can be an electrically scannable array, with a lens or objective assembly for focusing light generated by the array onto the object of interest. As the array is energized, its laser beams scan over the object, and light reflected at each point is returned by the lens to the element of the array from which it originated. A single photosensitive detector element can generate an intensity-representative signal for all lasers of the array. The intensity-representative signal from the photosensitive detector can be processed to provide an image of the object of interest.

  12. Expression of keratin 14 in the basal cells of the lingual epithelium of mice during the morphogenesis of filiform papillae: visualization by fluorescent immunostaining and confocal laser-scanning microscopy in the transmission mode.

    PubMed

    Iwasaki, Shin-Ichi; Aoyagi, Hidekazu

    2007-07-01

    We examined the expression of keratin 14 (K14) on the lingual epithelium by immunofluorescent staining while monitoring morphological changes in the filiform papillae of mice by confocal laser-scanning microscopy in the transmission mode of the same sections to define both the histology and the morphology of cells. It is difficult to visualize histological details of the fetal lingual epithelium of the mouse on semi-ultrathin sections by light microscopy after immunohistochemical staining because the histological structures in such sections cannot be distinguished by standard counterstaining. To solve this problem and to visualize the immunoreactivity specific for K14, we analyzed the results of immunofluorescent staining of semi-ultrathin sections in combination with an examination of the corresponding images by laser-scanning microscopy in the transmission mode after staining of specimens with toluidine blue. No immunoreactivity specific for K14 was detected on the lingual epithelium of fetuses on embryonic day 15 (E15), but immunoreactivity was distinct at all postnatal stages from postnatal day 0 (P0) to P21. PMID:17660983

  13. Adaptive optics parallel near-confocal scanning ophthalmoscopy.

    PubMed

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

    2016-08-15

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

  14. Line-scanning laser ophthalmoscope

    NASA Astrophysics Data System (ADS)

    Hammer, Daniel X.; Ferguson, R. Daniel; Ustun, Teoman E.; Bigelow, Chad E.; Iftimia, Nicusor V.; Webb, Robert H.

    2006-07-01

    Scanning laser ophthalmoscopy (SLO) is a powerful imaging tool with specialized applications limited to research and ophthalmology clinics due in part to instrument size, cost, and complexity. Conversely, low-cost retinal imaging devices have limited capabilities in screening, detection, and diagnosis of diseases. To fill the niche between these two, a hand-held, nonmydriatic line-scanning laser ophthalmoscope (LSLO) is designed, constructed, and tested on normal human subjects. The LSLO has only one moving part and uses a novel optical approach to produce wide-field confocal fundus images. Imaging modes include multiwavelength illumination and live stereoscopic imaging with a split aperture. Image processing and display functions are controlled with two stacked prototype compact printed circuit boards. With near shot-noise limited performance, the digital LSLO camera requires low illumination power (<500 µW) at near-infrared wavelengths. The line-scanning principle of operation is examined in comparison to SLO and other imaging modes. The line-scanning approach produces high-contrast confocal images with nearly the same performance as a flying-spot SLO. The LSLO may significantly enhance SLO utility for routine use by ophthalmologists, optometrists, general practitioners, and also emergency medical personnel and technicians in the field for retinal disease detection and other diverse applications.

  15. Compact high-speed line scanning quasi-confocal ophthalmoscope and retina imaging experiments

    NASA Astrophysics Data System (ADS)

    He, Yi; Wang, Zhibin; Wei, Ling; Li, Xiqi; Shi, Guohua; Zhang, Yudong

    2014-09-01

    A compact, high-speed line scanning quasi-confocal ophthalmoscope (LSO) for retina imaging is presented in this paper. By using a line beam to illuminate the retina, meanwhile a linear array sensor is used for imaging the retina, the LSO system significantly reduces the size, complexity, and cost comparing to a conventional confocal scanning laser ophthalmoscope (CSLO). With only one moving scanner to provide raster scanning of the line beam of the retina, the imaging frequency achieves 160 Hz and the lateral resolution is nearly 10 μm for 1024×330 pixels imaging mode. Preliminary experiments are performed for imaging the macula, the optic nerve head and other targets, providing high resolution and high speed videos of human retina.

  16. Needle-based confocal laser endomicroscopy

    PubMed Central

    Giovannini, Marc

    2015-01-01

    New applications of confocal laser endomicroscopy were developed as pCLE in the bile duct and nCLE for pancreatic cystic tumors, pancreatic masses and lymph nodes. The aim of this paper would be to give you an update in this new technology and to try to define its place in the diagnosis of cystic and solid pancreatic masses. The material used was a 19G EUS-needle in which the stylet was replaced by the Confocal mini-probe. The mini-probe (0.632 mm of diameter) is pre-loaded and screwed by a locking device in the EUS-Needle and guided endosonographically in the target. Regarding pancreatic cystic lesion, the presence of epithelial villous structures based on nCLE was associated with pancreatic cystic neoplasm (IPMN) (P = 0.004) and provided a sensitivity of 59%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 50%. A superficial vascular network pattern visualized on nCLE was identified in serous cystadenomas. It corresponded on pathological specimen to a dense and subepithelial capillary vascularization. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of this sign for the diagnosis of SCA were 87%, 69%, 100%, 100%, and 82%, respectively. In pancreatic adenocarcinomas, nCLE found vascular leakage with irregular vessels with leakage of fluorescein into the tumor, large dark clumps which correspond to humps of malignant cells. These criteria correlate with the histological structure of those tumors which are characterized by tumoral glands, surrounded by fibrosis in case of fibrous stroma tumor. Neuroendocrine tumors showed a dense network of small vessels on a dark background, which fits with the histological structure based on cord of cells surrounded by vessels and by fibrosis. nCLE is feasible during a EUS examination; these preliminary results are very encouraging and may be used in the future in case of inconclusive EUS-FNA. PMID:26643694

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

    PubMed

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

    2010-04-15

    In most deaths caused by explosive, the victim's body becomes a depot for fragments of explosive materials, so contributing to the collection of trace evidence which may provide clues about the specific type of device used with explosion. Improvised explosive devices are used which contain "homemade" explosives rather than high explosives because of the relative ease with which such components can be procured. Many methods such as chromatography-mass spectrometry, scanning electron microscopy, stereomicroscopy, capillary electrophoresis are available for use in the identification of explosive residues on objects and bomb fragments. Identification and reconstruction of the distribution of explosive residues on the decedent's body may give additional hints in assessing the position of the victim in relation to the device. Traditionally these residues are retrieved by swabbing the body and clothing during the early phase, at autopsy. Gas chromatography-mass spectrometry and other analytical methods may be used to analyze the material swabbed from the victim body. The histological examination of explosive residues on skin samples collected during the autopsy may reveal significant details. The information about type, quantity and particularly about anatomical distribution of explosive residues obtained utilizing confocal laser scanning microscope (CLSM) together with inductively coupled plasma atomic emission spectrometer (ICP-AES), may provide very significant evidence in the clarification and reconstruction of the explosive-related events. PMID:20047806

  18. Fluorescence imaging of reactive oxygen species by confocal laser scanning microscopy for track analysis of synchrotron X-ray photoelectric nanoradiator dose: X-ray pump-optical probe.

    PubMed

    Jeon, Jae Kun; Han, Sung Mi; Kim, Jong Ki

    2016-09-01

    Bursts of emissions of low-energy electrons, including interatomic Coulomb decay electrons and Auger electrons (0-1000 eV), as well as X-ray fluorescence produced by irradiation of large-Z element nanoparticles by either X-ray photons or high-energy ion beams, is referred to as the nanoradiator effect. In therapeutic applications, this effect can damage pathological tissues that selectively take up the nanoparticles. Herein, a new nanoradiator dosimetry method is presented that uses probes for reactive oxygen species (ROS) incorporated into three-dimensional gels, on which macrophages containing iron oxide nanoparticles (IONs) are attached. This method, together with site-specific irradiation of the intracellular nanoparticles from a microbeam of polychromatic synchrotron X-rays (5-14 keV), measures the range and distribution of OH radicals produced by X-ray emission or superoxide anions ({\\rm{O}}_2^-) produced by low-energy electrons. The measurements are based on confocal laser scanning of the fluorescence of the hydroxyl radical probe 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF) or the superoxide probe hydroethidine-dihydroethidium (DHE) that was oxidized by each ROS, enabling tracking of the radiation dose emitted by the nanoradiator. In the range 70 µm below the irradiated cell, ^\\bullet{\\rm{OH}} radicals derived mostly from either incident X-ray or X-ray fluorescence of ION nanoradiators are distributed along the line of depth direction in ROS gel. In contrast, {\\rm{O}}_2^- derived from secondary electron or low-energy electron emission by ION nanoradiators are scattered over the ROS gel. ROS fluorescence due to the ION nanoradiators was observed continuously to a depth of 1.5 mm for both oxidized APF and oxidized DHE with relatively large intensity compared with the fluorescence caused by the ROS produced solely by incident primary X-rays, which was limited to a depth of 600 µm, suggesting dose enhancement as well as more

  19. A microfabricated scanning confocal optical microscope for in situ imaging

    NASA Astrophysics Data System (ADS)

    Dickensheets, David Lee

    Scanning confocal optical microscopes are well suited for imaging living tissue because of their ability to 'cross section' intact tissue. They are not, however, well suited for imaging tissues in situ. This dissertation describes a new, miniature, mirror scanned, high resolution confocal optical microscope that operates in real time. It is small enough to fit into an endoscope, and may eventually be incorporated into a hypodermic needle. Such a device would provide immediate in-situ tissue assessment at the cellular level and may enable, for example, biopsy without tissue removal. Non-medical applications may include process monitoring and endoscopic inspection. The microfabricated confocal optical scanning microscope, or μCOSM, incorporates single mode optical fiber illumination, silicon torsional scan mirrors, and an off- axis micro diffractive lens. The prototype device is monochromatic, at 633 nm, with a 1.1 mm working distance and 0.25 NA. It achieves a line response of 0.98 μm FWHM, and an axial response of 11.1 μm FWHM. The first part of the dissertation describes the opto- mechanical design of the microscope, which was chosen to be compatible with the microfabrication technologies used for its construction. Then the imaging properties of the off-axis diffractive objective lens are developed, including the aberrations of second and third order which constrain its use. The lens is a surface relief phase grating, and a rigorous electromagnetic analysis is employed to specify the pupil function of the microscope. Then the image forming properties of the μCOSM are derived and compared to experimental results. The second part of the dissertation describes the fabrication of the individual elements of the μCOSM, and their assembly into an imaging instrument. The lens is constructed using electron beam lithography and reactive ion etching of a fused silica substrate. The scanning mirrors for the microscope, which comprise a single crystal silicon plate

  20. Hyperchromatic laser scanning cytometry

    NASA Astrophysics Data System (ADS)

    Tárnok, Attila; Mittag, Anja

    2007-02-01

    In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

  1. Laser Scanning In Inspection

    NASA Astrophysics Data System (ADS)

    West, Patricia; Baker, Lionel R.

    1989-03-01

    This paper is a review of the applications of laser scanning in inspection. The reasons for the choice of a laser in flying spot scanning and the optical properties of a laser beam which are of value in a scanning instrument will be given. The many methods of scanning laser beams in both one and two dimensions will be described. The use of one dimensional laser scanners for automatic surface inspection for transmitting and reflective products will be covered in detail, with particular emphasis on light collection techniques. On-line inspection applications which will be mentioned include: photographic film web, metal strip products, paper web, glass sheet, car body paint surfaces and internal cylinder bores. Two dimensional laser scanning is employed in applications where increased resolution, increased depth of focus, and better contrast are required compared with conventional vidicon TV or solid state array cameras. Such examples as special microscope laser scanning systems and a TV compatible system for use in restricted areas of a nuclear reactor will be described. The technical and economic benefits and limitations of laser scanning video systems will be compared with conventional TV and CCD array devices.

  2. Exploring the diversity of Listeria monocytogenes biofilm architecture by high-throughput confocal laser scanning microscopy and the predominance of the honeycomb-like morphotype.

    PubMed

    Guilbaud, Morgan; Piveteau, Pascal; Desvaux, Mickaël; Brisse, Sylvain; Briandet, Romain

    2015-03-01

    Listeria monocytogenes is involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation by L. monocytogenes isolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96 L. monocytogenes strains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improve L. monocytogenes biofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA). PMID:25548046

  3. Use of a white light supercontinuum laser for confocal interference-reflection microscopy

    PubMed Central

    Chiu, L-D; Su, L; Reichelt, S; Amos, WB

    2012-01-01

    Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460–700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser. PMID:22432542

  4. Withania somnifera prevents morphine withdrawal-induced decrease in spine density in nucleus accumbens shell of rats: a confocal laser scanning microscopy study.

    PubMed

    Kasture, Sanjay; Vinci, Stefania; Ibba, Federico; Puddu, Alessandro; Marongiu, Mara; Murali, Balasubramanian; Pisanu, Augusta; Lecca, Daniele; Zernig, Gerald; Acquas, Elio

    2009-11-01

    Opiate withdrawal is associated with morphological changes of dopamine neurons in the ventral tegmental area and with reduction of spine density of second-order dendrites of medium size spiny neurons in the nucleus accumbens shell but not core. Withania somnifera has long been used in the Middle East, Africa, and India as a remedy for different conditions and diseases and a growing body of evidence points to its beneficial effects on a number of experimental models of neurological disorders. Recently, many studies focused on the potential neuritic regeneration and synaptic reconstruction properties of its methanolic extract and its constituents (withanolides). This study investigates whether morphine withdrawal-induced spine reduction in the nucleus accumbens is affected by the administration of a Withania somnifera extract. To this end, rats were chronically treated with Withania somnifera extract along with morphine or saline and, upon spontaneous (1 and 3 days) or pharmacologically precipitated withdrawal, their brains were fixed in Golgi-Cox stain for confocal microscopic examination. In a separate group of animals, Withania somnifera extract was administered during three days of spontaneous withdrawal. Withania somnifera extract treatment reduced the severity of the withdrawal syndrome when given during chronic morphine but not during withdrawal. In addition, treatment with Withania somnifera extract during chronic morphine, but not during withdrawal, fully prevented the reduction of spine density in the nucleus accumbens shell in spontaneous and pharmacologically precipitated morphine withdrawal. These results indicate that pretreatment with Withania somnifera extract protects from the structural changes induced by morphine withdrawal potentially providing beneficial effects on the consequences related to this condition. PMID:19551457

  5. Shipborne hydrographic laser scanning

    NASA Astrophysics Data System (ADS)

    Pfennigbauer, Martin; Rieger, Peter; Schaich, Martin

    2011-11-01

    Applications like hydro-archeology, hydrobiology, or hydraulic engineering sometimes require accurate surveying of submerged areas with point densities usually only achieved with mobile or terrestrial laser scanning. For navigable waterbodies, hydrographic laser scanning from a floating platform represents a viable solution. RIEGL's new hydrographic laser scanner VQ-820-G with its exceptionally high measurement rate of up to 110,000 net measurements per second and its small laser footprint is optimally suited for such applications. We present results from a measurement campaign surveying prehistoric lake dwellings at Lake Constance in Germany. While the aim of typical hydrographic laser scanning applications is to roughly acquire the ground's shape and structure, in this case it was tried to determine the exact position, shape, and attitude of the remainders of the piles. The special requirements with respect to mission planning and data processing are discussed and the performance of the laser scanner is assessed.

  6. Laser confocal radius measurement method for unpolished spheres.

    PubMed

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

    2016-06-10

    A laser confocal radius measurement method for unpolished spheres (CRMUS) is proposed for measuring the radius of an unpolished sphere during optical sphere processing. CRMUS uses the laser confocal focusing technique to accurately identify the cat's eye and confocal positions of the unpolished sphere, and then uses the distance between the cat's eye and confocal positions measured by a distance measurement interferometer to derive the radius. The partially coherent optical theoretical model of the CRMUS derived indicates that the CRMUS is able to measure the radius of the unpolished sphere with a roughness of less than 0.15 μm. Using an unpolished sphere made of Schott BK7 as the test sphere, experimental results indicate that the CRMUS has a relative expanded uncertainty of less than 20 ppm. The CRMUS could greatly increase processing efficiency. PMID:27409012

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

    PubMed Central

    Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

    2008-01-01

    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. PMID:18627634

  8. Ultrasensitive and selective detection of mercury (II) in serum based on the gold film sensor using a laser scanning confocal imaging-surface plasmon resonance system in real time

    NASA Astrophysics Data System (ADS)

    Liu, Sha; Zhang, Hongyan; Liu, Weimin; Wang, Pengfei

    2015-10-01

    Hg2+ ions are one of the most toxic heavy metal ion pollutants, and are caustic and carcinogenic materials with high cellular toxicity. The Hg2+ ions can accumulate in the human body through the food chain and cause serious and permanent damage to the brain with both acute and chronic toxicity. According to the US Environment Protection Agency (EPA) guidelines, Hg2+ ions must be at concentrations below 1 ng/ml (10 nM) in drinking water. If the Hg2+ ions are higher than 2.5 ng/ml in serum, that will bring mercury poisoning. The traditional testing for Hg2+ ions includes atomic absorption, atomic fluorescence, and inductively coupled plasma mass spectrometry. These methods are usually coupled with gas chromatography, high-performance liquid chromatography, and capillary electrophoresis. However, these instrument-based techniques are rather complicated, time-consuming, costly, and unsuitable for online and portable use. An ultrasensitive and selective detection of mercury (II) in serum was investigated using a laser scanning confocal imaging-surface plasmon resonance system (LSCI-SPR). The detection limit was as low as 0.01 ng/ml for Hg2+ ions in fetal calf serum and that is lower than that was required Hg2+ ions must be at concentrations below 1 ng/ml by the US Environment Protection Agency (EPA) guidelines. This sensor was designed on a T-rich, single-stranded DNA (ssDNA)-modified gold film, which can be individually manipulated using specific T-Hg2+-T complex formation. The quenching intensity of the fluorescence images for rhodamine-labeled ssDNA fitted well with the changes in SPR. The changes varied with the Hg2+ ion concentration, which is unaffected by the presence of other metal ions. A good liner relation was got with the coefficients of 0.9116 in 30% fetal calf serums with the linear part over a range of 0.01 ng/ml to10 ng/ml.

  9. Laser excited confocal microscope fluorescence scanner and method

    DOEpatents

    Mathies, Richard A.; Peck, Konan

    1992-01-01

    A fluorescent scanner for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier including a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from said volume to provide a display of the separated sample.

  10. Laser excited confocal microscope fluorescence scanner and method

    DOEpatents

    Mathies, R.A.; Peck, K.

    1992-02-25

    A fluorescent scanner is designed for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier. The scanner includes a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from the volume to provide a display of the separated sample. 8 figs.

  11. Sheet-scanned dual-axis confocal (SS-DAC) microscopy using Richardson-Lucy deconvolution

    PubMed Central

    Wang, Danni; Meza, Daphne; Wang, Yu; Gao, Liang; Liu, Jonathan T.C.

    2015-01-01

    We have previously developed a line-scanned dual-axis confocal (LS-DAC) microscope with subcellular resolution suitable for high-frame-rate diagnostic imaging at shallow depths. Due to the loss of confocality along one dimension, the contrast (signal-to-background ratio) of a LS-DAC microscope is deteriorated compared to a point-scanned DAC microscope. However, by using a sCMOS camera for detection, a short oblique light-sheet is imaged at each scanned position. Therefore, by scanning the light sheet in only one dimension, a thin 3D volume is imaged. Both sequential two-dimensional deconvolution and three-dimensional deconvolution are performed on the thin image volume to improve the resolution and contrast of one en face confocal image section at the center of the volume, a technique we call sheet-scanned dual-axis confocal (SS-DAC) microscopy. PMID:26466290

  12. Sheet-scanned dual-axis confocal microscopy using Richardson-Lucy deconvolution.

    PubMed

    Wang, D; Meza, D; Wang, Y; Gao, L; Liu, J T C

    2014-09-15

    We have previously developed a line-scanned dual-axis confocal (LS-DAC) microscope with subcellular resolution suitable for high-frame-rate diagnostic imaging at shallow depths. Due to the loss of confocality along one dimension, the contrast (signal-to-background ratio) of a LS-DAC microscope is deteriorated compared to a point-scanned DAC microscope. However, by using a sCMOS camera for detection, a short oblique light-sheet is imaged at each scanned position. Therefore, by scanning the light sheet in only one dimension, a thin 3D volume is imaged. Both sequential two-dimensional deconvolution and three-dimensional deconvolution are performed on the thin image volume to improve the resolution and contrast of one en face confocal image section at the center of the volume, a technique we call sheet-scanned dual-axis confocal (SS-DAC) microscopy. PMID:26466290

  13. Confocal Fabry-Perot interferometer for frequency stabilization of laser

    NASA Astrophysics Data System (ADS)

    Pan, H.-J.; Ruan, P.; Wang, H.-W.; Li, F.

    2011-02-01

    The frequency shift of laser source of Doppler lidar is required in the range of a few megahertzs. To satisfy this demand, a confocal Fabry-Perot (F-P) interferometer was manufactured as the frequency standard for frequency stabilization. After analyzing and contrasting the center frequency shift of confocal Fabry-Perot interferometers that are made of three different types of material with the change of temperature, the zerodur material was selected to fabricate the interferometer, and the cavity mirrors were optically contacted onto the end of spacer. The confocal Fabry-Perot interferometer was situated within a double-walled chamber, and the change of temperature in the chamber was less than 0.01 K. The experimental results indicate that the free spectral range is 500 MHz, the full-width at half maximum is 3.33 MHz, and the finesse is 150.

  14. Imaging of whole tumor cut sections using a novel scanning beam confocal fluorescence MACROscope

    NASA Astrophysics Data System (ADS)

    Constantinou, Paul; Vukovic, Vojislav; Haugland, Hans K.; Nicklee, Trudey; Hedley, David W.; Wilson, Brian C.

    2001-07-01

    Hypoxia caused by inadequate structure and function of the tumor vasculature has been found to negatively determine the prognosis of cancer patients. Hence, understanding the biological basis of tumor hypoxia is of significant clinical interest. To study solid tumor microenvironments in sufficient detail, large areas (several mm in diameter) need to be imaged at micrometers resolutions. We have used a novel confocal scanning laser MACROscopeTM (CSLM) capable of acquiring images over fields of view up to 2 cm X 2 cm. To demonstrate its performance, frozen sections from a cervical carcinoma xenograft were triple labeled for tissue hypoxia, blood vessels and hypoxia-inducible transcription factor 1 alpha (HIF-1(alpha) ), imaged using the CSLM and compared to images obtained using a standard epifluorescence microscope imaging system. The results indicate that the CSLM is a useful instrument for imaging tissue-based fluorescence at resolutions comparable to standard low-power microscope objectives.

  15. Spectrally encoded slit confocal microscopy using a wavelength-swept laser

    NASA Astrophysics Data System (ADS)

    Kim, Soocheol; Hwang, Jaehyun; Heo, Jung; Ryu, Suho; Lee, Donghak; Kim, Sang-Hoon; Oh, Seung Jae; Joo, Chulmin

    2015-03-01

    We present an implementation of spectrally encoded slit confocal microscopy. The method employs a rapid wavelength-swept laser as the light source and illuminates a specimen with a line focus that scans through the specimen as the wavelength sweeps. The reflected light from the specimen is imaged with a stationary line scan camera, in which the finite pixel height serves as a slit aperture. This scanner-free operation enables a simple and cost-effective implementation in a small form factor, while allowing for the three-dimensional imaging of biological samples.

  16. Design and Performance of a Multi-Point Scan Confocal Microendoscope

    PubMed Central

    Risi, Matthew D.; Makhlouf, Houssine; Rouse, Andrew R.; Tanbakuchi, Anthony A.; Gmitro, Arthur F.

    2016-01-01

    Confocal fluorescence microendoscopy provides high-resolution cellular-level imaging via a minimally invasive procedure, but requires fast scanning to achieve real-time imaging in vivo. Ideal confocal imaging performance is obtained with a point scanning system, but the scan rates required for in vivo biomedical imaging can be difficult to achieve. By scanning a line of illumination in one direction in conjunction with a stationary confocal slit aperture, very high image acquisition speeds can be achieved, but at the cost of a reduction in image quality. Here, the design, implementation, and experimental verification of a custom multi-point aperture modification to a line-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution of a line-scan system while maintaining high imaging rates. In addition, compared to the line-scanning configuration, previously reported simulations predicted that the multi-point aperture geometry greatly reduces the effects of tissue scatter on image quality. Experimental results confirming this prediction are presented. PMID:26998478

  17. Identification of ex-vivo confocal scanning microscopic features and their histological correlates in human skin.

    PubMed

    Hartmann, Daniela; Ruini, Cristel; Mathemeier, Leonie; Dietrich, Andreas; Ruzicka, Thomas; von Braunmühl, Tanja

    2016-04-01

    Ex-vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. The first generation of ex-vivo devices have already shown promising results in the ex-vivo evaluation of basal cell carcinoma compared to Mohs surgery. Nevertheless, for the diagnostics of pathological skin lesions the knowledge of normal skin features is essential. Therefore we examined 50 samples of healthy skin from various donor sites including head and neck (n = 25), trunk (n = 10), upper (n = 10) and lower extremities (n = 5) using a new generation ex-vivo CLSM device offering three different laser wavelengths and compared the findings to the corresponding histological sections. In correlation with the histopathology we identified different layers of the epidermis, differentiated keratinocytes from melanocytes and described in detail skin appendages including hair follicle, sebaceous and sweat glands. Furthermore, structures of the dermis and subcutis were illustrated. Additionally, artefacts and pitfalls occurring with the use of ex-vivo CLSM have been documented. The study offers an overview of the main ex-vivo CLSM skin characteristics in comparison to the standard histological examination and helps to recognize and avoid common artefacts. Anatomy of a hair follicle in the reflectance mode (RM) CLSM, fluorescence mode (FM) CLSM and in a routine hematoxylin-eosin stained histological section (H). PMID:25996548

  18. Emulation and design of terahertz reflection-mode confocal scanning microscopy based on virtual pinhole

    NASA Astrophysics Data System (ADS)

    Yang, Yong-fa; Li, Qi

    2014-12-01

    In the practical application of terahertz reflection-mode confocal scanning microscopy, the size of detector pinhole is an important factor that determines the performance of spatial resolution characteristic of the microscopic system. However, the use of physical pinhole brings some inconvenience to the experiment and the adjustment error has a great influence on the experiment result. Through reasonably selecting the parameter of matrix detector virtual pinhole (VPH), it can efficiently approximate the physical pinhole. By using this approach, the difficulty of experimental calibration is reduced significantly. In this article, an imaging scheme of terahertz reflection-mode confocal scanning microscopy that is based on the matrix detector VPH is put forward. The influence of detector pinhole size on the axial resolution of confocal scanning microscopy is emulated and analyzed. Then, the parameter of VPH is emulated when the best axial imaging performance is reached.

  19. [Calibration Procedure of Laser Confocal Micro-Raman Spectrometer].

    PubMed

    Zhao, Ying-chun; Ren, Ling-ling; Wei, Wei-sheng; Yao, Ya-xuan

    2015-09-01

    As a common spectral characterization technique, Raman spectroscopy is widely used and has a specified calibration procedure. Based on laser confocal micro-Raman spectrometer, in this paper, we briefly introduced the principle, configuration and main components of Raman spectrometer. In addition, the calibration procedures were also presented, with an emphasis on the calibration of spectrometer (spectrograph) and that of excitation laser wavelength. On the basis of conventional calibration method, a novel and more accurate method was proposed to obtain the actual excitation wavelength, that is, calibration at the point of Raman shift Δν=0. Using this novel calibration method of excitation wavelength, Raman frequency shift values of sulfur were measured, and compared with the standard values from American Society Testing and Materials (ASTM). As a result, the measured values after calibration were consistent with those ASTM values, which indicated that the calibration method is accurate. Thus, a more reasonable calibration procedure of the laser confocal micro-Raman spectrometer was provided. PMID:26669164

  20. Fast scanning cavity offset lock for laser frequency drift stabilization

    NASA Astrophysics Data System (ADS)

    Seymour-Smith, Nicolas; Blythe, Peter; Keller, Matthias; Lange, Wolfgang

    2010-07-01

    We have implemented a compact setup for long-term laser frequency stabilization. Light from a stable reference laser and several slave lasers is coupled into a confocal Fabry-Pérot resonator. By stabilizing the position of the transmission peaks of the slave lasers relative to successive peaks of the master laser as the length of the cavity is scanned over one free spectral range, the long-term stability of the master laser is transferred to the slave lasers. By using fast analog peak detection and low-latency microcontroller-based digital feedback, with a scanning frequency of 3 kHz, we obtain a feedback bandwidth of 380 Hz and a relative stability of better than 10 kHz at timescales longer than 1 s, a significant improvement on previous scanning-cavity stabilization systems.

  1. Probe based confocal laser endomicroscopy of the pancreatobiliary system

    PubMed Central

    Almadi, Majid A; Neumann, Helmut

    2015-01-01

    AIM: To review applications of confocal laser endomicroscopy (CLE) in pancreatobiliary lesions and studies that assessed training and interpretation of images. METHODS: A computerized literature search was performed using OVID MEDLINE, EMBASE, Cochrane library, and the ISI Web of Knowledge from 1980 to October 2014. We also searched abstracts from major meetings that included the Digestive Disease Week, Canadian Digestive Disease Week and the United European Gastroenterology Week using a combination of controlled vocabulary and text words related to pCLE, confocal, endomicroscopy, probe-based confocal laser endomicroscopy, and bile duct to identify reports of trials. In addition, recursive searches and cross-referencing was performed, and manual searches of articles identified after the initial search was also completed. We included fully published articles and those in abstract form. Given the relatively recent introduction of CLE we included randomized trials and cohort studies. RESULTS: In the evaluation of indeterminate pancreatobiliary strictures CLE with ERCP compared to ERCP alone can increase the detection of cancerous strictures with a sensitivity of (98% vs 45%) and has a negative predictive value (97% vs 69%), but decreased the specificity (67% vs 100%) and the positive predictive value (71% vs 100%) when compared to index pathology. Modifications in the classification systems in indeterminate biliary strictures have increased the specificity of pCLE from 67% to 73%. In pancreatic cystic lesions there is a need to develop similar systems to interpret and characterize lesions based on CLE images obtained. The presence of superficial vascular network predicts serous cystadenomas accurately. Also training in acquiring and interpretation of images is feasible in those without any prior knowledge in CLE in a relatively simple manner and computer-aided diagnosis software is a promising innovation. CONCLUSION: The role of pCLE in the evaluation of

  2. Imaging inflammation in mouse colon using a rapid stage-scanning confocal fluorescence microscope

    PubMed Central

    Saldua, Meagan A.; Olsovsky, Cory A.; Callaway, Evelyn S.; Chapkin, Robert S.

    2012-01-01

    Abstract. Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333  lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7  mm/sec. A single 1×60 mm2 field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion. PMID:22352656

  3. Imaging inflammation in mouse colon using a rapid stage-scanning confocal fluorescence microscope.

    PubMed

    Saldua, Meagan A; Olsovsky, Cory A; Callaway, Evelyn S; Chapkin, Robert S; Maitland, Kristen C

    2012-01-01

    Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333 lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7 mm/sec. A single 1 × 60 mm(2) field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion. PMID:22352656

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

    PubMed

    Conchello, J A; Lichtman, J W

    1994-02-01

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

  5. Vital fluorescent labeling for confocal scanning microscopic study of living cell invasion

    NASA Astrophysics Data System (ADS)

    Wang, Allan Z.; Chen, Jian M.; Fisher, Gregory W.; Wang, Jane C.

    1997-07-01

    Invasion by cells with malignant or transformed phenotypes precedes destruction of adjacent tissue and fatal cell metastasis. State-of-the-art confocal laser scanning technology facilitates both in vitro and in vivo research into cell invasion and metastasis. In particular, studies performed with living cells yield more precise information than those with fixed cells, giving new insight into cell invasion and metastasis. We have tested a variety of vital florescent dyes and fluorogenic protease substrates in our studies of invasion of cartilage by transformed synoviocytes or osteosarcoma cells. The fluorescent dyes tested include Calcein acetoxy methyl-FITC (Calcein), Hoechst 33342 (Hoechst), CellTracker, DiI, DiO, DiD, and ethidium bromide (EB). The fluorogenic protease substrate used Meoxysuccinyl-Gly-Pro-Leu-Gly-Pro-AFC (MOS-GPLGP-AFC) for detection of collagenase activity. We found that Calcein-FITC labeling permitted the clearest direct observation of the penetration of transformed synoviocytes and osteosarcoma cells into cartilage. Even better results were obtained when chondrocyte nuclei were counter-stained with Hoechst 33342. During the invasion process, collagenase activity was observed around the synoviocyte in the cartilage matrix labeled with the fluorogenic collagenase substrate. We concluded that of the vital fluorescent dyes tested, a combined application of Calcein-FITC, Hoechst 23223, and MOS- GPLGP-AFC is most appropriate for the study of the cell invasion process.

  6. Laser multi-reflection confocal long focal-length measurement

    NASA Astrophysics Data System (ADS)

    Li, Zhigang; Qiu, Lirong; Zhao, Weiqian; Xiao, Yang

    2016-06-01

    We propose a new laser multi-reflection confocal focal-length measurement (MCFM) method to meet the requirements of a high-precision measurement for a long focal-length more than 2 m. It places an optical flat and a reflector behind the test lens for reflecting the measuring beam repeatedly, and then, uses the property that the peak points of confocal response curves precisely corresponds to the convergence points of a multi-reflected measuring beam to exactly identify the positions of the convergence points. Subsequently, it obtains the position variation of the reflector with a different number of reflections by a distance measuring instrument, and thereby achieving the high precise long focal-length measurement. The theoretical analyses and preliminary experimental results indicate that MCFM has a relative standard uncertainty of 0.066% for a test lens with the focal-length of 9.76 m. MCFM can provide a novel approach for the high-precision focal-length measurement.

  7. Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning

    PubMed Central

    Wang, Thomas D.; Contag, Christopher H.; Mandella, Michael J.; Chan, Ning Y.; Kino, Gordon S.

    2007-01-01

    We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution ≤4.4 μm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging. PMID:15250760

  8. Surface microstructure profilometry based on laser confocal feedback

    NASA Astrophysics Data System (ADS)

    Wang, Weiping; Zhang, Shulian; Li, Yan

    2015-10-01

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

  9. Laser confocal feedback tomography and nano-step height measurement

    PubMed Central

    Tan, Yidong; Wang, Weiping; Xu, Chunxin; Zhang, Shulian

    2013-01-01

    A promising method for tomography and step height measurement is proposed, which combines the high sensitivity of the frequency-shifted feedback laser and the axial positioning ability of confocal microscopy. By demodulating the feedback-induced intensity modulation signals, the obtained amplitude and phase information are used to respectively determine the coarse and fine measurement of the samples. Imaging the micro devices and biological samples by the demodulated amplitude, this approach is proved to be able to achieve the cross-sectional image in highly scattered mediums. And then the successful height measurement of nano-step on a glass-substrate grating by combination of both amplitude and phase information indicates its axial high resolution (better than 2 nm) in a non-ambiguous range of about ten microns. PMID:24145717

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

    SciTech Connect

    Apedo, K.L.; Munzer, C.; He, H.; Montgomery, P.; Serres, N.; Fond, C.; Feugeas, F.

    2015-02-15

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

  11. Improved scanning laser fundus imaging using polarimetry

    NASA Astrophysics Data System (ADS)

    Bueno, Juan M.; Hunter, Jennifer J.; Cookson, Christopher J.; Kisilak, Marsha L.; Campbell, Melanie C. W.

    2007-05-01

    We present a polarimetric technique to improve fundus images that notably simplifies and extends a previous procedure [Opt. Lett.27, 830 (2002)]. A generator of varying polarization states was incorporated into the illumination path of a confocal scanning laser ophthalmoscope. A series of four images, corresponding to independent incoming polarization states, were recorded. From these images, the spatially resolved elements of the top row of the Mueller matrix were computed. From these elements, images with the highest and lowest quality (according to different image quality metrics) were constructed, some of which provided improved visualization of fundus structures of clinical importance (vessels and optic nerve head). The metric values were better for these constructed images than for the initially recorded images and better than averaged images. Entropy is the metric that is most sensitive to differences in the image quality. Improved visualization of features could aid in the detection, localization, and tracking of ocular disease and may be applicable in other biomedical imaging.

  12. Effects of axial scanning in confocal microscopy employing adaptive lenses (CAL)

    NASA Astrophysics Data System (ADS)

    Koukourakis, N.; Finkeldey, M.; Stürmer, M.; Gerhardt, N. C.; Wallrabe, U.; Hofmann, M. R.; Czarske, J. W.; Fischer, A.

    2014-05-01

    We analyze axial scanning in Confocal microscopy based on Adaptive Lenses (CAL). A tunable lens located in the illumination path of a confocal setup enables scanning the focus position by applying an electrical voltage. This opens up the possibility to replace mechanical axial scanning which is commonly used. In our proof-of-principle experiment, we demonstrate a tuning range of about 380 μm. The range can easily be extended by using the whole possible tuning range. During the scan the axial resolution degrades by a factor of about 2.3. The deterioration is introduced by aberrations that strongly depend on the scanning process. Therefore a second lens is located in the detection path of the CAL setup to balance the aberration effects. Both experiments and simulations show that this approach allows creating a homogeneous axial resolution throughout the scan. This is at the cost of tuning range which halves to about 200 μm. The lateral resolution is not noticeably affected and amounts to 500 nm.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  14. Remote-access slit-scanning confocal microscope for in vivo tumor diagnosis

    NASA Astrophysics Data System (ADS)

    Sabharwal, Yashvinder Singh

    Microscopic fluorescence imaging of thick biological tissue has been successfully demonstrated with a fiber- based, slit-scanning, confocal microscope. The system developed under this research consists of an illumination arm, a fiber-optic imaging system, and a detection arm. The illumination arm is an anamorphic optical system that converts a circular, laser beam into a cylindrical beam forming a line image at the proximal face of the fiber- optic relay. This relay system is comprised of a fiber- optic imaging bundle, a miniature objective lens, and a miniature hydraulic positioning mechanism. It delivers illumination to a remote sample and simultaneously collects the fluorescence from the sample. The miniature objective lens and positioning mechanism were specially designed and fabricated for this system, allowing for high resolution imaging and optical sectioning in-vivo. The detection arm relays the fluorescence image at the proximal face of the fiber-optic relay with magnification onto a two-dimensional CCD. Characterization of the system has demonstrated a lateral resolution of three microns. The axial resolution when imaging a point object is 10 microns. When imaging a planar object, the axial resolution is 25 microns. Images are acquired at a rate of 2-4 frames per second and the imaging performance has been evaluated with different biological models including animal peritoneal tissue and human prostate tissue in-vitro. In-vivo images of human skin and rat peritoneum have also been acquired to demonstrate that patient motion does not adversely affect the performance of the system. These in-vitro and in vivo images demonstrate the capability of the system to resolve cell nuclear morphology, to visualize cell density and organization, and to image at selected depths below the tissue surface.

  15. Three-dimensional imaging of carbon nanostructures by scanning confocal electron microscopy

    NASA Astrophysics Data System (ADS)

    Hashimoto, Ayako; Shimojo, Masayuki; Mitsuishi, Kazutaka; Takeguchi, Masaki

    2009-10-01

    Although scanning confocal electron microscopy (SCEM) shows a promise for optical depth sectioning with high resolution, practical and theoretical problems have prevented its application to three-dimensional (3D) imaging. We employed a stage-scanning system in which only the specimen is moved three dimensionally under a fixed lens configuration, and an annular dark-field (ADF) aperture which blocks direct beams and selects only the scattered electrons. This ADF-SCEM improved depth resolution sufficiently to perform optical depth sectioning. Finally, we succeeded in demonstrating the 3D reconstruction of carbon nanocoils using ADF-SCEM.

  16. Femtosecond laser subsurface scleral treatment in cadaver human sclera and evaluation using two-photon and confocal microscopy

    NASA Astrophysics Data System (ADS)

    Sun, Hui; Fan, Zhongwei; Yan, Ying; Lian, Fuqiang; Kurtz, Ron; Juhasz, Tibor

    2016-03-01

    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial-thickness drainage channels can be created with femtosecond laser in the translucent sclera for the potential treatment of glaucoma. We demonstrate the creation of partial-thickness subsurface drainage channels with the femtosecond laser in the cadaver human eyeballs and describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. A femtosecond laser operating at a wavelength of 1700 nm was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of cadaver human eyes. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such partial thickness subsurface scleral channels. Our studies suggest that the confocal and two-photon microscopy can be used to investigate femtosecond-laser created partial-thickness drainage channels in the sclera of cadaver human eyes.

  17. Probe-based confocal laser endomicroscopy in head and neck malignancies: early preclinical experience

    NASA Astrophysics Data System (ADS)

    Englhard, Anna; Girschick, Susanne; Mack, Brigitte; Volgger, Veronika; Gires, Oliver; Conderman, Christian; Stepp, Herbert; Betz, Christian Stephan

    2013-06-01

    Background: Malignancies of the upper aerodigestive tract (UADT) are conventionally diagnosed by white light endoscopy, biopsy and histopathology. Probe-based Confocal Laser Endomicroscopy (pCLE) is a novel non-invasive technique which offers in vivo surface and sub-surface imaging of tissue. It produces pictures of cellular architecture comparable to histology without the need for biopsy. It has already been successfully used in different clinical subspecialties to help in the diagnosis and treatment planning of inflammatory and neoplastic diseases. PCLE needs to be used in combination with specific or non-specific contrast agents. In this study we evaluated the potential use of pCLE in combination with non-specific and specific contrast agents to distinguish between healthy mucosa and invasive carcinoma. Methods: Tissue samples from healthy mucosa and squamous cell carcinoma of the head and neck were taken during surgery. After topical application of three different contrast agents, samples were examined using different pCLE-probes and a Confocal Laser Scanning Microscope (CLSM). Images were then compared to the corresponding histological slides and cryosections. Results: Initial results show that pCLE in combination with fluorophores allows visualization of cellular and structural components. Imaging of different layers was possible using three distinct pCLEprobes. Conclusion: pCLE is a promising non-invasive technique that may be a useful adjunct in the evaluation, diagnosis and treatment planning of head and neck malignancies.

  18. The method of axial drift compensation of laser differential confocal microscopy based on zero-tracking

    NASA Astrophysics Data System (ADS)

    Wang, Yajie; Cui, Han; Wang, Yun; Qiu, Lirong; Zhao, Weiqian

    2015-08-01

    Laser differential confocal microscopy (DCM) has advantages of high axial resolution and strong ability of focus identification. However, the imaging mechanism of point scanning needs long measurement time, in the process due to itself mechanical instability and the influence of environment vibration the axial drift of object position is inevitable, which will reduce lateral resolution of the DCM. To ensure the lateral resolution we propose an axial drift compensation method based on zero-tracking in this paper. The method takes advantage of the linear region of differential confocal axial response curve, gets axial drift by detecting the laser intensity; uses grating sensor to monitor the real-time axial drift of lifting stage and realizes closed-loop control; uses capacitive sensor of objective driver to measure its position. After getting the axial drift of object, the lifting stage and objective driver will be driven to compensate position according to the axial drift. This method is realized by using Visual Studio 2010, and the experiment demonstrates that the compensation precision of the proposed method can reach 6 nm. It is not only easy to implement, but also can compensate the axial drift actively and real-timely. Above all, this method improves the system stability of DCM effectively.

  19. Confocal laser endomicroscopy in gastrointestinal and pancreatobiliary diseases.

    PubMed

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

    2014-01-01

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

  20. Confocal laser endomicroscopy features of sessile serrated adenomas/polyps

    PubMed Central

    Parikh, Neil D; Gibson, Joanna; Nagar, Anil; Ahmed, Ali A

    2015-01-01

    Background and aims Sessile serrated adenomas/polyps (SSA/Ps) are difficult to differentiate from non-neoplastic tissue on white-light endoscopy. Confocal laser endomicroscopy (CLE) provides subcellular imaging and real-time “optical biopsy”. The aim of this study was to prospectively describe CLE features of SSA/Ps. Patients and methods Consecutive patients with SSA/Ps were prospectively evaluated with probe-based CLE imaging. CLE images and polyp histology were independently reviewed by three endoscopists and an expert gastrointestinal (GI) pathologist. Distinguishing CLE features of SSA/Ps were identified in conjunction with pathologic correlation. Results In total, 260 CLE images were generated from nine SSA/Ps evaluated in seven patients. Four consensus CLE features of SSA/P were identified: (1) a mucus cap with a bright, cloud-like appearance; (2) thin, branching crypts; (3) increased number of goblet cells and microvesicular mucin-containing cells; and (4) architectural disarray, with dystrophic goblet cells and lack of regular circular crypts Conclusion This is a novel description of characteristic CLE features of SSA/Ps. The four features we identified are easy to detect and may allow for CLE to serve as a diagnostic modality. PMID:27536371

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  2. High speed, line-scanning, fiber bundle fluorescence confocal endomicroscopy for improved mosaicking

    PubMed Central

    Hughes, Michael; Yang, Guang-Zhong

    2015-01-01

    A significant limitation of fiber bundle endomicroscopy systems is that the field of view tends to be small, usually only several hundred micrometers in diameter. Image mosaicking techniques can increase the effective image size, but require careful manipulation of the probe to ensure sufficient overlap between adjacent frames. For confocal endomicroscopes, which typically have frame rates on the order of 10 fps, this is particularly challenging. In this paper we demonstrate that line-scanning confocal endomicroscopy can, by use of a high speed linear CCD camera, achieve a frame rate of 120 fps while maintaining sufficient resolution and signal-to-noise ratio to allow imaging of topically stained gastrointestinal tissues. This leads to improved performance of a cross-correlation based mosaicking algorithm when compared with lower frame-rate systems. PMID:25909008

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

    DOEpatents

    de Jonge, Niels [Oak Ridge, TN

    2010-08-17

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

  4. Laparoscopic Manipulation of a Probe-based Confocal Laser Endomicroscope Using a Steerable Intravascular Catheter

    PubMed Central

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

    2015-01-01

    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. PMID:25807277

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

    PubMed Central

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

    2015-01-01

    Zone-plate-based scanning transmission soft X-ray microspectroscopy (STXM) is a well established technique for high-contrast imaging of sufficiently transparent specimens (e.g. ultrathin biological tissues, polymer materials, archaeometric specimens or magnetic thin films) with spatial resolutions in the regime of 20 nm and high spectroscopic or chemical sensitivity. However, due to the relatively large depth of focus of zone plates, the resolution of STXM along the optical axis so far stays unambiguously behind for thicker X-ray transparent specimens. This challenge can be addressed by the implementation of a second zone plate in the detection pathway of the beam, resulting in a confocal arrangement. Within this paper a first proof-of-principle study for a confocal STXM (cSTXM) and an elaborate alignment procedure in transmission and fluorescence geometry are presented. Based on first confocal soft X-ray micrographs of well known specimens, the advantage and limitation of cSTXM as well as further development potentials for future applications are discussed. PMID:25537596

  6. Toward real-time virtual biopsy of oral lesions using confocal laser endomicroscopy interfaced with embedded computing

    NASA Astrophysics Data System (ADS)

    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

    2012-05-01

    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.

  7. Performance measurements of an infrared digital scanning laser ophthalmoscope.

    PubMed

    Manivannan, A; Sharp, P F; Forrester, J V

    1994-08-01

    Direct digital acquisition of images using a scanning laser ophthalmoscope (SLO) offers several advantages over the conventional fundus camera; in particular, the ability to produce tomographic images using a confocal aperture. This note describes measurements of the performance of an SLO. Spatial resolution, measured by the modulation transfer function, MTF, was shown to be worse along the direction of scan. As expected, image uniformity was good, with a coefficient of variation of 1.9%. While the effect of using a 100 microns diameter confocal aperture instead of one with a 400 microns diameter was to reduce slice thickness from 2600 microns to 975 microns, image intensity was reduced by a factor of 30. PMID:7994210

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

    PubMed

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

    2012-05-01

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

  9. Interobserver Agreement of Confocal Laser Endomicroscopy for Bladder Cancer

    PubMed Central

    Chang, Timothy C.; Liu, Jen-Jane; Hsiao, Shelly T.; Pan, Ying; Mach, Kathleen E.; Leppert, John T.; McKenney, Jesse K.; Rouse, Robert V.

    2013-01-01

    Abstract Background and Purpose Emerging optical imaging technologies such as confocal laser endomicroscopy (CLE) hold promise in improving bladder cancer diagnosis. The purpose of this study was to determine the interobserver agreement of image interpretation using CLE for bladder cancer. Methods Experienced CLE urologists (n=2), novice CLE urologists (n=6), pathologists (n=4), and nonclinical researchers (n=5) were recruited to participate in a 2-hour computer-based training consisting of a teaching and validation set of intraoperative white light cystoscopy (WLC) and CLE video sequences from patients undergoing transurethral resection of bladder tumor. Interobserver agreement was determined using the κ statistic. Results Of the 31 bladder regions analyzed, 19 were cancer and 12 were benign. For cancer diagnosis, experienced CLE urologists had substantial agreement for both CLE and WLC+CLE (90%, κ 0.80) compared with moderate agreement for WLC alone (74%, κ 0.46), while novice CLE urologists had moderate agreement for CLE (77%, κ 0.55), WLC (78%, κ 0.54), and WLC+CLE (80%, κ 0.59). Pathologists had substantial agreement for CLE (81%, κ 0.61), and nonclinical researchers had moderate agreement (77%, κ 0.49) in cancer diagnosis. For cancer grading, experienced CLE urologists had fair to moderate agreement for CLE (68%, κ 0.64), WLC (74%, κ 0.67), and WLC+CLE (53%, κ 0.33), as did novice CLE urologists for CLE (53%, κ 0.39), WLC (66%, κ 0.50), and WLC+CLE (61%, κ 0.49). Pathologists (65%, κ 0.55) and nonclinical researchers (61%, κ 0.56) both had moderate agreement for CLE in cancer grading. Conclusions CLE is an adoptable technology for cancer diagnosis in novice CLE observers after a short training with moderate interobserver agreement and diagnostic accuracy similar to WLC alone. Experienced CLE observers may be capable of achieving substantial levels of agreement for cancer diagnosis that is higher than with WLC alone. PMID:23072435

  10. Laser Scanning Applications in Fluvial Geomorphology

    NASA Astrophysics Data System (ADS)

    Alho, P.

    2014-12-01

    During recent decades, the use of high-resolution laser scanning data in fluvial studies has rapidly increased. Airborne laser scanning (ALS) can be used to extensively map riverine topography. Laser scanning data have great potential to improve the effectiveness of topographical data acquisition and the accuracy and resolution of DTMs (Digital Terrain Models) needed in fluvial geomorphology. Airborne Laser Scanning (ALS) is applicable for mapping areas varying from reach to catchment scale and these data are, therefore, particularly suitable, especially for hydraulic modelling, mapping of flood inundation, and the detection of macro-scale fluvial geomorphology. With Terrestrial Laser Scanning (TLS) a spatial resolution of less than 1 mm and a range accuracy of few millimetres can be achieved. Mobile Laser Scanning (MLS) enables a remarkably faster survey approach compared to the conventional TLS method. One of the newest applications of MLS approaches involves a boat/cart/backpack -based mobile mapping system. This set-up includes laser scanning and imaging from a platform moving along a river course or floodplain and may be used to expand the spatial extent of terrestrial scanning. Detailed DTMs derived from laser scanning data can be used to improve the recognition of fluvial landforms, the geometric data of hydraulic modelling, and the estimation of flood inundation extents and the associated fluvial processes. Fluvial environments also offer challenges for the application of laser scanning techniques. Factors such as vegetation cover, terrain undulation, coarse surface materials and water surfaces may distort a laser scanning survey.

  11. Compact two-photon laser-scanning microscope made from minimally modified commercial components

    NASA Astrophysics Data System (ADS)

    Iyer, Vijay; Hoogland, Tycho; Losavio, Bradley E.; McQuiston, A. R.; Saggau, Peter

    2002-06-01

    A compact two-photon laser-scanning microscope (TPLSM) was constructed using a diode-pumped, mode-locked Nd:YLF laser (Biolight 1000, Coherent Laser Group) and a small confocal laser scan-head (PCM2000, Nikon Bioscience). The laser emits at 1047nm and is fiber-coupled to a compact compressor unit producing a pulse-width of ~175fsec. Both the pulse compressor and confocal scan head were interfaced on a small optical breadboard that was directly attached to an upright research microscope (Eclipse E600FN, Nikon Bioscience). Two-photon fluorescence emitted from the specimen was collected into a multimode fiber and transmitted directly to an external PMT supplied with the Nikon confocal system. The modifications to the scanhead were minimal (a single mirror replacement) and did not interfere with its confocal function. The resulting system offers several advantages: compact size, turnkey operation, and the ability to translate the microscope rather than an often delicate specimen. In addition, it is possible to switch between confocal and two-photon operation, allowing for straightforward comparison. Using this compact TPLSM, we obtained structural and functional images from hippocampal neurons in living brain slices using commonly available fluorophores.

  12. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

    PubMed Central

    Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H.; Wouters, Fred S.; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

    2013-01-01

    We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions. PMID:24324140

  13. Quasi-confocal, multichannel parallel scan hyperspectral fluorescence imaging method optimized for analysis of multicolor microarrays.

    PubMed

    Liu, Zhiyi; Ma, Suihua; Ji, Yanhong; Liu, Le; Hu, Zhaoxu; Guo, Jihua; Ma, Hui; He, Yonghong

    2010-09-15

    The microarray technique, which can provide parallel detection with high throughput in biomedical research, has generated considerable interest since the end of the 20th century. A number of instruments have been reported for microarray detection. In this paper, we have developed a quasi-confocal, multichannel parallel scan hyperspectral fluorescence imaging system for multicolor microarray research. Hyperspectral imaging records the entire emission spectrum for every voxel within the imaged area in contrast to recording only fluorescence intensities of filter-based scanners. When coupled with data analysis, the recorded spectral information allows for quantitative identification of the contributions of multiple, spectrally overlapping fluorescent dyes and elimination of unwanted artifacts. This system is improved with a specifically designed, high performance spectrometer which can offer a spectral resolution of 0.2 nm and operates with spatial resolutions ranging from 2 to 30 μm. We demonstrate the application of the system by reading out arrays for identification of bacteria. PMID:20718427

  14. Fabrication of microgrooves on a curved surface by the confocal measurement system using pulse laser and continuous laser

    NASA Astrophysics Data System (ADS)

    Noh, Jiwhan; Cho, Ilhwan; Lee, Seungwoo; Na, Suckjoo; Lee, Jae-Hoon

    2012-03-01

    In order to fabricate microgrooves on a curved surface, the curved surface was measured with a confocal system and then it was used for laser microprocessing. This paper proposes a new method of using a pulse laser for the confocal system to measure the curved surface. It also compares the conventional way of using a continuous laser and a new way of using the pulse laser with the confocal system. Using the data measured with the pulse laser for fabrication, microgrooves were fabricated on a curved surface. The width of the fabricated microgroove was 10 μm and the depth was 27 μm. The microgroove fabricated on a curved surface as a part of this study can be used in injection molding to manufacture a micropatterned plastic surface at a low cost. This plastic surface can be applied for a superhydrophobic surface, a self-cleaning surface, or a biochip.

  15. Probe-based confocal laser endomicroscopy of the urinary tract: the technique.

    PubMed

    Chang, Timothy C; Liu, Jen-Jane; Liao, Joseph C

    2013-01-01

    Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory and gastrointestinal tracts, CLE has also been explored in the urinary tract for bladder cancer diagnosis. Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature. The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use. Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent-most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile. Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy. Recent availability of a < 1 mm imaging probe opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i

  16. Three-dimensional measurements with a novel technique combination of confocal and focus variation with a simultaneous scan

    NASA Astrophysics Data System (ADS)

    Matilla, A.; Mariné, J.; Pérez, J.; Cadevall, C.; Artigas, R.

    2016-04-01

    The most common optical measurement technologies used today for the three dimensional measurement of technical surfaces are Coherence Scanning Interferometry (CSI), Imaging Confocal Microscopy (IC), and Focus Variation (FV). Each one has its benefits and its drawbacks. FV will be the ideal technology for the measurement of those regions where the slopes are high and where the surface is very rough, while CSI and IC will provide better results for smoother and flatter surface regions. In this work we investigated the benefits and drawbacks of combining Interferometry, Confocal and focus variation to get better measurement of technical surfaces. We investigated a way of using Microdisplay Scanning type of Confocal Microscope to acquire on a simultaneous scan confocal and focus Variation information to reconstruct a three dimensional measurement. Several methods are presented to fuse the optical sectioning properties of both techniques as well as the topographical information. This work shows the benefit of this combination technique on several industrial samples where neither confocal nor focus variation is able to provide optimal results.

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

    PubMed Central

    Klauss, André; König, Marcelle; Hille, Carsten

    2015-01-01

    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. PMID:26091552

  18. Distribution of ALA metabolic products in esophageal carcinoma cells using spectrally resolved confocal laser microscopy

    NASA Astrophysics Data System (ADS)

    Smolka, Jozef; Mateasik, Anton

    2006-08-01

    Aminolevulinic acid (ALA) is an efficient substance used in photodynamic therapy (PDT). It is a precursor of light-sensitive products that can selectively accumulate in malignant cells following the altered activity of the heme biosynthetic pathway enzymes in such cells. These products are synthesized in mitochondria and distributed to various cellular structures [1]. The localization of ALA products in subcellular structures depends on their chemical characteristics as well as on the properties of the intracellular environment [2]. Characterization of such properties is possible by means of fluorescent probes like JC-1 and carboxy SNARF-1. However, the emission spectra of these probes are overlapped with spectral pattern of typical ALA product -protoporphyrin IX (PpIX). Spectral overlap of fluorescence signals prevents to clearly separate a distribution of probes from PpIX distribution what can completely mess the applicability of these probes in characterization of cell properties. The spectrally resolved confocal laser microscopy can be used to overcome this problem. In this study, a distribution of ALA metabolic products in relation to the mitochondrial membrane potential and intracellular pH was examined. Human cell lines (KYSE-450, KYSE-70) from esophageal squamous cell carcinoma were used. Cells were incubated with 1mM solution of ALA for four hours. Two fluorescent probes, carboxy SNARF-1 and JC-1 , were used to monitor intracellular pH levels and to determine membrane potential changes, respectively. The samples were scanned by spectrally resolved laser scanning microscope. Spectral linear unmixing method was used to discriminate and separate regions of accumulation of ALA metabolic products of JC-1 and carboxy SNARF-1.

  19. Effects of Fluorescein Staining on Laser In Vivo Confocal Microscopy Images of the Cornea

    PubMed Central

    Sindt, Christine W.; Critser, D. Brice; Grout, Trudy K.; Kern, Jami R.

    2012-01-01

    This study was designed to identify whether topical fluorescein, a common ophthalmic tool, affects laser in vivo confocal microscopy of the cornea, a tool with growing applications. Twenty-five eye care specialists were asked to identify presence or absence of fluorescein in 99 confocal micrographs of healthy corneas. Responses were statistically similar to guessing for the epithelium (48% ± 14% of respondents correct per image) and the subbasal nerve plexus (49% ± 11% correct), but results were less clear for the stroma. Dendritic immune cells were quantified in bilateral images from subjects who had been unilaterally stained with fluorescein. Density of dendritic immune cells was statistically similar between the unstained and contralateral stained eyes of 24 contact lens wearers (P = .72) and of 10 nonwearers (P = .53). Overall, the results indicated that fluorescein staining did not interfere with laser confocal microscopy of corneal epithelium, subbasal nerves, or dendritic immune cells. PMID:22363837

  20. Endothelial cell adhesion in real time. Measurements in vitro by tandem scanning confocal image analysis.

    PubMed

    Davies, P F; Robotewskyj, A; Griem, M L

    1993-06-01

    Real time measurements of cell-substratum adhesion in endothelial cells were obtained by tandem scanning confocal microscopy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radiance levels) in the living cell such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal determinant of cell-substratum adhesion. Measurements of (a) the focal contact area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time. The topography of focal contacts was estimated by calculating separation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm. Subtraction of consecutive images revealed continuous spontaneous remodeling of individual focal adhesions in unperturbed cells during periods of < 1 min. Despite extensive remodeling of focal contact sites, however, cell adhesion calculated for an entire cell over extended periods varied by < 10%. When cytoskeletal stability was impaired by exposure to cytochalasin or when cells were exposed to proteolytic enzyme, endothelial adhesion declined rapidly. Such changes were recorded at the level of single cells, groups of cells, and at single focal adhesions. In both unperturbed and manipulated cells, the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance of existing sites and appearance of new ones often occurred within minutes while adjacent sites underwent minimal remodelling. Tandem scanning confocal microscopy image analysis of

  1. Scanning afocal laser velocimeter projection lens system

    NASA Technical Reports Server (NTRS)

    Rhodes, D. B. (Inventor)

    1982-01-01

    A method and apparatus for projecting and focusing parallel laser light beams from a laser doppler velocimeter on a target area are described. The system includes three lenses. Two lenses work together as a fixed afocal lens combination. The third lens is a movable scanning lens. Parallel laser beams travel from the velocimeter through the scanning lens and through the afocal lens combination and converge, i.e., are focused, somewhere beyond. Moving the scanning lens relative to the fixed afocal combination results in a scanning of the focus area along the afocal combination's optical axis.

  2. Thermographic system with a laser scanning device

    SciTech Connect

    Skvortsov, L A; Kirillov, V M

    2007-11-30

    It is shown that laser photothermal radiometry (LPTR) in combination with laser beam scanning within the instantaneous field of view of a single-element photodetector can be used to develop a scanning thermal emission microscope. An expression is derived for estimating its temperature resolution. The results of calculations are presented and the factors influencing the spatial lateral resolution of the technique and the time of image formation with the help of an acousto-optical deflector are analysed. (laser applications)

  3. Extraction of ultra-high frequency retinal motions with a line scanning quasi-confocal ophthalmoscope

    NASA Astrophysics Data System (ADS)

    He, Yi; Wei, Ling; Wang, Zhibin; Yang, Jinsheng; Li, Xiqi; Shi, Guohua; Zhang, Yudong

    2015-01-01

    Ultra-high frequency motions of the retina severely affect the stabilization of images and result in significant imaging distortions. In this paper, a high speed line scanning quasi-confocal ophthalmoscope (LSO) capable of 160 frames per second was devised for generating stable undistorted retinal images. This technique resulted in minimal intra-frame motions, and a strip-based cross correlation algorithm with sub-pixel resolution was applied to extract retinal motions. Three retinal motion components at rates of up to 1600 Hz were clearly distinguished and extracted accurately for the first time using ophthalmic imaging methods. This was especially apparent for the fastest tremor and microsaccade movements that cannot be estimated from previously reported ophthalmic imaging instruments. Furthermore, these results were consistent with retinal motion characteristics obtained with optical lever methods, validating this technique. Actually, the LSO system has great potential to extract retinal motions, and some other tracking systems may be adopted to correct retinal motions in ophthalmic imaging modalities.

  4. Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope

    PubMed Central

    Dubra, Alfredo; Sulai, Yusufu; Norris, Jennifer L.; Cooper, Robert F.; Dubis, Adam M.; Williams, David R.; Carroll, Joseph

    2011-01-01

    The rod photoreceptors are implicated in a number of devastating retinal diseases. However, routine imaging of these cells has remained elusive, even with the advent of adaptive optics imaging. Here, we present the first in vivo images of the contiguous rod photoreceptor mosaic in nine healthy human subjects. The images were collected with three different confocal adaptive optics scanning ophthalmoscopes at two different institutions, using 680 and 775 nm superluminescent diodes for illumination. Estimates of photoreceptor density and rod:cone ratios in the 5°–15° retinal eccentricity range are consistent with histological findings, confirming our ability to resolve the rod mosaic by averaging multiple registered images, without the need for additional image processing. In one subject, we were able to identify the emergence of the first rods at approximately 190 μm from the foveal center, in agreement with previous histological studies. The rod and cone photoreceptor mosaics appear in focus at different retinal depths, with the rod mosaic best focus (i.e., brightest and sharpest) being at least 10 μm shallower than the cones at retinal eccentricities larger than 8°. This study represents an important step in bringing high-resolution imaging to bear on the study of rod disorders. PMID:21750765

  5. Confocal scanning optical microscopy of a 3-million-year-old Australopithecus afarensis femur.

    PubMed

    Bromage, T G; Goldman, H M; McFarlin, S C; Perez Ochoa, A; Boyde, A

    2009-01-01

    Portable confocal scanning optical microscopy (PCSOM) has been specifically developed for the noncontact and nondestructive imaging of early human fossil hard tissues, which here we describe and apply to a 3-million-year-old femur from the celebrated Ethiopian skeleton, "Lucy," referred to Australopithecus afarensis. We examine two bone tissue parameters that demonstrate the potential of this technology. First, subsurface reflection images from intact bone reveal bone cell spaces, the osteocyte lacunae, whose density is demonstrated to scale negatively with body size, reflecting aspects of metabolism and organismal life history. Second, images of a naturally fractured cross section near to Lucy's femoral mid-shaft, which match in sign those of transmitted circularly polarized light, reveal relative collagen fiber orientation patterns that are an important indicator of femoral biomechanical efficacy. Preliminary results indicate that Lucy was characterized by metabolic constraints typical for a primate her body size and that in her femur she was adapted to habitual bipedalism. Limitations imposed by the transport and invasive histology of unique or rare fossils motivated development of the PCSOM so that specimens may be examined wherever and whenever nondestructive imaging is required. PMID:19191265

  6. Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents

    NASA Astrophysics Data System (ADS)

    Higgins, Laura M.; Zevon, Margot; Ganapathy, Vidya; Sheng, Yang; Tan, Mei Chee; Riman, Richard E.; Roth, Charles M.; Moghe, Prabhas V.; Pierce, Mark C.

    2015-11-01

    Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4:Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic.

  7. Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents.

    PubMed

    Higgins, Laura M; Zevon, Margot; Ganapathy, Vidya; Sheng, Yang; Tan, Mei Chee; Riman, Richard E; Roth, Charles M; Moghe, Prabhas V; Pierce, Mark C

    2015-11-01

    Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4 : Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic. PMID:26603495

  8. EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE

    EPA Science Inventory

    BACKGROUND. The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by ea...

  9. Confocal laser endomicroscopy to monitor the colonic mucosa of mice.

    PubMed

    Mielke, Lisa; Preaudet, Adele; Belz, Gabrielle; Putoczki, Tracy

    2015-06-01

    The gastrointestinal tract is a unique organ system that provides an epithelial barrier between our underlying immune system and luminal pathogens. Disruption of gastrointestinal homeostasis, as a result of impaired barrier function, is associated with numerous pathologies including inflammatory bowel disease and colorectal cancer. In parallel to the clinical development of endoscopy technologies to monitor and diagnose these pathologies in humans, advanced mouse colonoscopy techniques are being developed. When these technologies are coupled with model systems of human disease, which are essential to our understanding of the pathophysiology of gastrointestinal diseases, the requirement for euthanasia of multiple cohorts of mice is eliminated. Here we highlight the suitability of white light endoscopy to monitor the progression of colitis in mice. We further outline the experimental power of combined standard endoscopy with confocal microendoscopy, which permits visualization of fluorescent markers in a single animal in real-time. Together, these technologies will enhance our understanding of the interplay between components of the gastrointestinal microenvironment and their role in disease. PMID:25960174

  10. Evaluation of human sclera after femtosecond laser ablation using two photon and confocal microscopy

    NASA Astrophysics Data System (ADS)

    Sun, Hui; Kurtz, Ronald; Juhasz, Tibor

    2012-08-01

    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.

  11. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide.

    PubMed

    Rodighiero, Simona; Torre, Bruno; Sogne, Elisa; Ruffilli, Roberta; Cagnoli, Cinzia; Francolini, Maura; Di Fabrizio, Enzo; Falqui, Andrea

    2015-06-01

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. PMID:25810353

  12. Application of laser differential confocal technique in back vertex power measurement for phoropters

    NASA Astrophysics Data System (ADS)

    Li, Fei; Li, Lin; Ding, Xiang; Liu, Wenli

    2012-10-01

    A phoropter is one of the most popular ophthalmic instruments used in optometry and the back vertex power (BVP) is one of the most important parameters to evaluate the refraction characteristics of a phoropter. In this paper, a new laser differential confocal vertex-power measurement method which takes advantage of outstanding focusing ability of laser differential confocal (LDC) system is proposed for measuring the BVP of phoropters. A vertex power measurement system is built up. Experimental results are presented and some influence factor is analyzed. It is demonstrated that the method based on LDC technique has higher measurement precision and stronger environmental anti-interference capability compared to existing methods. Theoretical analysis and experimental results indicate that the measurement error of the method is about 0.02m-1.

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

    PubMed

    Reeves, M; Musculus, M; Farrell, P

    1998-10-01

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

  14. Application of confocal laser microscopy for monitoring mesh implants in herniology

    SciTech Connect

    Zakharov, V P; Belokonev, V I; Bratchenko, I A; Timchenko, P E; Vavilov, A V; Volova, L T

    2011-04-30

    The state of the surface of mesh implants and their encapsulation region in herniology is investigated by laser confocal microscopy. A correlation between the probability of developing relapses and the size and density of implant microdefects is experimentally shown. The applicability limits of differential reverse scattering for monitoring the post-operation state of implant and adjacent tissues are established based on model numerical experiments. (optical technologies in biophysics and medicine)

  15. A prospective cohort study: probe based confocal laser endomicroscopy for peripheral pulmonary lesions (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Matsumoto, Yuji; Izumo, Takehiro; Hiraishi, Yoshihisa; Tsuchida, Takaaki

    2016-03-01

    Introduction: The diagnostic value of bronchoscopy for peripheral pulmonary lesions (PPLs) has improved since the application of radial endobronchial ultrasound (R-EBUS). Though R-EBUS indicates the position of the PPL, there is often a discrepancy between the obtained R-EBUS image and the diagnostic outcome. Meanwhile, probe based confocal laser endomicroscopy (pCLE) is a novel technique which provides in vivo real-time image of the contacted surface structures. However, its findings have not been established yet. Methods: Consecutive patients who have underwent bronchoscopy for PPLs were prospectively enrolled. R-EBUS with a guide sheath (GS) was inserted to the target PPL under X-ray fluoroscopic guidance. When an adequate R-EBUS image (within or adjacent to) was obtained, pCLE was sequentially inserted through the GS. Then pCLE image was scanned and biopsy was performed where an abnormal finding was estimated. The pCLE findings of PPLs and the background were recorded and analyzed exploratorily. Results: We analyzed 19 cases that we could get appropriate tissues. In all cases, bronchial walls showed longitudinal elastic fibers whereas alveolar walls formed grid-like elastic fiber networks. Conversely, discontinuous, crushed or aggregated alveolar structures accompanied by thickened and distorted fibers were detected in PPLs. Some cases showed dark hollow with fragmented or granular fluorescence. On the other hand, 11 cases (57.9%) indicated normal elastic fibers and needed the position change (3 cases; approached other bronchus, 6 cases; adjusted the position, 2 cases; penetrated the covered bronchial wall). Conclusion: The pCLE has a potential to improve the efficacy of diagnostic bronchoscopy for PPLs.

  16. Comparison of divided and full pupil configurations for line-scanning confocal microscopy in human skin and oral mucosa

    NASA Astrophysics Data System (ADS)

    Larson, Bjorg; Abeytunge, Sanjeewa; Glazowski, Chris; Rajadhyaksha, Milind

    2012-02-01

    Confocal point-scanning microscopy has been showing promise in the detection, diagnosing and mapping of skin lesions in clinical settings. The noninvasive technique allows provides optical sectioning and cellular resolution for in vivo diagnosis of melanoma and basal cell carcinoma and pre-operative and intra-operative mapping of margins. The imaging has also enabled more accurate "guided" biopsies while minimizing the otherwise large number of "blind" biopsies. Despite these translational advances, however, point-scanning technology remains relatively complex and expensive. Line-scanning technology may offer an alternative approach to accelerate translation to the clinic. Line-scanning, using fewer optical components, inexpensive linear-array detectors and custom electronics, may enable smaller, simpler and lower-cost confocal microscopes. A line is formed using a cylindrical lens and scanned through the back focal plane of the objective with a galvanometric scanner. A linear CCD is used for detection. Two pupil configurations were compared for performance in imaging human tissue. In the full-pupil configuration, illumination and detection is made through the full objective pupil. In the divided pupil approach, half the pupil is illuminated and the other half is used for detection. The divided pupil configuration loses spatial and axial resolution due to a diminished NA, but the sectioning capability and rejection of background is improved. Imaging in skin and oral mucosa illustrate the performance of the two configurations.

  17. Development and verification of a novel device for dental intra-oral 3D scanning using chromatic confocal technology

    NASA Astrophysics Data System (ADS)

    Zint, M.; Stock, K.; Graser, R.; Ertl, T.; Brauer, E.; Heyninck, J.; Vanbiervliet, J.; Dhondt, S.; De Ceuninck, P.; Hibst, R.

    2015-03-01

    The presented work describes the development and verification of a novel optical, powder-free intra-oral scanner based on chromatic confocal technology combined with a multifocal approach. The proof of concept for a chromatic confocal area scanner for intra-oral scanning is given. Several prototype scanners passed a verification process showing an average accuracy (distance deviation on flat surfaces) of less than 31μm +/- 21μm and a reproducibility of less than 4μm +/- 3μm. Compared to a tactile measurement on a full jaw model fitted with 4mm ceramic spheres the measured average distance deviation between the spheres was 49μm +/- 12μm for scans of up to 8 teeth (3- unit bridge, single Quadrant) and 104μm +/- 82μm for larger scans and full jaws. The average deviation of the measured sphere diameter compared to the tactile measurement was 27μm +/- 14μm. Compared to μCT scans of plaster models equipped with human teeth the average standard deviation on up to 3 units was less than 55μm +/- 49μm whereas the reproducibility of the scans was better than 22μm +/- 10μm.

  18. Laser lithography by photon scanning tunneling microscopy

    SciTech Connect

    Lee, I.; Warmack, R.J.; Ferrell, T.L.

    1993-06-01

    We have investigated the possibility of using a photon scanning tunneling microscope (PSTM) for laser lithography. A contrast enhancement material (CEM) is coated onto a sample slide and coupled to the prism of a PSTM. The CEM becomes transparent above a laser (HeCd at a wavelength of 442 nm) intensity threshold attained due to the proximity of the probe tip. The same surface can then be inspected using the given experimental configuration by replacing the HeCd laser line with a non-exposing 633-nm HeNe laser line. Direct patterns can be produced by varying the exposure time and the shape of the probe tip.

  19. In-Situ Observation of Crystallization and Growth in High-Temperature Melts Using the Confocal Laser Microscope

    NASA Astrophysics Data System (ADS)

    Sohn, Il; Dippenaar, Rian

    2016-08-01

    This review discusses the innovative efforts initiated by Emi and co-workers for in-situ observation of phase transformations at high temperatures for materials. By using the high-temperature confocal laser-scanning microscope (CLSM), a robust database of the phase transformation behavior during heating and cooling of slags, fluxes, and steel can be developed. The rate of solidification and the progression of solid-state phase transformations can be readily investigated under a variety of atmospheric conditions and be correlated with theoretical predictions. The various research efforts following the work of Emi and co-workers have allowed a deeper fundamental understanding of the elusive solidification and phase transformation mechanisms in materials beyond the ambit of steels. This technique continues to evolve in terms of its methodology, application to other materials, and its contribution to technology.

  20. In-Situ Observation of Crystallization and Growth in High-Temperature Melts Using the Confocal Laser Microscope

    NASA Astrophysics Data System (ADS)

    Sohn, Il; Dippenaar, Rian

    2016-04-01

    This review discusses the innovative efforts initiated by Emi and co-workers for in-situ observation of phase transformations at high temperatures for materials. By using the high-temperature confocal laser-scanning microscope (CLSM), a robust database of the phase transformation behavior during heating and cooling of slags, fluxes, and steel can be developed. The rate of solidification and the progression of solid-state phase transformations can be readily investigated under a variety of atmospheric conditions and be correlated with theoretical predictions. The various research efforts following the work of Emi and co-workers have allowed a deeper fundamental understanding of the elusive solidification and phase transformation mechanisms in materials beyond the ambit of steels. This technique continues to evolve in terms of its methodology, application to other materials, and its contribution to technology.

  1. Characterizing image quality in a scanning laser ophthalmoscope with differing pinholes and induced scattered light

    NASA Astrophysics Data System (ADS)

    Hunter, Jennifer J.; Cookson, Christopher J.; Kisilak, Marsha L.; Bueno, Juan M.; Campbell, Melanie C. W.

    2007-05-01

    We quantify the effects on scanning laser ophthalmoscope image quality of controlled amounts of scattered light, confocal pinhole diameter, and age. Optical volumes through the optic nerve head were recorded for a range of pinhole sizes in 12 subjects (19-64 years). The usefulness of various overall metrics in quantifying the changes in fundus image quality is assessed. For registered and averaged images, we calculated signal-to-noise ratio, entropy, and acutance. Entropy was best able to distinguish differing image quality. The optimum confocal pinhole diameter was found to be 50 μm (on the retina), providing improved axial resolution and image quality under all conditions.

  2. Laser scanning by rotating polarization gratings.

    PubMed

    Zhou, Yuan; Fan, Dapeng; Fan, Shixun; Chen, Ying; Liu, Guangcan

    2016-07-01

    Laser beam scanning can be realized using two independently rotating, inline polarization gratings, termed Risley gratings, in a fashion similar to Risley prisms. The analytical formulas of pointing position as well as their inverse solutions are described. On this basis, the beam scanning is investigated and the performance of scanning imaging is evaluated. It is shown that the scanning function in 1D scanning evolves from a sinusoidal to triangular scan and the duty cycle increases rapidly as the ratio of grating period to wavelength is reduced toward 2. The scan pattern in 2D scanning is determined by the ratio k of the gratings' rotatory frequency. In imaging applications, when k tends toward 1 or -1, the scan pattern becomes dense and is inclined to be spiral or rose-like, respectively, which is desirable for the purpose of enhancing spatial resolution. There is a direct trade-off between spatial resolution and frame rate. The spiral and rose scanning enable multiresolution imaging, providing a preview of the scanned area in a fraction of the overall scan time, which is extremely useful for fast, real-time imaging applications. PMID:27409203

  3. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY

    EPA Science Inventory

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

  4. Nondestructive 3D confocal laser imaging with deconvolution of seven whole stardust tracks with complementary XRF and quantitative analysis

    SciTech Connect

    Greenberg, M.; Ebel, D.S.

    2009-03-19

    We present a nondestructive 3D system for analysis of whole Stardust tracks, using a combination of Laser Confocal Scanning Microscopy and synchrotron XRF. 3D deconvolution is used for optical corrections, and results of quantitative analyses of several tracks are presented. The Stardust mission to comet Wild 2 trapped many cometary and ISM particles in aerogel, leaving behind 'tracks' of melted silica aerogel on both sides of the collector. Collected particles and their tracks range in size from submicron to millimeter scale. Interstellar dust collected on the obverse of the aerogel collector is thought to have an average track length of {approx}15 {micro}m. It has been our goal to perform a total non-destructive 3D textural and XRF chemical analysis on both types of tracks. To that end, we use a combination of Laser Confocal Scanning Microscopy (LCSM) and X Ray Florescence (XRF) spectrometry. Utilized properly, the combination of 3D optical data and chemical data provides total nondestructive characterization of full tracks, prior to flattening or other destructive analysis methods. Our LCSM techniques allow imaging at 0.075 {micro}m/pixel, without the use of oil-based lenses. A full textural analysis on track No.82 is presented here as well as analysis of 6 additional tracks contained within 3 keystones (No.128, No.129 and No.140). We present a method of removing the axial distortion inherent in LCSM images, by means of a computational 3D Deconvolution algorithm, and present some preliminary experiments with computed point spread functions. The combination of 3D LCSM data and XRF data provides invaluable information, while preserving the integrity of the samples for further analysis. It is imperative that these samples, the first extraterrestrial solids returned since the Apollo era, be fully mapped nondestructively in 3D, to preserve the maximum amount of information prior to other, destructive analysis.

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

    NASA Astrophysics Data System (ADS)

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

    1999-06-01

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

  6. Recommendations for the design and the installation of large laser scanning microscopy systems

    NASA Astrophysics Data System (ADS)

    Helm, P. Johannes

    2012-03-01

    Laser Scanning Microscopy (LSM) has since the inventions of the Confocal Scanning Laser Microscope (CLSM) and the Multi Photon Laser Scanning Microscope (MPLSM) developed into an essential tool in contemporary life science and material science. The market provides an increasing number of turn-key and hands-off commercial LSM systems, un-problematic to purchase, set up and integrate even into minor research groups. However, the successful definition, financing, acquisition, installation and effective use of one or more large laser scanning microscopy systems, possibly of core facility character, often requires major efforts by senior staff members of large academic or industrial units. Here, a set of recommendations is presented, which are helpful during the process of establishing large systems for confocal or non-linear laser scanning microscopy as an effective operational resource in the scientific or industrial production process. Besides the description of technical difficulties and possible pitfalls, the article also illuminates some seemingly "less scientific" processes, i.e. the definition of specific laboratory demands, advertisement of the intention to purchase one or more large systems, evaluation of quotations, establishment of contracts and preparation of the local environment and laboratory infrastructure.

  7. 3D Quantitative Confocal Laser Microscopy of Ilmenite Volume Distribution in Alpe Arami Olivine

    NASA Astrophysics Data System (ADS)

    Bozhilov, K. N.

    2001-12-01

    The deep origin of the Alpe Arami garnet lherzolite massif in the Swiss Alps proposed by Dobrzhinetskaya et al. (Science, 1996) has been a focus of heated debate. One of the lines of evidence supporting an exhumation from more than 200 km depth includes the abundance, distribution, and orientation of magnesian ilmenite rods in the oldest generation of olivine. This argument has been disputed in terms of the abundance of ilmenite and consequently the maximum TiO2 content in the discussed olivine. In order to address this issue, we have directly measured the volume fraction of ilmenite of the oldest generation of olivine by applying confocal laser scanning microscopy (CLSM). CLSM is a method which allows for three-dimensional imaging and quantitative volume determination by optical sectioning of the objects. The images for 3D reconstruction and measurements were acquired from petrographic thin sections in reflected laser light with 488 nm wavelength. Measurements of more than 80 olivine grains in six thin sections of our material yielded an average volume fraction of 0.31% ilmenite in the oldest generation of olivine from Alpe Arami. This translates into 0.23 wt.% TiO2 in olivine with error in determination of ±0.097 wt.%, a value significantly different from that of 0.02 to 0.03 wt.% TiO2 determined by Hacker et al. (Science, 1997) by a broad-beam microanalysis technique. During the complex geological history of the Alpe Arami massif, several events of metamorphism are recorded which all could have caused increased mobility of the mineral components. Evidence for loss of TiO2 from olivine is the tendency for high densities of ilmenite to be restricted to cores of old grains, the complete absence of ilmenite inclusions from the younger, recrystallized, generation of olivine, and reduction in ilmenite size and abundance in more serpentinized specimens. These observations suggest that only olivine grains with the highest concentrations of ilmenite are close to the

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

    NASA Astrophysics Data System (ADS)

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

    1999-04-01

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

  9. Reversible patterning of poly(methylmethacrylate) doped with disperse Red 1 by laser scanning

    SciTech Connect

    Tuma, J.; Lyutakov, O.; Huttel, I.; Slepicka, P.; Svorcik, V.

    2013-09-07

    Thin poly(methylmethacrylate) films doped by or covalently attached to disperse Red 1 acrylate (DR1) were patterned by laser scanning and simultaneous sample movement in confocal microscope. In both cases, periodical structure due to Marangoni effect is created. Modified polymers surfaces were analyzed by FTIR spectroscopy, X-ray photoelectron spectroscopy, and atomic force microscopy. After first stage of patterning, second stage with sample movement in perpendicular direction was applied. Depending on the method of DR1 dotation fishnet structure is obtained or pattern structure disappears. In the latter case, reversibility of pattern formation and erasure by laser scanning was studied.

  10. LASER APPLICATIONS: Thermographic system with a laser scanning device

    NASA Astrophysics Data System (ADS)

    Skvortsov, L. A.; Kirillov, V. M.

    2007-11-01

    It is shown that laser photothermal radiometry (LPTR) in combination with laser beam scanning within the instantaneous field of view of a single-element photodetector can be used to develop a scanning thermal emission microscope. An expression is derived for estimating its temperature resolution. The results of calculations are presented and the factors influencing the spatial lateral resolution of the technique and the time of image formation with the help of an acousto-optical deflector are analysed.

  11. The design of laser scanning galvanometer system

    NASA Astrophysics Data System (ADS)

    Sun, Xiaoling; Zhou, Bin; Xie, Weihao; Zhang, Yuangeng

    2015-02-01

    In this paper, we designed the laser scanning galvanometer system according to our requirements. Based on scanning range of our laser scanning galvanometer system, the design parameters of this system were optimized. During this work, we focused on the design of the f-θ field lens. An optical system of patent lens in the optical manual book, which had three glasses structure, was used in our designs. Combining the aberration theory, the aberration corrections and image quality evaluations were finished using Code V optical design software. An optimum f-θ field lens was designed, which had focal length of 434 mm, pupil diameter of 30 mm, scanning range of 160 mm × 160 mm, and half field angle of 18°×18°. At the last, we studied the influences of temperature changes on our system.

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

    SciTech Connect

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

    2010-05-21

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

  13. Microscopic tomography by laser scanning microscopy and its three-dimensional reconstruction.

    PubMed

    Takamatsu, T; Fujita, S

    1988-03-01

    We have developed a new confocal laser scanning microscope equipped with two galvanometer mirrors which swing the laser beam. With this set up we can observe large and fragile specimens. Using a focused laser beam as light source to minimize 'flare' and a pinhole in front of a photodetector to eliminate out-of-focus data, we could obtain a depth-discriminated fluorescence image. The scanning apparatus of our system can eliminate mechanical vibration and sweep widely, to obtain images at a low magnification. A thinly sectioned image with high resolution and high contrast could be obtained optically from an in situ thick specimen. We have called this technique 'microscopic tomography'. Combining the laser scanning microscope with the colour image analyser generated semi-automatically a three-dimensional picture of the biological material with information on its interior. PMID:3398041

  14. Laser-scanning photostimulation of optogenetically targeted forebrain circuits.

    PubMed

    Lee, Charles C; Lam, Ying-Wan; Imaizumi, Kazuo; Sherman, S Murray

    2013-01-01

    The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches. PMID:24430760

  15. Multiplatform Mobile Laser Scanning: Usability and Performance

    PubMed Central

    Kukko, Antero; Kaartinen, Harri; Hyyppä, Juha; Chen, Yuwei

    2012-01-01

    Mobile laser scanning is an emerging technology capable of capturing three-dimensional data from surrounding objects. With state-of-the-art sensors, the achieved point clouds capture object details with good accuracy and precision. Many of the applications involve civil engineering in urban areas, as well as traffic and other urban planning, all of which serve to make 3D city modeling probably the fastest growing market segment in this field. This article outlines multiplatform mobile laser scanning solutions such as vehicle- and trolley-operated urban area data acquisition, and boat-mounted equipment for fluvial environments. Moreover, we introduce a novel backpack version of mobile laser scanning equipment for surveying applications in the field of natural sciences where the requirements include precision and mobility in variable terrain conditions. In addition to presenting a technical description of the systems, we discuss the performance of the solutions in the light of various applications in the fields of urban mapping and modeling, fluvial geomorphology, snow-cover characterization, precision agriculture, and in monitoring the effects of climate change on permafrost landforms. The data performance of the mobile laser scanning approach is described by the results of an evaluation of the ROAMER on a permanent MLS test field. Furthermore, an in situ accuracy assessment using a field of spherical 3D targets for the newly-introduced Akhka backpack system is conducted and reported on.

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. Laser scan microscope and infrared laser scan microcope: two important tools for device testing

    NASA Astrophysics Data System (ADS)

    Ziegler, Eberhard

    1991-03-01

    The optical beam induced current (OBIC) produced in devices by a laser scan microscope (LSM) is used to localize hot spots, leakage currents, electrostatic discharge defects and weak points. The LSM also allows photoluminescence measurements with high spatial and energy resolution. Using the infrared laser scan microscope (IR LSM), defects in the metallization and latch-up sensitive region could be detected from the back of the device.

  20. Technique of laser confocal and Raman spectroscopy for living cell analysis

    NASA Astrophysics Data System (ADS)

    Meng, Xiaochen; Zhu, Lianqing

    2013-10-01

    Because of the shortcomings of the main methods used to analysis single cell, the need of single living cell analysis with no damage, unmarked and in situ dynamic multi-parameter measurement is urgent in the life sciences and biomedical advanced research field. And the method of for living cells analysis is proposed. The spectral pretreatment technology of living cell is the key work of laser confocal Raman spectroscopy. To study the spectrum processing methods for Raman spectrum on single living cell and develop the pre-process techniques to enhance the signal-to-noise ratio, sensitivity, and decrease the influence of fluorescence, elimination the cosmic rays was used to improve the spectrum. The classification, average and filtration of spectrum were applied to enhance signal-to-noise ratio. The fluorescence was depressed for quantity analysis or utilized for analysis by comparing the background and the spectrum. The results show that the proposed technique for laser confocal Raman spectrum of single cell can perform the sensitive and weak intensity peaks and reflect the information of molecules structures very well.

  1. EUS-Guided Needle-Based Confocal Laser Endomicroscopy: A Novel Technique With Emerging Applications

    PubMed Central

    Koduru, Pramoda; Joshi, Virendra; Karstensen, John G.; Saftoiu, Adrian; Vilmann, Peter; Giovannini, Marc

    2015-01-01

    Endoscopic ultrasound (EUS) has emerged as an excellent tool for imaging the gastrointestinal tract, as well as surrounding structures. EUS-guided fine-needle aspiration (EUS-FNA) has become the standard of care for the tissue sampling of a variety of masses and lymph nodes within and around the gut, providing further diagnostic and staging information. Confocal laser endomicroscopy (CLE) is a novel endoscopic method that enables imaging at a subcellular level of resolution during endoscopy, allowing up to 1000-fold magnification of tissue and providing an optical biopsy. A new procedure that has been developed in the past few years is needle-based confocal laser endomicroscopy (nCLE), which involves a mini-CLE probe that can be passed through a 1 9-gauge needle during EUS-FNA. This enables the real-time visualization of tissue at a microscopic level, with the potential to further improve the diagnostic accuracy of EUS-FNA. The device has been studied in animals as well as in humans, and the results so far have been promising. Recently, this method has also been used for the visualization of regulatory proteins and receptors in the pancreas, setting a cornerstone for nCLE in molecular imaging. The aim of this article is to review the role of EUS-guided nCLE in modern endoscopy and its implications in molecular imaging. PMID:27099595

  2. Confocal Laser Endomicroscopy of Bladder and Upper Tract Urothelial Carcinoma: A New Era of Optical Diagnosis?

    PubMed Central

    Chen, Stephanie P.; Liao, Joseph C.

    2014-01-01

    Urothelial carcinoma of the bladder and upper tract pose significant diagnostic and therapeutic challenges. White light endoscopy plays a central role in the management of urothelial carcinoma but has several well-recognized shortcomings. New optical imaging technologies may improve diagnostic accuracy, enhance local cancer control, and better stratify treatment options. Confocal laser endomicroscopy enables dynamic imaging of the cellular structures below the mucosal surface and holds promise in providing real time optical diagnosis and grading of urothelial carcinoma. A variety of imaging probes are available that are compatible with the full spectrum of cystoscopes and ureteroscopes. We review the underlying principles and technique of confocal laser endomicroscopy in the urinary tract, with emphasis on specific application towards urothelial carcinoma. While the available data are largely related to urothelial carcinoma of the bladder, the lessons learned are directly applicable to the upper tract, where the clinical needs are significant. Ongoing efforts to optimize this technology offer an exciting glimpse into future advances in optical imaging and intraoperative image guidance. PMID:25002073

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

    PubMed

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

    2011-03-01

    A confocal laser microscope system was developed for the measurement of radiophotoluminescence (RPL) photons emitted from a minute alpha-ray-irradiated area in an RPL glass dosemeter. The system was composed mainly of an inverted-type microscope, an ultraviolet laser, an XY movable stage and photon-counting circuits. The photon-counting circuits were effective in the reduction of the background noise level in the measurement of RPL photons. The performance of this microscope system was examined by the observation of standard RPL glass samples irradiated using (241)Am alpha rays. The spatial resolution of this system was ∼ 3 μm, and with regard to the sensitivity of this system, a hit of more than four to five alpha rays in unit area produced enough amount of RPL photons to construct the image. PMID:21212081

  4. Seeding requirements for scanning laser velocimetry

    NASA Technical Reports Server (NTRS)

    Hackett, C. E.

    1985-01-01

    To measure the velocity distributions within time dependent turbulent flow fields, a continuously scanning laser velocimeter system is being developed at Sandia National Laboratories Livermore (SNLL). A prototype of this system has produced results which show that spatial and temporal variations in particle seed distribution seriously compromise the overall performance and operation of this device. To alleviate some of these problems, alternate flow seeding concepts are explored. The most promising appear to be those that actively induce laser sparks within the gas flow, the velocity of which may be measured by a Fourier transformed velocimetry system.

  5. Embedding complementary imaging data in laser scanning microscopy micrographs by reversible watermarking

    PubMed Central

    Dragoi, Ioan-Catalin; Stanciu, Stefan G.; Hristu, Radu; Coanda, Henri-George; Tranca, Denis E.; Popescu, Marius; Coltuc, Dinu

    2016-01-01

    Complementary laser scanning microscopy micrographs are considered as pairs consisting in a master image (MI) and a slave image (SI), the latter with potential for facilitating the interpretation of the MI. We propose a strategy based on reversible watermarking for embedding a lossy compressed version of the SI into the MI. The use of reversible watermarking ensures the exact recovery of the host image. By storing and/or transmitting the watermarked MI in a single file, the information contained in both images that constitute the pair is made available to a potential end-user, which simplifies data association and transfer. Examples are presented using support images collected by two complementary techniques, confocal scanning laser microscopy and transmission laser scanning microscopy, on Hematoxylin and Eosin stained tissue fragments. A strategy for minimizing the watermarking distortions of the MI, while preserving the content of the SI, is discussed in detail.

  6. Embedding complementary imaging data in laser scanning microscopy micrographs by reversible watermarking.

    PubMed

    Dragoi, Ioan-Catalin; Stanciu, Stefan G; Hristu, Radu; Coanda, Henri-George; Tranca, Denis E; Popescu, Marius; Coltuc, Dinu

    2016-04-01

    Complementary laser scanning microscopy micrographs are considered as pairs consisting in a master image (MI) and a slave image (SI), the latter with potential for facilitating the interpretation of the MI. We propose a strategy based on reversible watermarking for embedding a lossy compressed version of the SI into the MI. The use of reversible watermarking ensures the exact recovery of the host image. By storing and/or transmitting the watermarked MI in a single file, the information contained in both images that constitute the pair is made available to a potential end-user, which simplifies data association and transfer. Examples are presented using support images collected by two complementary techniques, confocal scanning laser microscopy and transmission laser scanning microscopy, on Hematoxylin and Eosin stained tissue fragments. A strategy for minimizing the watermarking distortions of the MI, while preserving the content of the SI, is discussed in detail. PMID:27446641

  7. 100 kHz Mueller polarimeter for laser scanning polarimetric microscopy

    NASA Astrophysics Data System (ADS)

    Le Gratiet, A.; Dubreuil, M.; Rivet, S.; Le Grand, Y.

    2016-04-01

    A new setup was recently proposed to perform Mueller matrix polarimetry at 100 kHz using a swept laser source, high order retarders and a single channel photodetector. In this communication, we present the implementation of this setup on a laser scanning microscope to perform high speed scanning Mueller microscopy in transmission. Calibration of the instrument is briefly described and precision and stability over time are evaluated. Finally, Mueller images of a manufactured scene are reported. To our best knowledge, this is the first time that Mueller polarimetry is performed using a laser scanning microscope. We further plan to develop confocal/nonlinear/Mueller microscopy from the same setup in order to produce multimodal contrast images of biological samples.

  8. Multiplatform Approach to Mobile Laser Scanning

    NASA Astrophysics Data System (ADS)

    Kukko, A.; Kaartinen, H.; Hyyppä, J.; Chen, Y.

    2012-07-01

    Mobile laser scanning is an emerging technology for capturing three-dimensional information from the surrounding objects. With state of the art sensors the achieved point cloud could capture fine details of the surroundings with good accuracy and effectiveness. Many of the applications deal with the civil engineering purposes in urban areas for traffic and city planning and modelling. In this article we present multiplatform mobile laser scanning solutions for mapping applications that require mobility in various terrains and river environments but yet produce high density point clouds with good reliability and accuracy. The ROAMER mobile laser scanning system was deployed in multitude of tasks from urban areas to climate research. The paper also introduces a completely new backpack Akhka platform for mobile lidar mapping of areas where wheeled vehicles cannot operate. The sensor set is the same in all of the approaches, but the carrying vessel was selected according to the application. This was possible thanks to a relatively light and compact yet simple design of the system. ROAMER system is one of the few high-end MLS systems that are easily adaptable to various platforms. In addition to technical description of the system we discuss the practical performance of the solutions through various applications in the fields of urban mapping, fluvial geomorphology, snow cover characterization and climate change monitoring.

  9. Two-Photon Laser Scanning Microscopy

    NASA Astrophysics Data System (ADS)

    Nimmerjahn, A.; Theer, P.; Helmchen, F.

    Since its inception more than 15 years ago, two-photon laser scanning microscopy (2PLSM) has found widespread use in biological and medical research. Two-photon microscopy is based on simultaneous absorption of two photons by fluorophores and subsequent fluorescence emission, a process which under normal illumination conditions is highly improbable. Theoretically described around 1930 by Maria Göppert-Mayer [1], the first experimental demonstration of two-photon excitation had to await the invention of the laser, which produced sufficiently high light intensities to observe two-photon absorption events [2]. Only after the development of ultrafast lasers providing subpicosecond light pulses with high peak power intensities, however, two-photon-excited fluorescence became practical in a laser-scanning microscope [3]. Since then 2PLSM has developed into the method of choice for high-resolution imaging in living animals (reviewed in [4,5]). One of the main reasons is the low sensitivity of 2PLSM to light scattering, which enables imaging relatively deep inside biological tissue and direct observation of the dynamic behavior of cells in their native environment. In this chapter, we introduce the physical principles governing 2PLSM and briefly describe the key instrument components. We give an overview of fluorescence labeling techniques and how they are combined with 2PLSM for functional imaging and photomanipulation in living tissue. Finally, we discuss limitations and provide some future perspectives.

  10. Laser scanned image sensors using photoconductors with deep traps

    NASA Technical Reports Server (NTRS)

    Maserjian, J.

    1975-01-01

    Photoconductor records image when holes and electrons are trapped inside it due to incident photons. Image can be read out by exposing photoconductor to scanning laser beam. Photons from scanning laser empty traps, generating photocurrent. Image information is obtained by detecting this photocurrent synchronously with laser scan.

  11. Laser multi-reflection differential confocal long focal-length measurement.

    PubMed

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

    2016-06-20

    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. PMID:27409117

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

    PubMed

    Storm, Andrew C; Lee, Linda S

    2016-01-01

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

  13. Scanning tunneling microscope design with a confocal small field permanent magnet.

    SciTech Connect

    Messina, P.; Pearson, J.; Vasserman, I.; Sasaki, S.; Moog, E.; Fradin, F.

    2008-09-01

    The field of ultra-sensitive measurements with scanning probes requires the design and construction of novel instruments. For example, the combination of radio frequency detection and scanning probe can be exploited to measure thermal properties and mechanical resonances at a very low scale. Very recent results by Komeda and Manassen (2008 Appl. Phys. Lett. 92 212506) on the detection of spin noise with the scanning tunneling microscopy (STM) have further expanded previous results reported by one of the authors of this manuscript (Messina et al 2007 J. Appl. Phys. 101 053916). In a previous publication, one of the authors used a new STM instrument (Messina et al J. Appl. Phys. 2007 101 053916 and Mannini et al 2007 Inorg. Chim. Acta 360 3837-42) to obtain the detection of electron spin noise (ESN) from individual paramagnetic adsorbates. The magnetic field homogeneity at the STM tip-sample region was limited. Furthermore, vacuum operation of the STM microscope was limited by the heat dissipation at the electromagnet and the radio frequency (RF) recovery electronics. We report here on a new STM head that incorporates a specially designed permanent magnet and in-built RF amplification system. The magnet provides both a better field homogeneity and freedom to operate the instrument in vacuum. The STM microscope is vacuum compatible, and vertical stability has been improved over the previous design (Messina et al 2007 J. Appl. Phys. 101 053916), despite the presence of a heat dissipative RF amplifier in the close vicinity of the STM tip.

  14. Laser scanning system for object monitoring

    DOEpatents

    McIntyre, Timothy James [Knoxville, TN; Maxey, Lonnie Curtis [Powell, TN; Chiaro, Jr; John, Peter [Clinton, TN

    2008-04-22

    A laser scanner is located in a fixed position to have line-of-sight access to key features of monitored objects. The scanner rapidly scans pre-programmed points corresponding to the positions of retroreflecting targets affixed to the key features of the objects. The scanner is capable of making highly detailed scans of any portion of the field of view, permitting the exact location and identity of targets to be confirmed. The security of an object is verified by determining that the cooperative target is still present and that its position has not changed. The retroreflecting targets also modulate the reflected light for purposes of returning additional information back to the location of the scanner.

  15. Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

    NASA Astrophysics Data System (ADS)

    Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

    2015-10-01

    Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

  16. Scan-less, line-field confocal microscopy by combination of wavelength/space conversion with dual optical comb

    NASA Astrophysics Data System (ADS)

    Yasui, Takeshi; Hase, Eiji; Miyamoto, Shuji; Hsieh, Yi-Da; Minamikawa, Takeo; Yamamoto, Hirotsugu

    2016-03-01

    Optical frequency comb (OFC) has attracted attentions for optical frequency metrology in visible and infrared regions because the mode-resolved OFC spectrum can be used as a precise frequency ruler due to both characteristics of broadband radiation and narrow-line CW radiation. Furthermore, the absolute accuracy of all frequency modes in OFC is secured by phase-locking a repetition frequency frep and a carrier-envelope-offset frequency fceo to a frequency standard. However, application fields of OFC other than optical frequency metrology are still undeveloped. One interesting aspect of OFC except for the frequency ruler is optical carrier having a huge number of discrete frequency channels because OFC is composed of a series of frequency spikes regularly separated by frep in the broad spectral range. If a certain quantity to be measured is encoded on each comb mode by dimensional conversion, a huge number of data for the measured quantity can be obtained from a single mode-resolved spectrum of OFC. In this paper, we encode the confocal microscopic line-image of a sample on the mode-resolved OFC spectrum by the dimensional conversion between wavelength and 1D-space. The resulting image-encoded OFC spectrum is acquired by an optical spectrum analyzer or dual comb spectrometer. Finally, the line image of the sample is decoded from the spectral amplitude of the mode-resolved OFC spectrum. The combination of OFC with the dimensional conversion enables to establish both confocal modality and line-field imaging under the scan-less condition.

  17. Mobile Laser Scanning for Indoor Modelling

    NASA Astrophysics Data System (ADS)

    Thomson, C.; Apostolopoulos, G.; Backes, D.; Boehm, J.

    2013-10-01

    The process of capturing and modelling buildings has gained increased focus in recent years with the rise of Building Information Modelling (BIM). At the heart of BIM is a process change for the construction and facilities management industries whereby a BIM aids more collaborative working through better information exchange, and as a part of the process Geomatic/Land Surveyors are not immune from the changes. Terrestrial laser scanning has been proscribed as the preferred method for rapidly capturing buildings for BIM geometry. This is a process change from a traditional measured building survey just with a total station and is aided by the increasing acceptance of point cloud data being integrated with parametric building models in BIM tools such as Autodesk Revit or Bentley Architecture. Pilot projects carried out previously by the authors to investigate the geometry capture and modelling of BIM confirmed the view of others that the process of data capture with static laser scan setups is slow and very involved requiring at least two people for efficiency. Indoor Mobile Mapping Systems (IMMS) present a possible solution to these issues especially in time saved. Therefore this paper investigates their application as a capture device for BIM geometry creation over traditional static methods through a fit-for-purpose test.

  18. A scanning laser velocimeter for turbulence research

    NASA Technical Reports Server (NTRS)

    Duen, F. K.

    1984-01-01

    Turbulent and unsteady separated flows occur on most practical flight vehicles, but are not yet sufficiently understood for designs to provide safe margins of performance without recourse to extensive experiment and computation. In date, reliable experimental data for even basic flows is severely limited and does not yet provide a satisfactory data base with which to assess current design and calculation methods. Although the laser velocimeter (LV) has become a proven, nonintrusive instrument for the measurement of local mean velocities and turbulence properties, measurements have been of a mean, statistical nature derived from averages accumulated independently at different positions in the flow. Thus, the measurements do not give an instantaneous dynamic, picture of the flow-field structures. Accordingly, a new technique for rapid LV scans of turbulent flow fields was proposed. The potential of this new instrument for fundamental fluid mechanical measurements of turbulent flows has been demonstrated. The results clearly show that significant unsteady flow features are hidden by conventional measurements and that the scanning laser velocimeter should prove an invauable tool in future studies of the structure of turbulent flows.

  19. Laser scanning endoscope for diagnostic medicine

    NASA Astrophysics Data System (ADS)

    Ouimette, Donald R.; Nudelman, Sol; Spackman, Thomas; Zaccheo, Scott

    1990-07-01

    A new type of endoscope is being developed which utilizes an optical raster scanning system for imaging through an endoscope. The optical raster scanner utilizes a high speed, multifaceted, rotating polygon mirror system for horizontal deflection, and a slower speed galvanometer driven mirror as the vertical deflection system. When used in combination, the optical raster scanner traces out a raster similar to an electron beam raster used in television systems. This flying spot of light can then be detected by various types of photosensitive detectors to generate a video image of the surface or scene being illuminated by the scanning beam. The optical raster scanner has been coupled to an endoscope. The raster is projected down the endoscope, thereby illuminating the object to be imaged at the distal end of the endoscope. Elemental photodetectors are placed at the distal or proximal end of the endoscope to detect the reflected illumination from the flying spot of light. This time sequenced signal is captured by an image processor for display and processing. This technique offers the possibility for very small diameter endoscopes since illumination channel requirements are eliminated. Using various lasers, very specific spectral selectivity can be achieved to optimum contrast of specific lesions of interest. Using several laser lines, or a white light source, with detectors of specific spectral response, multiple spectrally selected images can be acquired simultaneously. The potential for co-linear therapy delivery while imaging is also possible.

  20. Real time diagnosis of bladder cancer with probe-based confocal laser endomicroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Jen-Jane; Wu, Katherine; Adams, Winifred; Hsiao, Shelly T.; Mach, Kathleen E.; Beck, Andrew H.; Jensen, Kristin C.; Liao, Joseph C.

    2011-02-01

    Probe-based confocal laser endomicroscopy (pCLE) is an emerging technology for in vivo optical imaging of the urinary tract. Particularly for bladder cancer, real time optical biopsy of suspected lesions will likely lead to improved management of bladder cancer. With pCLE, micron scale resolution is achieved with sterilizable imaging probes (1.4 or 2.6 mm diameter), which are compatible with standard cystoscopes and resectoscopes. Based on our initial experience to date (n = 66 patients), we have demonstrated the safety profile of intravesical fluorescein administration and established objective diagnostic criteria to differentiate between normal, benign, and neoplastic urothelium. Confocal images of normal bladder showed organized layers of umbrella cells, intermediate cells, and lamina propria. Low grade bladder cancer is characterized by densely packed monomorphic cells with central fibrovascular cores, whereas high grade cancer consists of highly disorganized microarchitecture and pleomorphic cells with indistinct cell borders. Currently, we are conducting a diagnostic accuracy study of pCLE for bladder cancer diagnosis. Patients scheduled to undergo transurethral resection of bladder tumor are recruited. Patients undergo first white light cystocopy (WLC), followed by pCLE, and finally histologic confirmation of the resected tissues. The diagnostic accuracy is determined both in real time by the operative surgeon and offline after additional image processing. Using histology as the standard, the sensitivity, specificity, positive and negative predictive value of WLC and WLC + pCLE are calculated. With additional validation, pCLE may prove to be a valuable adjunct to WLC for real time diagnosis of bladder cancer.

  1. MEMS scanned laser head-up display

    NASA Astrophysics Data System (ADS)

    Freeman, Mark O.

    2011-03-01

    Head-up displays (HUD) in automobiles and other vehicles have been shown to significantly reduce accident rates by keeping the driver's eyes on the road. The requirements for automotive HUDs are quite demanding especially in terms of brightness, dimming range, supplied power, and size. Scanned laser display technology is particularly well-suited to this application since the lasers can be very efficiently relayed to the driver's eyes. Additionally, the lasers are only turned on where the light is needed in the image. This helps to provide the required brightness while minimizing power and avoiding a background glow that disturbs the see-through experience. Microvision has developed a couple of HUD architectures that are presented herein. One design uses an exit pupil expander and relay optics to produce a high quality virtual image for built-in systems where the image appears to float above the hood of the auto. A second design uses a patented see-through screen technology and pico projector to make automotive HUDs available to anyone with a projector. The presentation will go over the basic designs for the two types of HUD and discuss design tradeoffs.

  2. Laser microbeam CT scanning of dosimetry gels

    NASA Astrophysics Data System (ADS)

    Maryanski, Marek J.; Ranade, Manisha K.

    2001-06-01

    A novel design of an optical tomographic scanner is described that can be used for 3D mapping of optical attenuation coefficient within translucent cylindrical objects with spatial resolution on the order of 100 microns. Our scanner design utilizes the cylindrical geometry of the imaged object to obtain the desired paths of the scanning light rays. A rotating mirror and a photodetector are placed at two opposite foci of the translucent cylinder that acts as a cylindrical lens. A He-Ne laser beam passes first through a focusing lens and then is reflected by the rotating mirror, so as to scan the interior of the cylinder with focused and parallel paraxial rays that are subsequently collected by the photodetector to produce the projection data, as the cylinder rotates in small angle increments between projections. Filtered backprojection is then used to reconstruct planar distributions of optical attenuation coefficient in the cylinder. Multiplanar scans are used to obtain a complete 3D tomographic reconstruction. Among other applications, the scanner can be used in radiation therapy dosimetry and quality assurance for mapping 3D radiation dose distributions in various types of tissue-equivalent gel phantoms that change their optical attenuation coefficients in proportion to the absorbed radiation dose.

  3. A first approach for digital representation and automated classification of toolmarks on locking cylinders using confocal laser microscopy

    NASA Astrophysics Data System (ADS)

    Clausing, Eric; Kraetzer, Christian; Dittmann, Jana; Vielhauer, Claus

    2012-10-01

    An important part of criminalistic forensics is the analysis of toolmarks. Such toolmarks often consist of plenty of single striations, scratches and dents which can allow for conclusions in regards to the sequence of events or used tools. To receive qualified results with an automated analysis and contactless acquisition of such toolmarks, a detailed digital representation of these and their orientation as well as placing to each other is required. For marks of firearms and tools the desired result of an analysis is a conclusion whether or not a mark has been generated by a tool under suspicion. For toolmark analysis on locking cylinders, the aim is not an identification of the used tool but rather an identification of the opening method. The challenge of such an identification is that a one-to-one comparison of two images is not sufficient - although two marked objects look completely different in regards to the specific location and shape of found marks they still can represent a sample for the identical opening method. This paper provides the first approach for modelling toolmarks on lock pins and takes into consideration the different requirements necessary to generate a detailed and interpretable digital representation of these traces. These requirements are 'detail', i.e. adequate features which allow for a suitable representation and interpretation of single marks, 'meta detail', i.e. adequate representation of the context and connection between all marks and 'distinctiveness', i.e. the possibility to reliably distinguish different sample types by the according model. The model is evaluated with a set of 15 physical samples (resulting in 675 digital scans) of lock pins from cylinders opened with different opening methods, contactlessly scanned with a confocal laser microscope. The presented results suggest a high suitability for the aspired purpose of opening method determination.

  4. Utilizing confocal laser endomicroscopy for evaluating the adequacy of laparoscopic liver ablation

    PubMed Central

    Johnson, Sean P.; Walker‐Samuel, Simon; Gurusamy, Kurinchi; Clarkson, Matthew J.; Thompson, Stephen; Song, Yi; Totz, Johannes; Cook, Richard J.; Desjardins, Adrien E.; Hawkes, David J.; Davidson, Brian R.

    2015-01-01

    Background Laparoscopic liver ablation therapy can be used for the treatment of primary and secondary liver malignancy. The increased incidence of cancer recurrence associated with this approach, has been attributed to the inability of monitoring the extent of ablated liver tissue. Methods The feasibility of assessing liver ablation with probe‐based confocal laser endomicroscopy (CLE) was studied in a porcine model of laparoscopic microwave liver ablation. Following the intravenous injection of the fluorophores fluorescein and indocyanine green, CLE images were recorded at 488 nm and 660 nm wavelength and compared to liver histology. Statistical analysis was performed to assess if fluorescence intensity change can predict the presence of ablated liver tissue. Results CLE imaging of fluorescein at 488 nm provided good visualization of the hepatic microvasculature; whereas, CLE imaging of indocyanine green at 660 nm enabled detailed visualization of hepatic sinusoid architecture and interlobular septations. Fluorescence intensity as measured in relative fluorescence units was found to be 75–100% lower in ablated compared to healthy liver regions. General linear mixed modeling and ROC analysis found the decrease in fluorescence to be statistically significant. Conclusion Laparoscopic, dual wavelength CLE imaging using two different fluorophores enables clinically useful visualization of multiple liver tissue compartments, in greater detail than is possible at a single wavelength. CLE imaging may provide valuable intraoperative information on the extent of laparoscopic liver ablation. Lasers Surg. Med. 48:299–310, 2016. © 2015 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc. PMID:26718623

  5. Confocal microscopy imaging of solid tissue

    EPA Science Inventory

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

  6. Multimodal ophthalmic imaging using swept source spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Malone, Joseph D.; El-Haddad, Mohamed T.; Tye, Logan A.; Majeau, Lucas; Godbout, Nicolas; Rollins, Andrew M.; Boudoux, Caroline; Tao, Yuankai K.

    2016-03-01

    Scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) benefit clinical diagnostic imaging in ophthalmology by enabling in vivo noninvasive en face and volumetric visualization of retinal structures, respectively. Spectrally encoding methods enable confocal imaging through fiber optics and reduces system complexity. Previous applications in ophthalmic imaging include spectrally encoded confocal scanning laser ophthalmoscopy (SECSLO) and a combined SECSLO-OCT system for image guidance, tracking, and registration. However, spectrally encoded imaging suffers from speckle noise because each spectrally encoded channel is effectively monochromatic. Here, we demonstrate in vivo human retinal imaging using a swept source spectrally encoded scanning laser ophthalmoscope and OCT (SSSESLO- OCT) at 1060 nm. SS-SESLO-OCT uses a shared 100 kHz Axsun swept source, shared scanner and imaging optics, and are detected simultaneously on a shared, dual channel high-speed digitizer. SESLO illumination and detection was performed using the single mode core and multimode inner cladding of a double clad fiber coupler, respectively, to preserve lateral resolution while improving collection efficiency and reducing speckle contrast at the expense of confocality. Concurrent en face SESLO and cross-sectional OCT images were acquired with 1376 x 500 pixels at 200 frames-per-second. Our system design is compact and uses a shared light source, imaging optics, and digitizer, which reduces overall system complexity and ensures inherent co-registration between SESLO and OCT FOVs. En face SESLO images acquired concurrent with OCT cross-sections enables lateral motion tracking and three-dimensional volume registration with broad applications in multivolume OCT averaging, image mosaicking, and intraoperative instrument tracking.

  7. Building reconstruction from images and laser scanning

    NASA Astrophysics Data System (ADS)

    Brenner, Claus

    2005-03-01

    The automatic extraction of objects from laser scans and images has been a topic of research for decades. Nowadays, with new services expected, especially in the area of navigation systems, location based services, and augmented reality, the need for automated, efficient extraction systems becomes more urgent than ever. This paper reviews a number of automatic and semi-automatic reconstruction methods in more detail in order to reveal their underlying principles. It then discusses some general properties of reconstruction approaches which have evolved. This shows that, although research is still far from the goal of the initially envisioned fully automatic reconstruction systems, there is now a much better understanding of the problem and the ways it can be tackled.

  8. High-resolution adaptive optics scanning laser ophthalmoscope with multiple deformable mirrors

    DOEpatents

    Chen, Diana C.; Olivier, Scot S.; Jones; Steven M.

    2010-02-23

    An adaptive optics scanning laser ophthalmoscopes is introduced to produce non-invasive views of the human retina. The use of dual deformable mirrors improved the dynamic range for correction of the wavefront aberrations compared with the use of the MEMS mirror alone, and improved the quality of the wavefront correction compared with the use of the bimorph mirror alone. The large-stroke bimorph deformable mirror improved the capability for axial sectioning with the confocal imaging system by providing an easier way to move the focus axially through different layers of the retina.

  9. Automatic change detection using mobile laser scanning

    NASA Astrophysics Data System (ADS)

    Hebel, M.; Hammer, M.; Gordon, M.; Arens, M.

    2014-10-01

    Automatic change detection in 3D environments requires the comparison of multi-temporal data. By comparing current data with past data of the same area, changes can be automatically detected and identified. Volumetric changes in the scene hint at suspicious activities like the movement of military vehicles, the application of camouflage nets, or the placement of IEDs, etc. In contrast to broad research activities in remote sensing with optical cameras, this paper addresses the topic using 3D data acquired by mobile laser scanning (MLS). We present a framework for immediate comparison of current MLS data to given 3D reference data. Our method extends the concept of occupancy grids known from robot mapping, which incorporates the sensor positions in the processing of the 3D point clouds. This allows extracting the information that is included in the data acquisition geometry. For each single range measurement, it becomes apparent that an object reflects laser pulses in the measured range distance, i.e., space is occupied at that 3D position. In addition, it is obvious that space is empty along the line of sight between sensor and the reflecting object. Everywhere else, the occupancy of space remains unknown. This approach handles occlusions and changes implicitly, such that the latter are identifiable by conflicts of empty space and occupied space. The presented concept of change detection has been successfully validated in experiments with recorded MLS data streams. Results are shown for test sites at which MLS data were acquired at different time intervals.

  10. High-speed multispectral confocal biomedical imaging

    PubMed Central

    Carver, Gary E.; Locknar, Sarah A.; Morrison, William A.; Krishnan Ramanujan, V.; Farkas, Daniel L.

    2014-01-01

    Abstract. A new approach for generating high-speed multispectral confocal images has been developed. The central concept is that spectra can be acquired for each pixel in a confocal spatial scan by using a fast spectrometer based on optical fiber delay lines. This approach merges fast spectroscopy with standard spatial scanning to create datacubes in real time. The spectrometer is based on a serial array of reflecting spectral elements, delay lines between these elements, and a single element detector. The spatial, spectral, and temporal resolution of the instrument is described and illustrated by multispectral images of laser-induced autofluorescence in biological tissues. PMID:24658777

  11. Confocal Laser Endomicroscopy in Gastrointestinal and Pancreatobiliary Diseases: A Systematic Review and Meta-Analysis.

    PubMed

    Fugazza, Alessandro; Gaiani, Federica; Carra, Maria Clotilde; Brunetti, Francesco; Lévy, Michaël; Sobhani, Iradj; Azoulay, Daniel; Catena, Fausto; de'Angelis, Gian Luigi; de'Angelis, Nicola

    2016-01-01

    Confocal laser endomicroscopy (CLE) is an endoscopic-assisted technique developed to obtain histopathological diagnoses of gastrointestinal and pancreatobiliary diseases in real time. The objective of this systematic review is to analyze the current literature on CLE and to evaluate the applicability and diagnostic yield of CLE in patients with gastrointestinal and pancreatobiliary diseases. A literature search was performed on MEDLINE, EMBASE, Scopus, and Cochrane Oral Health Group Specialized Register, using pertinent keywords without time limitations. Both prospective and retrospective clinical studies that evaluated the sensitivity, specificity, or accuracy of CLE were eligible for inclusion. Of 662 articles identified, 102 studies were included in the systematic review. The studies were conducted between 2004 and 2015 in 16 different countries. CLE demonstrated high sensitivity and specificity in the detection of dysplasia in Barrett's esophagus, gastric neoplasms and polyps, colorectal cancers in inflammatory bowel disease, malignant pancreatobiliary strictures, and pancreatic cysts. Although CLE has several promising applications, its use has been limited by its low availability, high cost, and need of specific operator training. Further clinical trials with a particular focus on cost-effectiveness and medicoeconomic analyses, as well as standardized institutional training, are advocated to implement CLE in routine clinical practice. PMID:26989684

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

    SciTech Connect

    LAGASSE,ROBERT R.; THOMPSON,KYLE R.

    2000-06-12

    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.

  13. Confocal Laser Endomicroscopy for In Vivo Diagnosis of Clostridium difficile Associated Colitis — A Pilot Study

    PubMed Central

    Neumann, Helmut; Günther, Claudia; Vieth, Michael; Grauer, Martin; Wittkopf, Nadine; Mudter, Jonas; Becker, Christoph; Schoerner, Christoph; Atreya, Raja; Neurath, Markus F.

    2013-01-01

    Background Clostridium difficile infection (CDI) is one of the most dreaded causes of hospital-acquired diarrhea. Main objective was to investigate whether confocal laser endomicroscopy (CLE) has the capability for in vivo diagnosis of C. difficile associated histological changes. Second objective was to prove the presence of intramucosal bacteria using CLE. Methods 80 patients were prospectively included, 10 patients were diagnosed with CDI based on toxigenic culture. To validate the presence of intramucosal bacteria ex vivo, CLE was performed in pure C. difficile culture; additionally fluorescence in situ hybridization (FISH) was performed. Finally, CLE with fluorescence labelled oligonucleotide probe specific for C. difficile was performed ex vivo in order to prove the presence of bacteria. Results CLE identified CDI-associated histological changes in vivo (sensitivity and accuracy of 88.9% and 96.3%). In addition, intramucosal bacteria were visualized. The presence of these bacteria could be proven by CLE with labeled, specific molecular C. difficile probe and FISH-technique. Based on comparison between CLE and FISH analyses, sensitivity and specificity for the presence of intramucosal bacteria were 100%. Conclusion CLE has the potential for in vivo diagnosis of CDI associated colitis. In addition, CLE allowed the detection of intramucosal bacteria in vivo. PMID:23527018

  14. Multipass cell based on confocal mirrors for sensitive broadband laser spectroscopy in the near infrared.

    PubMed

    Mohamed, T; Zhu, F; Chen, S; Strohaber, J; Kolomenskii, A A; Bengali, A A; Schuessler, H A

    2013-10-10

    We report on broadband absorption spectroscopy in the near IR using a multipass cell design based on highly reflecting mirrors in a confocal arrangement having the particular aim of achieving long optical paths. We demonstrate a path length of 314 m in a cell consisting of two sets of highly reflecting mirrors with identical focal length, spaced 0.5 m apart. The multipass cell covers this path length in a relatively small volume of 1.25 l with the light beam sampling the whole volume. In a first application, the absorption spectra of the greenhouse gases CO(2), CH(4), and CO were measured. In these measurements we used a femtosecond fiber laser with a broadband spectral range spanning the near IR from 1.5 to 1.7 μm. The absorption spectra show a high signal-to-noise ratio, from which we derive a sensitivity limit of 6 ppmv for methane observed in a mixture with air. PMID:24217732

  15. Optical Biopsy of Peripheral Nerve Using Confocal Laser Endomicroscopy: A New Tool for Nerve Surgeons?

    PubMed

    Crowe, Christopher S; Liao, Joseph C; Curtin, Catherine M

    2015-09-01

    Peripheral nerve injuries remain a challenge for reconstructive surgeons with many patients obtaining suboptimal results. Understanding the level of injury is imperative for successful repair. Current methods for distinguishing healthy from damaged nerve are time consuming and possess limited efficacy. Confocal laser endomicroscopy (CLE) is an emerging optical biopsy technology that enables dynamic, high resolution, sub-surface imaging of live tissue. Porcine sciatic nerve was either left undamaged or briefly clamped to simulate injury. Diluted fluorescein was applied topically to the nerve. CLE imaging was performed by direct contact of the probe with nerve tissue. Images representative of both damaged and undamaged nerve fibers were collected and compared to routine H&E histology. Optical biopsy of undamaged nerve revealed bands of longitudinal nerve fibers, distinct from surrounding adipose and connective tissue. When damaged, these bands appear truncated and terminate in blebs of opacity. H&E staining revealed similar features in damaged nerve fibers. These results prompt development of a protocol for imaging peripheral nerves intraoperatively. To this end, improving surgeons' ability to understand the level of injury through real-time imaging will allow for faster and more informed operative decisions than the current standard permits. PMID:26430636

  16. Usefulness and Future Prospects of Confocal Laser Endomicroscopy for Gastric Premalignant and Malignant Lesions.

    PubMed

    Lee, Sang Kil

    2015-11-01

    Confocal laser endomicroscopy (CLE) is a new technology enabling endoscopists to visualize tissue at the cellular level. CLE has the fundamental potential to provide a histologic diagnosis, and may theoretically replace or reduce the need for performing biopsy for histology. The clinical benefits of CLE are more obvious in esophageal disease, including Barrett's esophagus. Currently, this technology has been adapted to the diagnosis and surveillance of Barrett's esophagus and related neoplasia. Standard white light endoscopy is the primary tool for gastric cancer screening. Currently, the only method available to precisely diagnose these lesions is upper endoscopy with an appropriate biopsy. A recent study showed that CLE could characterize dysplasia or cancer and identify the risk factors for gastric cancer, such as intestinal metaplasia and the presence of Helicobacter pylori in vivo, although fewer studies on CLE were performed on the stomach than on Barrett's esophagus and other esophageal diseases. However, the application of CLE to routine clinical endoscopy continues to be refined. This review focused on the usefulness and future prospects of CLE for gastric premalignant and malignant lesions. PMID:26668797

  17. Collagen and Elastic Fibers in Odontogenic Entities: Analysis Using Light and Confocal Laser Microscopic Methods

    PubMed Central

    Moure, Sabrina P; Carrard, Vinicius C; Lauxen, Isabel S; Manso, Pedro Paulo A; Oliveira, Marcia G; Martins, Manoela D; Sant´Ana Filho, Manoel

    2011-01-01

    Dentigerous cyst (DC) and keratocystic odontogenic tumor (KOT) are odontogenic lesions arising from epithelial elements, such as those observed in dental follicles (DF), that have been part of the tooth forming apparatus. These lesions show different clinical and histological characteristics, as well as distinct biological behavior. This study aimed to qualify and quantify collagen and elastic fibers by means of histochemical techniques with light and confocal laser microscopic methods in three odontogenic entities. Eleven DF, 13 DC (n=10 with inflammation, n=3 without inflammation) and 13 KOT were processed to the following techniques: Hematoxylin and Eosin, Masson’s Trichrome, Picrosirius, Direct Blue, and Orcein. DF and DC without inflammation exhibited collagen with similar characteristics: no parallel pattern of fiber orientation, thick fibers with dense arrangement, and absence of distinct layers. A comparison between DC with inflammation and KOT revealed similar collagen organization, showing distinct layers: thin collagen fibers with loose arrangement near the epithelium and thick fibers with dense arrangement in distant areas. The only difference found was that KOT exhibited a parallel collagen orientation in relation to the odontogenic epithelia. It may be suggested that the connective tissue of DC is a reactive tissue, inducing an expansive growth associated with fluid accumulation and inflammatory process, which in turn may be present as part of the lesion itself. In KOT, loosely arranged collagen may be associated with the behavior of the neoplastic epithelium. PMID:21760864

  18. Confocal Laser Endomicroscopy in Gastrointestinal and Pancreatobiliary Diseases: A Systematic Review and Meta-Analysis

    PubMed Central

    Fugazza, Alessandro; Gaiani, Federica; Carra, Maria Clotilde; Brunetti, Francesco; Lévy, Michaël; Sobhani, Iradj; Azoulay, Daniel; Catena, Fausto; de'Angelis, Gian Luigi; de'Angelis, Nicola

    2016-01-01

    Confocal laser endomicroscopy (CLE) is an endoscopic-assisted technique developed to obtain histopathological diagnoses of gastrointestinal and pancreatobiliary diseases in real time. The objective of this systematic review is to analyze the current literature on CLE and to evaluate the applicability and diagnostic yield of CLE in patients with gastrointestinal and pancreatobiliary diseases. A literature search was performed on MEDLINE, EMBASE, Scopus, and Cochrane Oral Health Group Specialized Register, using pertinent keywords without time limitations. Both prospective and retrospective clinical studies that evaluated the sensitivity, specificity, or accuracy of CLE were eligible for inclusion. Of 662 articles identified, 102 studies were included in the systematic review. The studies were conducted between 2004 and 2015 in 16 different countries. CLE demonstrated high sensitivity and specificity in the detection of dysplasia in Barrett's esophagus, gastric neoplasms and polyps, colorectal cancers in inflammatory bowel disease, malignant pancreatobiliary strictures, and pancreatic cysts. Although CLE has several promising applications, its use has been limited by its low availability, high cost, and need of specific operator training. Further clinical trials with a particular focus on cost-effectiveness and medicoeconomic analyses, as well as standardized institutional training, are advocated to implement CLE in routine clinical practice. PMID:26989684

  19. Usefulness and Future Prospects of Confocal Laser Endomicroscopy for Gastric Premalignant and Malignant Lesions

    PubMed Central

    Lee, Sang Kil

    2015-01-01

    Confocal laser endomicroscopy (CLE) is a new technology enabling endoscopists to visualize tissue at the cellular level. CLE has the fundamental potential to provide a histologic diagnosis, and may theoretically replace or reduce the need for performing biopsy for histology. The clinical benefits of CLE are more obvious in esophageal disease, including Barrett’s esophagus. Currently, this technology has been adapted to the diagnosis and surveillance of Barrett’s esophagus and related neoplasia. Standard white light endoscopy is the primary tool for gastric cancer screening. Currently, the only method available to precisely diagnose these lesions is upper endoscopy with an appropriate biopsy. A recent study showed that CLE could characterize dysplasia or cancer and identify the risk factors for gastric cancer, such as intestinal metaplasia and the presence of Helicobacter pylori in vivo, although fewer studies on CLE were performed on the stomach than on Barrett’s esophagus and other esophageal diseases. However, the application of CLE to routine clinical endoscopy continues to be refined. This review focused on the usefulness and future prospects of CLE for gastric premalignant and malignant lesions. PMID:26668797

  20. Appraisal of needle-based confocal laser endomicroscopy in the diagnosis of pancreatic cysts

    PubMed Central

    Krishna, Somashekar G; Lee, Jeffery H

    2016-01-01

    Nearly 2.5% of cross-sectional imaging studies will report a finding of a cystic pancreatic lesion. Even though most of these are incidental findings, it remains very concerning for both patients and treating clinicians. Differentiating and predicting malignant transformation in pancreatic cystic lesions is clinically challenging. Current evaluation of suspicious cystic lesions includes a combination of radiologic imaging, endoscopic ultrasound (EUS) and cyst fluid analyses. Despite these attempts, precise diagnostic stratification among non-mucinous, mucinous, and malignant cystic lesions is often not possible until surgical resection. EUS-guided needle based confocal laser endomicroscopy (nCLE) for evaluation of pancreatic cysts is emerging as a powerful technique with remarkable potential. Though limited imaging data from 3 large clinical trials (INSPECT, DETECT and CONTACT) are currently the reference standard for nCLE imaging, nonetheless these have not been validated in large studies. The aim of this review article is to review the evolving role of EUS-guided nCLE in management of pancreatic cystic lesions in terms of its significance, adverse events, limitations, and implications. PMID:26819534

  1. Optical Biopsy of Peripheral Nerve Using Confocal Laser Endomicroscopy: A New Tool for Nerve Surgeons?

    PubMed Central

    Liao, Joseph C; Curtin, Catherine M

    2015-01-01

    Peripheral nerve injuries remain a challenge for reconstructive surgeons with many patients obtaining suboptimal results. Understanding the level of injury is imperative for successful repair. Current methods for distinguishing healthy from damaged nerve are time consuming and possess limited efficacy. Confocal laser endomicroscopy (CLE) is an emerging optical biopsy technology that enables dynamic, high resolution, sub-surface imaging of live tissue. Porcine sciatic nerve was either left undamaged or briefly clamped to simulate injury. Diluted fluorescein was applied topically to the nerve. CLE imaging was performed by direct contact of the probe with nerve tissue. Images representative of both damaged and undamaged nerve fibers were collected and compared to routine H&E histology. Optical biopsy of undamaged nerve revealed bands of longitudinal nerve fibers, distinct from surrounding adipose and connective tissue. When damaged, these bands appear truncated and terminate in blebs of opacity. H&E staining revealed similar features in damaged nerve fibers. These results prompt development of a protocol for imaging peripheral nerves intraoperatively. To this end, improving surgeons' ability to understand the level of injury through real-time imaging will allow for faster and more informed operative decisions than the current standard permits. PMID:26430636

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

    PubMed Central

    Giovannini, Marc; Jamidar, Priya; Gan, S. Ian; Cesaro, Paola; Caillol, Fabrice; Filoche, Bernard; Karia, Kunal; Smith, Ioana; Slivka, Adam

    2015-01-01

    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. PMID:25866506

  3. Confocal fluorescence microendoscopy of bronchial epithelium

    NASA Astrophysics Data System (ADS)

    Lane, Pierre M.; Lam, Stephen; McWilliams, Annette; Leriche, Jean C.; Anderson, Marshall W.; Macaulay, Calum E.

    2009-03-01

    Confocal microendoscopy permits the acquisition of high-resolution real-time confocal images of bronchial mucosa via the instrument channel of an endoscope. We report here on the construction and validation of a confocal fluorescence microendoscope and its use to acquire images of bronchial epithelium in vivo. Our objective is to develop an imaging method that can distinguish preneoplastic lesions from normal epithelium to enable us to study the natural history of these lesions and the efficacy of chemopreventive agents without biopsy removal of the lesion that can introduce a spontaneous regression bias. The instrument employs a laser-scanning engine and bronchoscope-compatible confocal probe consisting of a fiber-optic image guide and a graded-index objective lens. We assessed the potential of topical application of physiological pH cresyl violet (CV) as a fluorescence contrast-enhancing agent for the visualization of tissue morphology. Images acquired ex vivo with the confocal microendoscope were first compared with a bench-top confocal fluorescence microscope and conventional histology. Confocal images from five sites topically stained with CV were then acquired in vivo from high-risk smokers and compared to hematoxylin and eosin stained sections of biopsies taken from the same site. Sufficient contrast in the confocal imagery was obtained to identify cells in the bronchial epithelium. However, further improvements in the miniature objective lens are required to provide sufficient axial resolution for accurate classification of preneoplastic lesions.

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

    PubMed Central

    Zehri, Aqib H.; Ramey, Wyatt; Georges, Joseph F.; Mooney, Michael A.; Martirosyan, Nikolay L.; Preul, Mark C.; Nakaji, Peter

    2014-01-01

    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. PMID:24872922

  5. Dermoscopy, confocal laser microscopy, and hi-tech evaluation of vascular skin lesions: diagnostic and therapeutic perspectives.

    PubMed

    Grazzini, Marta; Stanganelli, Ignazio; Rossari, Susanna; Gori, Alessia; Oranges, Teresa; Longo, Anna Sara; Lotti, Torello; Bencini, Pier Luca; De Giorgi, Vincenzo

    2012-01-01

    Vascular skin lesions comprise a wide and heterogeneous group of malformations and tumors that can be correctly diagnosed based on natural history and physical examination. However, considering the high incidence of such lesions, a great number of them can be misdiagnosed. In addition, it is not so rare that an aggressive amelanotic melanoma can be misdiagnosed as a vascular lesion. In this regard, dermoscopy and confocal laser microscopy examination can play a central role in increasing the specificity of the diagnosis of such lesions. In fact, the superiority of these tools over clinical examination has encouraged dermatologists to adopt these devices for routine clinical practice, with a progressive spread of their use. In this review, we will go through the dermoscopic and the confocal laser microscopy of diagnosis of most frequent vascular lesions (i.e., hemangiomas angiokeratoma, pyogenic granuloma, angiosarcoma) taking into particular consideration the differential diagnosis with amelanotic melanoma. PMID:22950556

  6. A confocal microscope position sensor for micron-scale target alignment in ultra-intense laser-matter experiments

    NASA Astrophysics Data System (ADS)

    Willis, Christopher; Poole, Patrick L.; Akli, Kramer U.; Schumacher, Douglass W.; Freeman, Richard R.

    2015-05-01

    A diagnostic tool for precise alignment of targets in laser-matter interactions based on confocal microscopy is presented. This device permits precision alignment of targets within the Rayleigh range of tight focusing geometries for a wide variety of target surface morphologies. This confocal high-intensity positioner achieves micron-scale target alignment by selectively accepting light reflected from a narrow range of target focal planes. Additionally, the design of the device is such that its footprint and sensitivity can be tuned for the desired chamber and experiment. The device has been demonstrated to position targets repeatably within the Rayleigh range of the Scarlet laser system at The Ohio State University, where use of the device has provided a marked increase in ion yield and maximum energy.

  7. Writing of rare-earth ion doped lithium niobate line patterns in glass by laser scanning

    NASA Astrophysics Data System (ADS)

    Honma, T.; Komatsu, T.; Zhao, D.; Jain, H.

    2009-02-01

    A glass of Er3+ doped Li2O-Nb2O5-SiO2-B2O3 with an addition of CuO or Sm2O3 crystallizing nonlinear optical lithium niobate LiNbO3 (LN) is developed. Crystalline lines of LN have been fabricated on the glass surface by continuous wave Yb fiber laser irradiations with a wavelength of 1080 nm. The laser written LN crystalline lines have been found, by means of electron back scattering method, micro-Raman and second harmonic experiments, to be well oriented along the laser scanning direction. For the testing of optical waveguides crystal lines exhibit light confinements due to the refractive index (n) changes between the patterned line (n~2.2) and the glass matrix (n=1.7). The analysis of the confocal micro-luminescence spectra obtained for the crystalline line indicates the incorporation of Er3+ ions into LN crystals.

  8. Speckle averaging system for laser raster-scan image projection

    DOEpatents

    Tiszauer, D.H.; Hackel, L.A.

    1998-03-17

    The viewers` perception of laser speckle in a laser-scanned image projection system is modified or eliminated by the addition of an optical deflection system that effectively presents a new speckle realization at each point on the viewing screen to each viewer for every scan across the field. The speckle averaging is accomplished without introduction of spurious imaging artifacts. 5 figs.

  9. Speckle averaging system for laser raster-scan image projection

    DOEpatents

    Tiszauer, Detlev H.; Hackel, Lloyd A.

    1998-03-17

    The viewers' perception of laser speckle in a laser-scanned image projection system is modified or eliminated by the addition of an optical deflection system that effectively presents a new speckle realization at each point on the viewing screen to each viewer for every scan across the field. The speckle averaging is accomplished without introduction of spurious imaging artifacts.

  10. Digital confocal microscopy using a virtual 4f-system based on numerical beam propagation for depth measurement without mechanical scanning

    NASA Astrophysics Data System (ADS)

    Goto, Yuta; Okamoto, Atsushi; Toda, Masataka; Kuno, Yasuyuki; Nozawa, Jin; Ogawa, Kazuhisa; Tomita, Akihisa

    2016-08-01

    We propose a digital confocal microscope using a virtual 4f-system based on numerical beam propagation for depth measurement without mechanical scanning. In our technique, the information in the sample target along the depth direction is obtained by defocusing the virtual 4f-system, which consists of two virtual lenses arranged in a computer simulation. The principle of our technique is completely different from that of the mechanical scanning method used in the conventional confocal microscope based on digital holography. By using the virtual 4f-system, the measurement and exposure time can be markedly reduced because multilayered tomographic images are generated using a single measurement. In this study, we tested the virtual depth imaging technique by measuring cover glasses arranged along the depth direction.

  11. Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery

    PubMed Central

    Liu, Jonathan T.C.; Mandella, Michael J.; Loewke, Nathan O.; Haeberle, Henry; Ra, Hyejun; Piyawattanametha, Wibool; Solgaard, Olav; Kino, Gordon S.; Contag, Christopher H.

    2010-01-01

    A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500×500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections. PMID:20459274

  12. Tree Height Growth Measurement with Single-Scan Airborne, Static Terrestrial and Mobile Laser Scanning

    PubMed Central

    Lin, Yi; Hyyppä, Juha; Kukko, Antero; Jaakkola, Anttoni; Kaartinen, Harri

    2012-01-01

    This study explores the feasibility of applying single-scan airborne, static terrestrial and mobile laser scanning for improving the accuracy of tree height growth measurement. Specifically, compared to the traditional works on forest growth inventory with airborne laser scanning, two issues are regarded: “Can the new technique characterize the height growth for each individual tree?” and “Can this technique refine the minimum growth-discernable temporal interval further?” To solve these two puzzles, the sampling principles of the three laser scanning modes were first examined, and their error sources against the task of tree-top capturing were also analyzed. Next, the three-year growths of 58 Nordic maple trees (Crimson King) for test were intermittently surveyed with one type of laser scanning each time and then analyzed by statistics. The evaluations show that the height growth of each individual tree still cannot be reliably characterized even by single-scan terrestrial laser scanning, and statistical analysis is necessary in this scenario. After Gaussian regression, it is found that the minimum temporal interval with distinguishable tree height growths can be refined into one month based on terrestrial laser scanning, far better than the two years deduced in the previous works based on airborne laser scanning. The associated mean growth was detected to be about 0.12 m. Moreover, the parameter of tree height generally under-estimated by airborne and even mobile laser scanning can be relatively revised by means of introducing static terrestrial laser scanning data. Overall, the effectiveness of the proposed technique is primarily validated. PMID:23112743

  13. High performance FBG interrogation technology with scan fiber laser

    NASA Astrophysics Data System (ADS)

    Yang, Yuanhong; Ma, Youchun; Yang, Minwei

    2010-11-01

    A Fiber Bragg gratings (FBG) Interrogation scheme with scan fiber laser was demonstrated. The ring cavity scan fiber laser was investigated and the scan fiber laser module was made and test, the 200Hz scan frequency, ~0.02nm line width, more than 40nm scan range and more than 1 mW output power were obtained. A 12 channels, 20 FBGs per channel FBG interrogator was made with this laser module and the high speed signal process circuit base on FPGA. The centroid finding method which has advantage on interrogation speed and accurate was taken for finding the peak of the return FBG spectrum. The FBG interrogator was test and less than 3pm standard deviation with 200Hz scan frequency were obtained.

  14. Applications of Adaptive Optics Scanning Laser Ophthalmoscopy

    PubMed Central

    Roorda, Austin

    2010-01-01

    Adaptive optics (AO) describes a set of tools to correct or control aberrations in any optical system. In the eye, AO allows for precise control of the ocular aberrations. If used to correct aberrations over a large pupil, for example, cellular level resolution in retinal images can be achieved. AO systems have been demonstrated for advanced ophthalmoscopy as well as for testing and/or improving vision. In fact, AO can be integrated to any ophthalmic instrument where the optics of the eye is involved, with a scope of applications ranging from phoropters to optical coherence tomography systems. In this paper, I discuss the applications and advantages of using AO in a specific system, the adaptive optics scanning laser ophthalmoscope, or AOSLO. Since the Borish award was, in part, awarded to me because of this effort, I felt it appropriate to select this as the topic for this paper. Furthermore, users of AOSLO continue to appreciate the benefits of the technology, some of which were not anticipated at the time of development, and so it is time to revisit this topic and summarize them in a single paper. PMID:20160657

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

    EPA Science Inventory

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The reliability of the CLSM to obtain specific measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. For man...

  16. In vivo probe-based confocal laser endomicroscopy in amiodarone-related pneumonia.

    PubMed

    Salaün, Mathieu; Roussel, Francis; Bourg-Heckly, Geneviève; Vever-Bizet, Christine; Dominique, Stéphane; Genevois, Anne; Jounieaux, Vincent; Zalcman, Gérard; Bergot, Emmanuel; Vergnon, Jean-Michel; Thiberville, Luc

    2013-12-01

    Probe-based confocal laser endomicroscopy (pCLE) allows microscopic imaging of the alveoli during bronchoscopy. The objective of the study was to assess the diagnostic accuracy of pCLE for amiodarone-related pneumonia (AMR-IP). Alveolar pCLE was performed in 36 nonsmoking patients, including 33 consecutive patients with acute or subacute interstitial lung disease (ILD), of which 17 were undergoing treatment with amiodarone, and three were amiodarone-treated patients without ILD. Nine out of 17 patients were diagnosed with high-probability AMR-IP (HP-AMR-IP) by four experts, and three separate observers. Bronchoalveolar lavage findings did not differ between HP-AMR-IP and low-probability AMR-IP (LP-AMR-IP) patients. In HP-AMR-IP patients, pCLE showed large (>20 μm) and strongly fluorescent cells in 32 out of 38 alveolar areas. In contrast, these cells were observed in only two out of 39 areas from LP-AMR-IP patients, in one out of 59 areas from ILD patients not receiving amiodarone and in none of the 10 areas from amiodarone-treated patients without ILD (p<0.001; HP-AMR-IP versus other groups). The presence of at least one alveolar area with large and fluorescent cells had a sensitivity, specificity, negative predictive value and positive predictive value for the diagnosis of AMR-IP of 100%, 88%, 100% and 90%, respectively. In conclusion, pCLE appears to be a valuable tool for the in vivo diagnosis of AMR-IP in subacute ILD patients. PMID:23018901

  17. Detection of superficial esophageal squamous cell neoplasia by chromoendoscopy-guided confocal laser endomicroscopy

    PubMed Central

    Huang, Jin; Yang, Yun-Sheng; Lu, Zhong-Sheng; Wang, Shuang-Fang; Yang, Jing; Yuan, Jing

    2015-01-01

    AIM: To evaluate the diagnostic potential of Lugol’s chromoendoscopy-guided confocal laser endomicroscopy (CLE) in detecting superficial esophageal squamous cell neoplasia (ESCN). METHODS: Between December 2008 and September 2010, a total of 52 patients were enrolled at the Chinese PLA General Hospital in Beijing, China. First, Lugol’s chromoendoscopy-guided CLE was performed in these patients and the CLE in vivo histological diagnosis was recorded. Then, chromoendoscopy-guided biopsy was performed in the same patients by another endoscopist who was blinded to the CLE findings. Based on the biopsy and CLE diagnosis, en bloc endoscopic resection was performed. The CLE in vivo diagnosis and the histological diagnosis of biopsy of ESCN were compared, using a histological examination of the endoscopic resection specimens as the standard reference. RESULTS: A total of 152 chromoendoscopy-guided biopsies were obtained from 56 lesions. In the 56 lesions of 52 patients, a total of 679 CLE images were obtained vs 152 corresponding biopsies. The sensitivity, specificity, negative predictive value and positive predictive value of chromoendoscopy-guided CLE compared with biopsy were 95.7% vs 82% (P < 0.05), 90% vs 70% (P < 0.05), 81.8% vs 46.7% (P < 0.05), and 97.8% vs 92.7% (P > 0.05), respectively. There was a significant improvement in sensitivity, specificity, negative predictive value, and accuracy when comparing chromoendoscopy-guided CLE with biopsy. CONCLUSION: Lugol’s chromoendoscopy-guided CLE is a real-time, non-invasive endoscopic diagnostic technology; the accuracy of the detection of superficial ESCN is equivalent to or may be superior to biopsy histology. PMID:26078575

  18. Automatic classification of small bowel mucosa alterations in celiac disease for confocal laser endomicroscopy

    NASA Astrophysics Data System (ADS)

    Boschetto, Davide; Di Claudio, Gianluca; Mirzaei, Hadis; Leong, Rupert; Grisan, Enrico

    2016-03-01

    Celiac disease (CD) is an immune-mediated enteropathy triggered by exposure to gluten and similar proteins, affecting genetically susceptible persons, increasing their risk of different complications. Small bowels mucosa damage due to CD involves various degrees of endoscopically relevant lesions, which are not easily recognized: their overall sensitivity and positive predictive values are poor even when zoom-endoscopy is used. Confocal Laser Endomicroscopy (CLE) allows skilled and trained experts to qualitative evaluate mucosa alteration such as a decrease in goblet cells density, presence of villous atrophy or crypt hypertrophy. We present a method for automatically classifying CLE images into three different classes: normal regions, villous atrophy and crypt hypertrophy. This classification is performed after a features selection process, in which four features are extracted from each image, through the application of homomorphic filtering and border identification through Canny and Sobel operators. Three different classifiers have been tested on a dataset of 67 different images labeled by experts in three classes (normal, VA and CH): linear approach, Naive-Bayes quadratic approach and a standard quadratic analysis, all validated with a ten-fold cross validation. Linear classification achieves 82.09% accuracy (class accuracies: 90.32% for normal villi, 82.35% for VA and 68.42% for CH, sensitivity: 0.68, specificity 1.00), Naive Bayes analysis returns 83.58% accuracy (90.32% for normal villi, 70.59% for VA and 84.21% for CH, sensitivity: 0.84 specificity: 0.92), while the quadratic analysis achieves a final accuracy of 94.03% (96.77% accuracy for normal villi, 94.12% for VA and 89.47% for CH, sensitivity: 0.89, specificity: 0.98).

  19. Confocal Laser Endomicroscopy for Diagnosis and Monitoring of Pulmonary Alveolar Proteinosis

    PubMed Central

    Averyanov, Alexander; Lesnyak, Viktor; Chernyaev, Andrey; Sorokina, Anastasia

    2015-01-01

    Background: The diagnosis of pulmonary alveolar proteinosis (PAP) is based on computed tomography, histology, and antibodies to granulocyte-macrophage colony-stimulating factor. The role of a novel technique for imaging cells and elastin during endoscopy, probe-based confocal laser endomicroscopy (pCLE), has not yet been investigated in PAP patients. The aim of the present study was to estimate the value of pCLE in the PAP diagnosis and treatment in comparison with the findings of high-resolution computed tomography (HRCT) before and after whole-lung lavage. Methods: In vivo pCLE was performed during bronchoscopy in 6 male patients with PAP before and after whole-lung lavage. In certain lung segments, pCLE was followed by HRCT. Results: During the in vivo pCLE, we found characteristic signs of PAP: a fluorescent floating amorphous substance in the alveoli lumen sticking to conglomerates along with alveolar macrophages. These features were present to a lesser extent after a whole-lung lavage. pCLE revealed specific PAP features not only in segments with crazy-paving and ground-glass opacity, but also in segments without HRCT findings. Conclusions: The alveolar imaging in PAP patients is able to reveal characteristic changes, both in the presence and in the absence of HRCT findings. Therefore, pCLE may be a helpful tool for the diagnosis and whole-lung lavage therapy. Our data prove that accumulation of lipoproteinaceous substances within the alveoli at PAP is a diffuse but not a patchy process. PMID:25590481

  20. Monitoring Pancreatic Carcinogenesis by the Molecular Imaging of Cathepsin E In Vivo Using Confocal Laser Endomicroscopy

    PubMed Central

    Cui, Lei; Wang, Biyuan; Cui, Wenli; Li, Minghua; Cheng, Yingsheng

    2014-01-01

    The monitoring of pancreatic ductal adenocarcinoma (PDAC) in high-risk populations is essential. Cathepsin E (CTSE) is specifically and highly expressed in PDAC and pancreatic intraepithelial neoplasias (PanINs), and its expression gradually increases along with disease progression. In this study, we first established an in situ 7,12-dimethyl-1,2-benzanthracene (DMBA)-induced rat model for PanINs and PDAC and then confirmed that tumorigenesis properties in this model were consistent with those of human PDAC in that CTSE expression gradually increased with tumor development using histology and immunohistochemistry. Then, using in vivo imaging of heterotopically implanted tumors generated from CTSE- overexpressing cells (PANC-1-CTSE) in nude mice and in vitro imaging of PanINs and PDAC in DMBA-induced rats, the specificity of the synthesized CTSE-activatable probe was verified. Quantitative determination identified that the fluorescence signal ratio of pancreatic tumor to normal pancreas gradually increased in association with progressive pathological grades, with the exception of no significant difference between PanIN-II and PanIN-III grades. Finally, we monitored pancreatic carcinogenesis in vivo using confocal laser endomicroscopy (CLE) in combination with the CTSE-activatable probe. A prospective double-blind control study was performed to evaluate the accuracy of this method in diagnosing PDAC and PanINs of all grades (>82.7%). This allowed us to establish effective diagnostic criteria for CLE in PDAC and PanINs to facilitate the monitoring of PDAC in high-risk populations. PMID:25184278

  1. Quality Analysis and Correction of Mobile Backpack Laser Scanning Data

    NASA Astrophysics Data System (ADS)

    Rönnholm, P.; Liang, X.; Kukko, A.; Jaakkola, A.; Hyyppä, J.

    2016-06-01

    Backpack laser scanning systems have emerged recently enabling fast data collection and flexibility to make measurements also in areas that cannot be reached with, for example, vehicle-based laser scanners. Backpack laser scanning systems have been developed both for indoor and outdoor use. We have developed a quality analysis process in which the quality of backpack laser scanning data is evaluated in the forest environment. The reference data was collected with an unmanned aerial vehicle (UAV) laser scanning system. The workflow included noise filtering, division of data into smaller patches, ground point extraction, ground data decimation, and ICP registration. As a result, we managed to observe the misalignments of backpack laser scanning data for 97 patches each including data from circa 10 seconds period of time. This evaluation revealed initial average misalignments of 0.227 m, 0.073 and -0.083 in the easting, northing and elevation directions, respectively. Furthermore, backpack data was corrected according to the ICP registration results. Our correction algorithm utilized the time-based linear transformation of backpack laser scanning point clouds. After the correction of data, the ICP registration was run again. This revealed remaining misalignments between the corrected backpack laser scanning data and the original UAV data. We found average misalignments of 0.084, 0.020 and -0.005 meters in the easting, northing and elevation directions, respectively.

  2. Laser-scanning techniques for rapid ballistics identification

    NASA Technical Reports Server (NTRS)

    Woodburgy, R. C.; Nakich, R. B.

    1974-01-01

    Two different laser-scanning methods may be utilized. In each case scanned cylindrical bullet surface is displayed ""unwrapped'' on oscilloscope screen. Bullets are compared by photographing each display and superimposing negatives of two images. With some modifications bullets can be scanned and compared by superimposing images on screen of dual-beam oscilloscope.

  3. Role of the scanning laser ophthalmoscope in photodynamic therapy of macular disease

    NASA Astrophysics Data System (ADS)

    Van de Velde, Frans J.

    2000-06-01

    Photodynamic therapy (PDT) is a new treatment modality for exudative forms of age-related maculopathy. It can be combined with others types of selective or conventional laser therapy. Imaging and functional testing with the scanning laser ophthalmoscope (SLO) are important for detailed diagnostic information as well as for the interpretation of the long term outcome of different treatment strategies. For example, infrared imaging in a confocal mode superbly outlines areas of minimal edema due to slow leakage and switching of wavelengths enables simultaneous and repeated angiographic studies of the retina with the same instrument. Visual acuities are strongly influenced by background illuminance and binocular fixation patterns, and absolute but not incremental microperimetric thresholds measure correctly the functional status of the photoreceptor-pigment epithelium complex. The scanning laser ophthalmoscope has been adapted for use as a delivery system in microphotocoagulation and photodynamic therapy. A non- scanning external therapeutic laser source uses the same Maxwellian view entrance location into the eye as the SLO. Advantages include a non-contact delivery, fixation control, registration of treatment locations, and the possibility to spatially modulate the area being treated.

  4. Concurrent Reflectance Confocal Microscopy and Laser Doppler Flowmetry to Improve Skin Cancer Imaging: A Monte Carlo Model and Experimental Validation.

    PubMed

    Mowla, Alireza; Taimre, Thomas; Lim, Yah Leng; Bertling, Karl; Wilson, Stephen J; Prow, Tarl W; Soyer, H Peter; Rakić, Aleksandar D

    2016-01-01

    Optical interrogation of suspicious skin lesions is standard care in the management of skin cancer worldwide. Morphological and functional markers of malignancy are often combined to improve expert human diagnostic power. We propose the evaluation of the combination of two independent optical biomarkers of skin tumours concurrently. The morphological modality of reflectance confocal microscopy (RCM) is combined with the functional modality of laser Doppler flowmetry, which is capable of quantifying tissue perfusion. To realize the idea, we propose laser feedback interferometry as an implementation of RCM, which is able to detect the Doppler signal in addition to the confocal reflectance signal. Based on the proposed technique, we study numerical models of skin tissue incorporating two optical biomarkers of malignancy: (i) abnormal red blood cell velocities and concentrations and (ii) anomalous optical properties manifested through tissue confocal reflectance, using Monte Carlo simulation. We also conduct a laboratory experiment on a microfluidic channel containing a dynamic turbid medium, to validate the efficacy of the technique. We quantify the performance of the technique by examining a signal to background ratio (SBR) in both the numerical and experimental models, and it is shown that both simulated and experimental SBRs improve consistently using this technique. This work indicates the feasibility of an optical instrument, which may have a role in enhanced imaging of skin malignancies. PMID:27598157

  5. Apparatus for controlling the scan width of a scanning laser beam

    DOEpatents

    Johnson, Gary W.

    1996-01-01

    Swept-wavelength lasers are often used in absorption spectroscopy applications. In experiments where high accuracy is required, it is desirable to continuously monitor and control the range of wavelengths scanned (the scan width). A system has been demonstrated whereby the scan width of a swept ring-dye laser, or semiconductor diode laser, can be measured and controlled in real-time with a resolution better than 0.1%. Scan linearity, or conformity to a nonlinear scan waveform, can be measured and controlled. The system of the invention consists of a Fabry-Perot interferometer, three CAMAC interface modules, and a microcomputer running a simple analysis and proportional-integral control algorithm. With additional modules, multiple lasers can be simultaneously controlled. The invention also includes an embodiment implemented on an ordinary PC with a multifunction plug-in board.

  6. Apparatus for controlling the scan width of a scanning laser beam

    DOEpatents

    Johnson, G.W.

    1996-10-22

    Swept-wavelength lasers are often used in absorption spectroscopy applications. In experiments where high accuracy is required, it is desirable to continuously monitor and control the range of wavelengths scanned (the scan width). A system has been demonstrated whereby the scan width of a swept ring-dye laser, or semiconductor diode laser, can be measured and controlled in real-time with a resolution better than 0.1%. Scan linearity, or conformity to a nonlinear scan waveform, can be measured and controlled. The system of the invention consists of a Fabry-Perot interferometer, three CAMAC interface modules, and a microcomputer running a simple analysis and proportional-integral control algorithm. With additional modules, multiple lasers can be simultaneously controlled. The invention also includes an embodiment implemented on an ordinary PC with a multifunction plug-in board. 8 figs.

  7. Confocal and Atomic Force Microscopies of Color Centers Produced by Ultrashort Laser Irradiation in LiF Crystals

    NASA Astrophysics Data System (ADS)

    Courrol, Lilia Coronato; Martinez, Oscar; Samad, Ricardo Elgul; Gomes, Laércio; Ranieri, Izilda Márcia; Baldochi, Sonia Licia; de Freitas, Anderson Zanardi; Junior, Nilson Dias Vieira

    2008-04-01

    We report properties of the spatial and spectral distribution of color centers produced in LiF single crystals by ultrashort high intensity laser pulses (60 fs, 10 GW) using confocal spectral microscopy and atomic force microscopy. We could identify a large amount of F centers that gave rise to aggregates such as F2, F4, F2+ and F3+ distributed in cracked shape brownish areas. We have taken a 3D image using confocal microscopy of the sample (luminescent image) and no difference is observed in the different planes. The atomic force microscopy image clearly shows the presence of defects on the modified surface. The formation of micrometer or sub-micrometer voids, filaments and void strings was observed and related to filamentation process.

  8. Boresight alignment method for mobile laser scanning systems

    NASA Astrophysics Data System (ADS)

    Rieger, P.; Studnicka, N.; Pfennigbauer, M.; Zach, G.

    2010-06-01

    Mobile laser scanning (MLS) is the latest approach towards fast and cost-efficient acquisition of 3-dimensional spatial data. Accurately evaluating the boresight alignment in MLS systems is an obvious necessity. However, recent systems available on the market may lack of suitable and efficient practical workflows on how to perform this calibration. This paper discusses an innovative method for accurately determining the boresight alignment of MLS systems by employing 3D laser scanners. Scanning objects using a 3D laser scanner operating in a 2D line-scan mode from various different runs and scan directions provides valuable scan data for determining the angular alignment between inertial measurement unit and laser scanner. Field data is presented demonstrating the final accuracy of the calibration and the high quality of the point cloud acquired during an MLS campaign.

  9. Any Way You Slice It—A Comparison of Confocal Microscopy Techniques

    PubMed Central

    Jonkman, James

    2015-01-01

    The confocal fluorescence microscope has become a popular tool for life sciences researchers, primarily because of its ability to remove blur from outside of the focal plane of the image. Several different kinds of confocal microscopes have been developed, each with advantages and disadvantages. This article will cover the grid confocal, classic confocal laser-scanning microscope (CLSM), the resonant scanning-CLSM, and the spinning-disk confocal microscope. The way each microscope technique works, the best applications the technique is suited for, the limitations of the technique, and new developments for each technology will be presented. Researchers who have access to a range of different confocal microscopes (e.g., through a local core facility) should find this paper helpful for choosing the best confocal technology for specific imaging applications. Others with funding to purchase an instrument should find the article helpful in deciding which technology is ideal for their area of research. PMID:25802490

  10. Diagnosis of gastric intraepithelial neoplasia by narrow-band imaging and confocal laser endomicroscopy

    PubMed Central

    Wang, Shu-Fang; Yang, Yun-Sheng; Wei, Li-Xin; Lu, Zhong-Sheng; Guo, Ming-Zhou; Huang, Jin; Peng, Li-Hua; Sun, Gang; Ling-Hu, En-Qiang; Meng, Jiang-Yun

    2012-01-01

    AIM: To evaluate the diagnosis of different differentiated gastric intraepithelial neoplasia (IN) by magnification endoscopy combined with narrow-band imaging (ME-NBI) and confocal laser endomicroscopy (CLE). METHODS: Eligible patients with suspected gastric IN lesions previously diagnosed by endoscopy in secondary hospitals and scheduled for further diagnosis and treatment were recruited for this study. Excluded from the study were patients who had liver cirrhosis, impaired renal function, acute gastrointestinal (GI) bleeding, coagulopathy, esophageal varices, jaundice, and GI post-surgery. Also excluded were those who were pregnant, breastfeeding, were younger than 18 years old, or were unable to provide informed consent. All patients had all mucus and bile cleared from their stomachs. They then received upper GI endoscopy. When a mucosal lesion is found during observation with white-light imaging, the lesion is visualized using maximal magnification, employing gradual movement of the tip of the endoscope to bring the image into focus. Saved images are analyzed. Confocal images were evaluated by two endoscopists (Huang J and Li MY), who were familiar with CLE, blinded to the related information about the lesions, and asked to classify each lesion as either a low grade dysplasia (LGD) or high grade dysplasia (HGD) according to given criteria. The results were compared with the final histopathologic diagnosis. ME-NBI images were evaluated by two endoscopists (Lu ZS and Ling-Hu EQ) who were familiar with NBI, blinded to the related information about the lesions and CLE images, and were asked to classify each lesion as a LGD or HGD according to the “microvascular pattern and surface pattern” classification system. The results were compared with the final histopathologic diagnosis. RESULTS: The study included 32 pathology-proven low grade gastric IN and 26 pathology-proven high grade gastric IN that were detected with any of the modalities. CLE and ME-NBI enabled

  11. Multiply scattered light tomography and confocal imaging: detecting neovascularization in age-related macular degeneration

    NASA Astrophysics Data System (ADS)

    Elsner, Ann E.; Miura, Masahiro; Burns, Stephen Allan; Beausencourt, E.; Kunze, C.; Kelley, L. M.; Walker, J. P.; Wing, G. L.; Raskauskas, P. A.; Fletcher, D. C.; Zhou, Qienyuan; Dreher, Andreas W.

    2000-07-01

    A novel technique, Multiply Scattered Light Tomography (MSLT), and confocal Infrared Imaging are used to provide diagnostic information using a comfortable, rapid, and noninvasive method. We investigated these techniques in detecting neovascularization in age-related macular degeneration. The MSLT used a Vertical Cavity Surface Emitting Laser (VCSEL) at 850 nm, while the confocal imaging technique used either the VCSEL or a 790 nm laser diode. Both were implemented into the topographical scanning system (TopSS, Laser Diagnostic Technologies, Inc.) Confocal imaging with both lasers provided different information about neovascularization as a function of focal plane, and different also from MSLT.

  12. Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy.

    PubMed

    Miyazaki, Jun; Iida, Tadatsune; Tanaka, Shinji; Hayashi-Takagi, Akiko; Kasai, Haruo; Okabe, Shigeo; Kobayashi, Takayoshi

    2016-05-01

    A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 μs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. PMID:27231615

  13. Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy

    PubMed Central

    Miyazaki, Jun; Iida, Tadatsune; Tanaka, Shinji; Hayashi-Takagi, Akiko; Kasai, Haruo; Okabe, Shigeo; Kobayashi, Takayoshi

    2016-01-01

    A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 μs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. PMID:27231615

  14. Multidepth imaging by chromatic dispersion confocal microscopy

    NASA Astrophysics Data System (ADS)

    Olsovsky, Cory A.; Shelton, Ryan L.; Saldua, Meagan A.; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

    2012-03-01

    Confocal microscopy has shown potential as an imaging technique to detect precancer. Imaging cellular features throughout the depth of epithelial tissue may provide useful information for diagnosis. However, the current in vivo axial scanning techniques for confocal microscopy are cumbersome, time-consuming, and restrictive when attempting to reconstruct volumetric images acquired in breathing patients. Chromatic dispersion confocal microscopy (CDCM) exploits severe longitudinal chromatic aberration in the system to axially disperse light from a broadband source and, ultimately, spectrally encode high resolution images along the depth of the object. Hyperchromat lenses are designed to have severe and linear longitudinal chromatic aberration, but have not yet been used in confocal microscopy. We use a hyperchromat lens in a stage scanning confocal microscope to demonstrate the capability to simultaneously capture information at multiple depths without mechanical scanning. A photonic crystal fiber pumped with a 830nm wavelength Ti:Sapphire laser was used as a supercontinuum source, and a spectrometer was used as the detector. The chromatic aberration and magnification in the system give a focal shift of 140μm after the objective lens and an axial resolution of 5.2-7.6μm over the wavelength range from 585nm to 830nm. A 400x400x140μm3 volume of pig cheek epithelium was imaged in a single X-Y scan. Nuclei can be seen at several depths within the epithelium. The capability of this technique to achieve simultaneous high resolution confocal imaging at multiple depths may reduce imaging time and motion artifacts and enable volumetric reconstruction of in vivo confocal images of the epithelium.

  15. Analysis of micro-structural relaxation phenomena in laser-modified fused silica using confocal Raman microscopy

    SciTech Connect

    Matthews, M; Vignes, R; Cooke, J; Yang, S; Stolken, J

    2009-12-15

    Fused silica micro-structural changes associated with localized 10.6 {micro}m CO{sub 2} laser heating are reported. Spatially-resolved shifts in the high-frequency asymmetric stretch transverse-optic (TO) phonon mode of SiO{sub 2} were measured using confocal Raman microscopy, allowing construction of axial fictive temperature (T{sub f}) maps for various laser heating conditions. A Fourier conduction-based finite element model was employed to compute on-axis temperature-time histories, and, in conjunction with a Tool-Narayanaswamy form for structural relaxation, used to fit T{sub f}(z) profiles to extract relaxation parameters. Good agreement between the calculated and measured T{sub f} was found, yielding reasonable values for relaxation time and activation enthalpy in the laser-modified silica.

  16. Laser safety in design of near-infrared scanning LIDARs

    NASA Astrophysics Data System (ADS)

    Zhu, X.; Elgin, D.

    2015-05-01

    3D LIDARs (Light Detection and Ranging) with 1.5μm nanosecond pulse lasers have been increasingly used in different applications. The main reason for their popularity is that these LIDARs have high performance while at the same time can be made eye-safe. Because the laser hazard effect on eyes or skin at this wavelength region (<1.4μm) is mainly from the thermal effect accumulated from many individual pulses over a period of seconds, scanning can effectively reduce the laser beam hazard effect from the LIDARs. Neptec LIDARs have been used in docking to the International Space Station, military helicopter landing and industrial mining applications. We have incorporated the laser safety requirements in the LIDAR design and conducted laser safety analysis for different operational scenarios. While 1.5μm is normally said to be the eye-safe wavelength, in reality a high performance 3D LIDAR needs high pulse energy, small beam size and high pulse repetition frequency (PRF) to achieve long range, high resolution and high density images. The resulting radiant exposure of its stationary beam could be many times higher than the limit for a Class 1 laser device. Without carefully choosing laser and scanning parameters, including field-of-view, scan speed and pattern, a scanning LIDAR can't be eye- or skin-safe based only on its wavelength. This paper discusses the laser safety considerations in the design of eye-safe scanning LIDARs, including laser pulse energy, PRF, beam size and scanning parameters in two basic designs of scanning mechanisms, i.e. galvanometer based scanner and Risley prism based scanner. The laser safety is discussed in terms of device classification, nominal ocular hazard distance (NOHD) and safety glasses optical density (OD).

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

    PubMed Central

    Ştefănescu, Daniela; Streba, Costin; Cârţână, Elena Tatiana; Săftoiu, Adrian; Gruionu, Gabriel; Gruionu, Lucian Gheorghe

    2016-01-01

    Introduction Confocal laser endomicroscopy (CLE) is becoming a popular method for optical biopsy of digestive mucosa for both diagnostic and therapeutic procedures. Computer aided diagnosis of CLE images, using image processing and fractal analysis can be used to quantify the histological structures in the CLE generated images. The aim of this study is to develop an automatic diagnosis algorithm of colorectal cancer (CRC), based on fractal analysis and neural network modeling of the CLE-generated colon mucosa images. Materials and Methods We retrospectively analyzed a series of 1035 artifact-free endomicroscopy images, obtained during CLE examinations from normal mucosa (356 images) and tumor regions (679 images). The images were processed using a computer aided diagnosis (CAD) medical imaging system in order to obtain an automatic diagnosis. The CAD application includes image reading and processing functions, a module for fractal analysis, grey-level co-occurrence matrix (GLCM) computation module, and a feature identification module based on the Marching Squares and linear interpolation methods. A two-layer neural network was trained to automatically interpret the imaging data and diagnose the pathological samples based on the fractal dimension and the characteristic features of the biological tissues. Results Normal colon mucosa is characterized by regular polyhedral crypt structures whereas malignant colon mucosa is characterized by irregular and interrupted crypts, which can be diagnosed by CAD. For this purpose, seven geometric parameters were defined for each image: fractal dimension, lacunarity, contrast correlation, energy, homogeneity, and feature number. Of the seven parameters only contrast, homogeneity and feature number were significantly different between normal and cancer samples. Next, a two-layer feed forward neural network was used to train and automatically diagnose the malignant samples, based on the seven parameters tested. The neural network

  18. Laser Scanning and Simulation at Kennedy Space Center

    NASA Technical Reports Server (NTRS)

    Kickbusch, Tracey E.

    2012-01-01

    We perform simulations of ground operations leading up launch at Kennedy Space Center and Vandenberg Air Force Base in CA. We use Laser Scanning, Modeling and Simulations to make sure operations are feasible, efficient, and safe.

  19. A novel multimodal laser scanning microscope control system

    NASA Astrophysics Data System (ADS)

    Lai, Zhenhua; Gu, Zetong; Karasek, Stephen; McLean, James; Zhang, Xi; DiMarzio, Charles; Yin, Jihao; Xiong, Daxi

    2015-03-01

    Traditional laser scanning microscopes require complex control systems to synchronize and control image acquisition. The control system is especially cumbersome in the multimodal laser scanning microscope. We have developed a novel multimodal laser scanning microscope control system based on a National Instruments multifunction data acquisition device (DAQ), which serves as both a data acquisition device and a programmable signal generator. The novel control system is low-cost and easy-to-build, with all components off-the-shelf. We have applied the control system in a multimodal laser scanning microscope. The control system has not only significantly decreased the complexity of the microscope, but also increased the system flexibility. We have demonstrated that the system can be easily customized for various applications.

  20. Endoscopic Ultrasound-Guided Needle-Based Probe Confocal Laser Endomicroscopy (nCLE) of Intrapancreatic Ectopic Spleen.

    PubMed

    Bastidas, Amanda B; Holloman, David; Lankarani, Ali; Nieto, Jose M

    2016-04-01

    Accessory spleens and splenosis represent the congenital and acquired type of ectopic splenic tissue. Generally, they are asymptomatic entities posing as solid hypervascular masses at the splenic hilum or in other organs, such as the pancreas. Intrapancreatic ectopic spleen mimics pancreatic neoplasms on imaging studies, and due to the lack of radiological diagnostic criteria, patients undergo unnecessary distal pancreatectomy. We present the first case of intrapancreatic ectopic spleen in which the concomitant use of needle-based probe confocal laser endomicroscopy and fine-needle aspiration supported the final diagnosis. PMID:27144203

  1. Endoscopic Ultrasound-Guided Needle-Based Probe Confocal Laser Endomicroscopy (nCLE) of Intrapancreatic Ectopic Spleen

    PubMed Central

    Bastidas, Amanda B.; Holloman, David; Lankarani, Ali

    2016-01-01

    Accessory spleens and splenosis represent the congenital and acquired type of ectopic splenic tissue. Generally, they are asymptomatic entities posing as solid hypervascular masses at the splenic hilum or in other organs, such as the pancreas. Intrapancreatic ectopic spleen mimics pancreatic neoplasms on imaging studies, and due to the lack of radiological diagnostic criteria, patients undergo unnecessary distal pancreatectomy. We present the first case of intrapancreatic ectopic spleen in which the concomitant use of needle-based probe confocal laser endomicroscopy and fine-needle aspiration supported the final diagnosis. PMID:27144203

  2. Software for visualization, analysis, and manipulation of laser scan images

    NASA Astrophysics Data System (ADS)

    Burnsides, Dennis B.

    1997-03-01

    The recent introduction of laser surface scanning to scientific applications presents a challenge to computer scientists and engineers. Full utilization of this two- dimensional (2-D) and three-dimensional (3-D) data requires advances in techniques and methods for data processing and visualization. This paper explores the development of software to support the visualization, analysis and manipulation of laser scan images. Specific examples presented are from on-going efforts at the Air Force Computerized Anthropometric Research and Design (CARD) Laboratory.

  3. Architectural stability analysis of the rotary-laser scanning technique

    NASA Astrophysics Data System (ADS)

    Xue, Bin; Yang, Xiaoxia; Zhu, Jigui

    2016-03-01

    The rotary-laser scanning technique is an important method in scale measurements due to its high accuracy and large measurement range. This paper first introduces a newly designed measurement station which is able to provide two-dimensional measurement information including the azimuth and elevation by using the rotary-laser scanning technique, then presents the architectural stability analysis of this technique by detailed theoretical derivations. Based on the designed station, a validation using both experiment and simulation is presented in order to verify the analytic conclusion. The results show that the architectural stability of the rotary-laser scanning technique is only affected by the two scanning angles' difference. And the difference which brings the best architectural stability can be calculated by using pre-calibrated parameters of the two laser planes. This research gives us an insight into the rotary-laser scanning technique. Moreover, the measurement accuracy of the rotary-laser scanning technique can be further improved based on the results of the study.

  4. Possibilities of holographic techniques in laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Zakharov, Yu.; Muravyeva, M.; Dudenkova, V.; Mukhina, I.; Meglinski, I.

    2015-07-01

    Holographic scanning microscopy - novel technique both in laser scanning microscopy and digital holographic microscopy allow multimodal approach to cell and tissue investigation in biomedical applications promising new advantages (quantitative phase imaging, superresolution, computerized tomography), but regular reconstruction leads to incorrectness. Analysis of light propagation through the schematics allows to offer reconstruction procedures depending on recording conditions.

  5. Confocal microscopy in microgravity research

    NASA Astrophysics Data System (ADS)

    Goede, A. P. H.; Brakenhoff, G. J.; Woldringh, C. L.; Aalders, J. W. G.; Imhof, J. P.; van Kralingen, P.; Mels, W. A.; Schreinemakers, P.; Zegers, A.

    We have studied the application and the feasibility of confocal scanning laser microscopy (CSLM) in microgravity research. Its superior spatial resolution and 3D imaging capabilities and its use of light as a probe, render this instrument ideally suited for the study of living biological material on a (sub-)cellular level. In this paper a number of pertinent biological microgravity experiments is listed, concentrating on the direct observation of developing cells and cellular structures under microgravity condition. A conceptual instrument design is also presented, aimed at sounding rocket application followed by Biorack/Biolab application at a later stage.

  6. Automatic Classification of Trees from Laser Scanning Point Clouds

    NASA Astrophysics Data System (ADS)

    Sirmacek, B.; Lindenbergh, R.

    2015-08-01

    Development of laser scanning technologies has promoted tree monitoring studies to a new level, as the laser scanning point clouds enable accurate 3D measurements in a fast and environmental friendly manner. In this paper, we introduce a probability matrix computation based algorithm for automatically classifying laser scanning point clouds into 'tree' and 'non-tree' classes. Our method uses the 3D coordinates of the laser scanning points as input and generates a new point cloud which holds a label for each point indicating if it belongs to the 'tree' or 'non-tree' class. To do so, a grid surface is assigned to the lowest height level of the point cloud. The grids are filled with probability values which are calculated by checking the point density above the grid. Since the tree trunk locations appear with very high values in the probability matrix, selecting the local maxima of the grid surface help to detect the tree trunks. Further points are assigned to tree trunks if they appear in the close proximity of trunks. Since heavy mathematical computations (such as point cloud organization, detailed shape 3D detection methods, graph network generation) are not required, the proposed algorithm works very fast compared to the existing methods. The tree classification results are found reliable even on point clouds of cities containing many different objects. As the most significant weakness, false detection of light poles, traffic signs and other objects close to trees cannot be prevented. Nevertheless, the experimental results on mobile and airborne laser scanning point clouds indicate the possible usage of the algorithm as an important step for tree growth observation, tree counting and similar applications. While the laser scanning point cloud is giving opportunity to classify even very small trees, accuracy of the results is reduced in the low point density areas further away than the scanning location. These advantages and disadvantages of two laser scanning point

  7. Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

    PubMed Central

    Lawrence, J. R.; Swerhone, G. D. W.; Leppard, G. G.; Araki, T.; Zhang, X.; West, M. M.; Hitchcock, A. P.

    2003-01-01

    Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications. PMID:12957944

  8. Effects of scanning orientation on outlier formation in 3D laser scanning of reflective surfaces

    NASA Astrophysics Data System (ADS)

    Wang, Yutao; Feng, Hsi-Yung

    2016-06-01

    Inspecting objects with reflective surfaces using 3D laser scanning is a demanded but challenging part inspection task due to undesirable specular reflections, which produce extensive outliers in the scanned point cloud. These outliers need to be removed in order to alleviate subsequent data processing issues. Many existing automatic outlier removal methods do not detect outliers according to the outlier formation properties. As a result, these methods only offer limited capabilities in removing extensive and complex outliers from scanning objects with reflective surfaces. This paper reports an empirical study which experimentally investigates the outlier formation characteristics in relation to the scanning orientation of the laser probe. The objective is to characterize the scanning orientation effects on outlier formation in order to facilitate the development of an effective outlier detection and removal method. Such an experimental investigation was hardly done before. It has been found in this work that scanning orientation can directly affect outlier extensity and occurrence in 3D laser scanning. A general guidance on proper scan path planning can then be provided with an aim to reduce the occurrence of outliers. Further, the observed dependency of outlier formation on scanning orientation can be exploited to facilitate effective and automatic outlier detection and removal.

  9. Use of endoscopic distal attachment cap to enhance image stabilization in probe-based confocal laser endomicroscopy in colorectal lesions*

    PubMed Central

    Ussui, Vivian; Xu, Can; Crook, Julia E.; Diehl, Nancy N.; Hardee, Joy; Staggs, Estela G.; Shahid, Muhammad W.; Wallace, Michael B.

    2015-01-01

    Background and study aims: Colorectal cancer can be prevented through the use of colonoscopy with polypectomy. Most colon polyps are benign or low grade adenomas. However, currently all lesions need histopathologic analysis, which increases diagnostic costs and delays the final diagnosis. Confocal laser endomicroscopy (CLE) is a new technology that enables real-time endomicroscopy. However, there are challenges to maintaining a stable image with currently available systems. We conducted a small study to obtain a preliminary assessment of whether the use of an endoscopic distal attachment cap may enhance image quality of CLE in comparison with images obtained with free-hand acquisition. Patients and methods: Forty outpatients underwent colonoscopy for evaluation of colon polyps in a single academic medical center. Patients were assigned randomly to 1 of 2 study arms on the basis of whether an endoscopic distal attachment cap was used (n = 21, Cap Used) or not used (n = 19, No Cap) in the procedure. The quality of confocal images and probe stabilization was summarized. Results: A total of 81 polyps were identified. The proportion of polyps with images of high quality was 74 % (28/38) in the Cap Used group and 79 % (30/38) in the No Cap arm. Image stability was also similar with and without a cap. Diagnostic accuracy was estimated to be slightly higher in the Cap Used group for probe-based confocal laser endomicroscopy (pCLE; 78 % vs 70 %). This was also true for white-light and narrow-band imaging. Conclusions: This preliminary study did not yield any evidence to support that the use of an endoscopic distal attachment cap improves the quality of images obtained during CLE. PMID:26528511

  10. Laser-scanning optical-resolution photoacoustic microscopy.

    PubMed

    Xie, Zhixing; Jiao, Shuliang; Zhang, Hao F; Puliafito, Carmen A

    2009-06-15

    We have developed a laser-scanning optical-resolution photoacoustic microscopy method that can potentially fuse with existing optical microscopic imaging modalities. To acquire an image, the ultrasonic transducer is kept stationary during data acquisition, and only the laser light is raster scanned by an x-y galvanometer scanner. A lateral resolution of 7.8 microm and a circular field of view with a diameter of 6 mm were achieved in an optically clear medium. Using a laser system working at a pulse repetition rate of 1,024 Hz, the data acquisition time for an image consisting of 256 x 256 pixels was less than 2 min. PMID:19529698

  11. The influence of scanning speed and number of scans on the properties of laser formed steel

    NASA Astrophysics Data System (ADS)

    Sanusi, Kazeem O.; Akinlabi, Stephen; Akinlabi, Esther T.

    2016-03-01

    Laser Beam Forming (LBF) process is an emerging and new forming method that generally requires brute force to forge the steel into the desired shape instead of using conventional methods. This study investigates the changes that occur in low carbon steel through the laser beam forming process. The parameters under investigation include variable scanning speed and number of scans at fixed laser intensity. The effect of these laser parameters on the chemical composition and properties of low carbon steel is assessed through characterisation of both the as received and LBF formed specimens. Characterizations of the laser formed steels were studied using microstructural analysis and micro hardness profiling. The results show that there is a significant increase in the mechanical properties of the LBF formed materials. Scanning power and the number of scans have a noticeable effect on the curvature achieved in the formed samples. The results obtained will contribute towards the further optimization of laser forming methods for steel for the optimization of the properties of steel using Laser Beam Forming process.

  12. Assessment of possibilities of ceramic biomaterial fracture surface reconstruction using laser confocal microscopy and long working distance objective lenses.

    PubMed

    Stach, Sebastian; Sapota, Wiktoria; Wróbel, Zygmunt; Ţălu, Ştefan

    2016-05-01

    A numerical description of fracture is an important step in the search of the correlation between specific micromechanisms of decohesion and material characteristics designated with the use of fracture mechanics methods. This issue is essential for the proper orientation of the search for basic relationships between chemical composition, technology, structure, and properties of materials. It often happens that fracture surfaces are well developed, which can significantly hinder or even prevent the measurement and reconstruction of the tested material surface geometry. In this article, comparative measurements of a biomaterial surface were performed using laser confocal microscopy. To this end, short working distance lenses dedicated to a focused UV laser beam and long working distance objective lenses were used. The article includes a quantitative comparative analysis and interpretation of the obtained results. Microsc. Res. Tech. 79:385-392, 2016. © 2016 Wiley Periodicals, Inc. PMID:26918261

  13. Confocal microscopy to guide Erbium:yttrium aluminum garnet laser ablation of basal cell carcinoma: an ex vivo feasibility study

    PubMed Central

    Larson, Bjorg A.; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

    2013-01-01

    Abstract. For the removal of superficial and nodular basal cell carcinomas (BCCs), laser ablation provides certain advantages relative to other treatment modalities. However, efficacy and reliability tend to be variable because tissue is vaporized such that none is available for subsequent histopathological examination for residual BCC (and to confirm complete removal of tumor). Intra-operative reflectance confocal microscopy (RCM) may provide a means to detect residual tumor directly on the patient and guide ablation. However, optimization of ablation parameters will be necessary to control collateral thermal damage and preserve sufficient viability in the underlying layer of tissue, so as to subsequently allow labeling of nuclear morphology with a contrast agent and imaging of residual BCC. We report the results of a preliminary study of two key parameters (fluence, number of passes) vis-à-vis the feasibility of labeling and RCM imaging in human skin ex vivo, following ablation with an erbium:yttrium aluminum garnet laser. PMID:24045654

  14. [Opportunities for confocal and laser biomicroscopy of corneal nerves in diabetic polyneuropathy].

    PubMed

    Surnina, Z V

    2015-01-01

    The review concerns corneal nerves involvement in diabetes mellitus (DM), a pressing issue for ophthalmology and endocrinology. The history of research in this field along with anatomical, physiological, and biochemical features of corneal nerves is provided. Corneal nerves anatomy is described in accordance with Soviet scientific school and contemporary foreign sources. The most part of the paper is devoted to technical description of a confocal microscope and Heidelberg Retina Tomograph with corneal module as well as the feasibility of corneal nerves visualization. Diabetic neuropathy, a threatening complication of DM that can result in lower limb amputations, is discussed. A number of authors suggest confocal biomicroscopy for early diagnosis of polyneuropathy, yet few relevant publications can be found. If effective, confocal biomicroscopy can be considered as a possible screening tool able to detect early signs of diabetes complications and thus to ensure the treatment initiated in a timely manner. The latter is crucial to prevent DM progression to its terminal stage--diabetic polyneuropathy, which is dangerous of lower limb amputations. PMID:25872394

  15. 3D Laser Scanning in Technology Education.

    ERIC Educational Resources Information Center

    Flowers, Jim

    2000-01-01

    A three-dimensional laser scanner can be used as a tool for design and problem solving in technology education. A hands-on experience can enhance learning by captivating students' interest and empowering them with creative tools. (Author/JOW)

  16. Benchmarking Mobile Laser Scanning Systems Using a Permanent Test Field

    NASA Astrophysics Data System (ADS)

    Kaartinen, H.; Kukko, A.; Hyyppä, J.; Jaakkola, A.

    2012-07-01

    The objective of the study was to benchmark the geometric accuracy of mobile laser scanning (MLS) systems using a permanent test field under good coverage of GNSS. Mobile laser scanning, also called mobile terrestrial laser scanning, is currently a rapidly developing area in laser scanning where laser scanners, GNSS and IMU are mounted onboard a moving vehicle. MLS can be considered to fill the gap between airborne and terrestrial laser scanning. Data provided by MLS systems can be characterized with the following technical parameters: a) point density in the range of 100-1000 points per m2 at 10 m distance, b) distance measurement accuracy of 2-5 cm, and c) operational scanning range from 1 to 100 m. Several commercial, including e.g. Riegl, Optech and others, and some research mobile laser scanning systems surveyed the test field using predefined driving speed and directions. The acquired georeferenced point clouds were delivered for analyzing. The geometric accuracy of the point clouds was determined using the reference targets that could be identified and measured from the point cloud. Results show that in good GNSS conditions most systems can reach an accuracy of 2 cm both in plane and elevation. The accuracy of a low cost system, the price of which is less than tenth of the other systems, seems to be within a few centimetres at least in ground elevation determination. Inaccuracies in the relative orientation of the instruments lead to systematic errors and when several scanners are used, in multiple reproductions of the objects. Mobile laser scanning systems can collect high density point cloud data with high accuracy. A permanent test field suits well for verifying and comparing the performance of different mobile laser scanning systems. The accuracy of the relative orientation between the mapping instruments needs more attention. For example, if the object is seen double in the point cloud due to imperfect boresight calibration between two scanners, this

  17. Laser beam scanning by rotary mirrors. I. Modeling mirror-scanning devices.

    PubMed

    Li, Y; Katz, J

    1995-10-01

    Avector approach to tracing the path of a laser beam through an optical system containing movable plane mirrors is described, which permits a unified treatment of a number of basic mirror-scanning devices. We show that the scan field produced by the mirror-scanning system is a curved surface with a straight line as its generating element. The cross section of the scan field can be a circle, an ellipse, or a curve in the shape of an egg. Based on this understanding, some advanced topics are addressed, e.g., the relationship between the scan field and the scan pattern, the dependence of the scan pattern on the location and orientation of the observation surface, optical distortions in a scan pattern, spot-size enlargement caused by non-normal incidence of the scan beam on the observation plane, and so on. Design equations and curves are derived for the mirror-scanning devices that most frequently exist in linear and circular scan technology. Part II contains an analysis of the galvanometer-based optical scanner paddle scanner and the regular polygon. In Part III, X-Y scanning systems are studied. PMID:21060488

  18. Repeat scanning technology for laser ultrasonic propagation imaging

    NASA Astrophysics Data System (ADS)

    Lee, Jung-Ryul; Yenn Chong, See; Sunuwar, Nitam; Park, Chan Yik

    2013-08-01

    Laser ultrasonic scanning in combination with contact or non-contact sensors provides new paradigms in structural health management (SHM) and non-destructive in-process quality control (IPQC) for large composite structures. Wave propagation imaging technology based on laser ultrasonic scanning and fixed-point sensing shows remarkable advantages, such as minimal need for embedded sensors in SHM, minimum invasive defect visualization in IPQC and general capabilities of curved and complex target inspection, and temporal reference-free inspection. However, as with other SHM methods and non-destructive evaluation based on ultrasound, the signal-to-noise ratio (SNR) is a prevalent issue in real structural applications, especially with non-contact thin-composite sensing or with thick and heterogeneous composites. This study proposes a high-speed repeat scanning technique for laser ultrasonic propagation imaging (UPI) technology, which is realized with the scanning speed of 1 kHz of a Q-switched continuous wave laser, and precise control of the laser beam pulses for identical point scanning. As a result, the technique enables the achievement of significant improvement in the SNR to inspect real-world composite structures. The proposed technique provides enhanced results for impact damage detection in a 2 mm thick wing box made of carbon-fiber-reinforced plastic, despite the low sensitivity of non-contact laser ultrasonic sensing. A field-applicable pure laser UPI system has been developed using a laser Doppler vibrometer as the non-contact ultrasonic sensor. The proposed technique enables the visualization of the disbond defect in a 15 mm thick wind blade specimen made of glass-fiber-reinforced plastic, despite the high dissipation of ultrasound in the thick composite.

  19. In vivo quantification of microglia dynamics with a scanning laser ophthalmoscope in a mouse model of focal laser injury

    NASA Astrophysics Data System (ADS)

    Alt, Clemens; Lin, Charles P.

    2012-03-01

    Microglia are the resident immune cells of the central nervous system and play a crucial role in maintaining neuronal health and function. Their dynamic behavior, that is, the constant extension and retraction of microglia processes, is thought to be critical for communication between microglia and their cellular neighbors, such as neurons, astrocytes and vascular endothelial cells. Here, we investigated the morphology and dynamics of retinal microglia in vivo under normal conditions and in response to focal laser injury of blood vessel endothelial wall, using a scanning laser ophthalmoscope (SLO) designed specifically for imaging the retina of live mice. The multichannel confocal imaging system allows retinal microstructure, such as the processes of microglia and retinal vasculature, to be visualized simultaneously. In order to generate focal laser injury, a photocoagulator based on a continuous wave (cw) laser was coupled into the SLO. An acousto-optic modulator chopped pulses from the cw laser. A tip-tilt-scanner was used to direct the laser beam into a blood vessel of interest under SLO image guidance. Mild coagulation was produced using millisecond-long pulses. Microglia react dynamically to focal laser injury of blood vessel endothelial walls. Under normal conditions, microglia somas remain stationary and the processes probe a territory of their immediate environment. In response to local injury, process movement velocity approximately doubles within minutes after injury. Moreover, the previously unpolarized process movement assumes a distinct directionality towards the injury site, indicating signaling between the injured tissue and the microglia. In vivo retinal imaging is a powerful tool for understanding the dynamic behavior of retinal cells.

  20. Virtual pinhole confocal microscope

    SciTech Connect

    George, J.S.; Rector, D.M.; Ranken, D.M.; Peterson, B.; Kesteron, J.

    1999-06-01

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

  1. Analysis of the melanin distribution in different ethnic groups by in vivo laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Antoniou, C.; Lademann, J.; Richter, H.; Astner, S.; Patzelt, A.; Zastrow, L.; Sterry, W.; Koch, S.

    2009-05-01

    The aim of this study was to determine whether Laser scanning confocal microscopy (LSM) is able to visualize differences in melanin content and distribution in different Skin Phototypes. The investigations were carried out on six healthy volunteers with Skin Phototypes II, IV, and VI. Representative skin samples of Skin Phototypes II, V, and VI were obtained for histological analysis from remaining tissue of skin grafts and were used for LSM-pathologic correlation. LSM evaluation showed significant differences in melanin distribution in Skin Phototypes II, IV, and VI, respectively. Based on the differences in overall reflectivity and image brightness, a visual evaluation scheme showed increasing brightness of the basal and suprabasal layers with increasing Skin Phototypes. The findings correlated well with histological analysis. The results demonstrate that LSM may serve as a promising adjunctive tool for real time assessment of melanin content and distribution in human skin, with numerous clinical applications and therapeutic and preventive implications.

  2. Designing a laser scanning picoprojector. Part 2: laser-safety-related issues.

    PubMed

    Wallhead, Ian; Ocaña, Roberto; Quinzá, Paula

    2012-08-10

    Laser scanning picoprojectors present a new challenge in the field of laser safety with methods of calculating accessible emission limits still in their infancy. We present a laser safety analysis and a calculation of an example picoprojector. We show that, due to its scanning operation, a picoprojector should be considered an extended laser source, and we also show that a picoprojector with two separate one-axis microelectromechanical systems mirrors offers a higher safe power limit than a projector with a single scanning mirror. Finally, a safety analysis is done under conditions of mirror failure. We show that, if the projector fails to scan in just one of the axes, the ocular hazard rises sharply, highlighting the need for a fail-safe system to be built into laser scanning picoprojectors. PMID:22885573

  3. In situ laser processing in a scanning electron microscope

    SciTech Connect

    Roberts, Nicholas A.; Magel, Gregory A.; Hartfield, Cheryl D.; Moore, Thomas M.; Fowlkes, Jason D.; Rack, Philip D.

    2012-07-15

    Laser delivery probes using multimode fiber optic delivery and bulk focusing optics have been constructed and used for performing materials processing experiments within scanning electron microscope/focused ion beam instruments. Controlling the current driving a 915-nm semiconductor diode laser module enables continuous or pulsed operation down to sub-microsecond durations, and with spot sizes on the order of 50 {mu}m diameter, achieving irradiances at a sample surface exceeding 1 MW/cm{sup 2}. Localized laser heating has been used to demonstrate laser chemical vapor deposition of Pt, surface melting of silicon, enhanced purity, and resistivity via laser annealing of Au deposits formed by electron beam induced deposition, and in situ secondary electron imaging of laser induced dewetting of Au metal films on SiO{sub x}.

  4. In situ laser processing in a scanning electron microscope

    SciTech Connect

    Roberts, Nicholas; Fowlkes, Jason Davidson; Rack, Prof. Philip; Moore, Tom; Magel, Greg; Hartfield, Cheryl

    2012-01-01

    Laser delivery probes using multimode fiber optic delivery and bulk focusing optics have been constructed and used for performing materials processing experiments within scanning electron microscope/focused ion beam instruments. Controlling the current driving a 915-nm semiconductor diode laser module enables continuous or pulsed operation down to sub-microsecond durations, and with spot sizes on the order of 50 {micro}m diameter, achieving irradiances at a sample surface exceeding 1 MW/cm{sup 2}. Localized laser heating has been used to demonstrate laser chemical vapor deposition of Pt, surface melting of silicon, enhanced purity, and resistivity via laser annealing of Au deposits formed by electron beam induced deposition, and in situ secondary electron imaging of laser induced dewetting of Au metal films on SiO{sub x}.

  5. Scanning Laser Infrared Molecular Spectrometer (SLIMS)

    NASA Technical Reports Server (NTRS)

    Scott, David C.; Rickey, Kelly; Ksendzov, Alexander; George, Warren P.; Aljabri, Abdullah S.; Steinkraus, Joel M.

    2012-01-01

    This prototype innovation is a novel design that achieves very long, effective laser path lengths that are able to yield ppb (parts per billion) and sub-ppb measurements of trace gases. SLIMS can also accommodate multiple laser channels covering a wide range of wavelengths, resulting in detection of more chemicals of interest. The mechanical design of the mirror cell allows for the large effective path length within a small footprint. The same design provides a robust structure that lends itself to being immune to some of the alignment challenges that similar cells face. By taking a hollow cylinder and by cutting an elliptically or spherically curved surface into its inner wall, the basic geometry of a reflecting ring is created. If the curved, inner surface is diamond-turned and highly polished, a surface that is very highly reflective can be formed. The surface finish can be further improved by adding a thin chrome or gold film over the surface. This creates a high-quality, curved, mirrored surface. A laser beam, which can be injected from a small bore hole in the wall of the cylinder, will be able to make many low-loss bounces around the ring, creating a large optical path length. The reflecting ring operates on the same principle as the Herriott cell. The difference exists in the mirror that doesn't have to be optically aligned, and which has a relatively large, internal surface area that lends itself to either open air or evacuated spectroscopic measurements. This solid, spherical ring mirror removes the possibility of mirror misalignment caused by thermal expansion or vibrations, because there is only a single, solid reflecting surface. Benefits of the reflecting ring come into play when size constraints reduce the size of the system, especially for space missions in which mass is at a premium.

  6. Defect Imaging Technique Using a Scanning Laser Source

    NASA Astrophysics Data System (ADS)

    Hayashi, T.; Murase, M.; Kitayama, T.

    2011-06-01

    Considering the applications to in-line testing of products, water-free imaging technique is required. This study described defect imaging technique using a scanning laser source. Laser beam was emitted onto a surface of a plate, and then guided waves excited at the laser source were detected by ultrasonic transducers fixed on the plate surface. For a plate with rounded defects, amplitude distributions obtained by scanning the laser source corresponded to thickness distributions, but for plates with rectangular notches, unwanted artifacts were seen due to reflection, diffraction, and ultrasonic attenuation. Then, using multiple receiving transducers and synthesizing multiple images, distinct defect images were obtained for a flat plate and a curved plate with rectangular notches.

  7. Velocity gradients in spatially resolved laser Doppler flowmetry and dynamic light scattering with confocal and coherence gating

    NASA Astrophysics Data System (ADS)

    Uribe-Patarroyo, Néstor; Bouma, Brett E.

    2016-08-01

    Dynamic light scattering (DLS) is widely used to characterize diffusive motion to obtain precise information on colloidal suspensions by calculating the autocorrelation function of the signal from a heterodyne optical system. DLS can also be used to determine the flow velocity field in systems that exhibit mass transport by incorporating the effects of the deterministic motion of scatterers on the autocorrelation function, a technique commonly known as laser Doppler flowmetry. DLS measurements can be localized with confocal and coherence gating techniques such as confocal microscopy and optical coherence tomography, thereby enabling the determination of the spatially resolved velocity field in three dimensions. It has been thought that spatially resolved DLS can determine the axial velocity as well as the lateral speed in a single measurement. We demonstrate, however, that gradients in the axial velocity of scatterers exert a fundamental influence on the autocorrelation function even in well-behaved, nonturbulent flow. By obtaining the explicit functional relation between axial-velocity gradients and the autocorrelation function, we show that the velocity field and its derivatives are intimately related and their contributions cannot be separated. Therefore, a single DLS measurement cannot univocally determine the velocity field. Our extended theoretical model was found to be in good agreement with experimental measurements.

  8. Velocity gradients in spatially resolved laser Doppler flowmetry and dynamic light scattering with confocal and coherence gating.

    PubMed

    Uribe-Patarroyo, Néstor; Bouma, Brett E

    2016-08-01

    Dynamic light scattering (DLS) is widely used to characterize diffusive motion to obtain precise information on colloidal suspensions by calculating the autocorrelation function of the signal from a heterodyne optical system. DLS can also be used to determine the flow velocity field in systems that exhibit mass transport by incorporating the effects of the deterministic motion of scatterers on the autocorrelation function, a technique commonly known as laser Doppler flowmetry. DLS measurements can be localized with confocal and coherence gating techniques such as confocal microscopy and optical coherence tomography, thereby enabling the determination of the spatially resolved velocity field in three dimensions. It has been thought that spatially resolved DLS can determine the axial velocity as well as the lateral speed in a single measurement. We demonstrate, however, that gradients in the axial velocity of scatterers exert a fundamental influence on the autocorrelation function even in well-behaved, nonturbulent flow. By obtaining the explicit functional relation between axial-velocity gradients and the autocorrelation function, we show that the velocity field and its derivatives are intimately related and their contributions cannot be separated. Therefore, a single DLS measurement cannot univocally determine the velocity field. Our extended theoretical model was found to be in good agreement with experimental measurements. PMID:27627357

  9. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

    The confocal laser-scanning microscopy (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of the emitted signal. The accuracy of these measurements demands t...

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

    EPA Science Inventory

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

  11. Tuning and scanning control system for high resolution alexandrite lasers

    NASA Technical Reports Server (NTRS)

    Smith, James C.; Schwemmer, Geary K.

    1988-01-01

    An alexandrite laser is spectrally narrowed and tuned by the use of three optical elements. Each element provides a successively higher degree of spectral resolution. The digitally controlled tuning and scanning control servo system simultaneously positions all three optical elements to provide continuous high resolution laser spectral tuning. The user may select manual, single, or continuous modes of automated scanning of ranges up to 3.00/cm and at scan rates up to 3.85/cm/min. Scanning over an extended range of up to 9.999/cm may be achieved if the highest resolution optic is removed from the system. The control system is also capable of being remotely operated by another computer or controller via standard RS-232 serial data link.

  12. Optimal lens design and use in laser-scanning microscopy

    PubMed Central

    Negrean, Adrian; Mansvelder, Huibert D.

    2014-01-01

    In laser-scanning microscopy often an off-the-shelf achromatic doublet is used as a scan lens which can reduce the available diffraction-limited field-of-view (FOV) by a factor of 3 and introduce chromatic aberrations that are scan angle dependent. Here we present several simple lens designs of superior quality that fully make use of high-NA low-magnification objectives, offering diffraction-limited imaging over a large FOV and wavelength range. We constructed a two-photon laser-scanning microscope with optimized custom lenses which had a near diffraction limit point-spread-function (PSF) with less than 3.6% variation over a 400 µm FOV and less than 0.5 µm lateral color between 750 and 1050 nm. PMID:24877017

  13. Application of in vivo laser scanning microscope in dermatology

    NASA Astrophysics Data System (ADS)

    Lademann, Juergen; Richter, H.; Otberg, N.; Lawrenz, F.; Blume-Peytavi, U.; Sterry, W.

    2003-10-01

    The state of the art of in-vivo and in-vitro penetration measurements of topically applied substances is described. Only optical techniques represent online measuring methods based on the absorption or scattering properties of the topically applied substances. Laser scanning microscopy (LSM) has become a promising method for investigations in dermatology and skin physiology, after it was possible to analyze the skin surface on any body side in-vivo. In the present paper the application of a dermatological laser scanning microscope for penetration and distribution measurements of topically applied substances is described. The intercellular and follicular penetration pathways were studied.

  14. Material characterization with a simple laser scanning microscope.

    PubMed

    Krug, R; Würfel, P; Ruppel, W

    1993-11-10

    The design of a computer-controlled laser scanning microscope is described. It is capable of inspecting a 1 mm × 1 mm area in less than 1 s with an optical resolution of 2 µm. Three applications of the laser scanning microscope are presented: the observation of the ferroelectric-domain structure of sodium nitrite layers, the observation of the spatial distribution of the photocurrent in polycrystalline solar cells, and the observation of the lateral distribution of thermoelectric currents in a thermal IR detector for the determination of the thermal properties of its absorber foil.

    PACS: 0760P, 7240, 7780D.

    PMID:20856484

  15. Street-Scene Tree Segmentation from Mobile Laser Scanning Data

    NASA Astrophysics Data System (ADS)

    Guan, H.; Cao, S.; Yu, Y.; Li, J.; Liu, N.; Chen, P.; Li, Y.

    2016-06-01

    Our work addresses the problem of extracting trees from mobile laser scanning data. The work is a two step-wise strategy, including terrain point removal and tree segmentation. First, a voxel-based upward growing filtering is proposed to remove terrain points from the mobile laser scanning data. Then, a tree segmentation is presented to extract individual trees via a Euclidean distance clustering approach and Voxel-based Normalized Cut (VNCut) segmentation approach. A road section data acquired by a RIEGL VMX-450 system are selected for evaluating the proposed tree segmentation method. Qualitative analysis shows that our algorithm achieves a good performance.

  16. Planar Projection of Mobile Laser Scanning Data in Tunnels

    NASA Astrophysics Data System (ADS)

    Gonçalves, J. A.; Mendes, R.; Araújo, E.; Oliveira, A.; Boavida, J.

    2012-07-01

    Laser scanning is now a common technology in the surveying and monitoring of large engineering infrastructures, such as tunnels, both in motorways and railways. Extended possibilities exist now with the mobile terrestrial laser scanning systems, which produce very large data sets that need efficient processing techniques in order to facilitate their exploitation and usability. This paper deals with the implementation of a methodology for processing and presenting 3D point clouds acquired by laser scanning in tunnels, making use of the approximately cylindrical shape of tunnels. There is a need for a 2D presentation of the 3D point clouds, in order to facilitate the inspection of important features as well as to easily obtain their spatial location. An algorithm was developed to treat automatically point clouds obtained in tunnels in order to produce rectified images that can be analysed. Tests were carried with data acquired with static and mobile Riegl laser scanning systems, by Artescan company, in highway tunnels in Portugal and Spain, with very satisfactory results. The final planar image is an alternative way of data presentation where image analysis tools can be used to analyze the laser intensity in order to detect problems in the tunnel structure.

  17. Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

    PubMed Central

    Amor, Rumelo; Mahajan, Sumeet; Amos, William Bradshaw; McConnell, Gail

    2014-01-01

    Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report here a variation in the intensity of fluorescence of successive planes related to the Stokes shift of the dye. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ≈90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria. PMID:25483987

  18. Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

    NASA Astrophysics Data System (ADS)

    Amor, Rumelo; Mahajan, Sumeet; Amos, William Bradshaw; McConnell, Gail

    2014-12-01

    Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report here a variation in the intensity of fluorescence of successive planes related to the Stokes shift of the dye. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ~90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria.

  19. Micromixing visualization and quantification in a microscale multi-inlet vortex nanoprecipitation reactor using confocal-based reactive micro laser-induced fluorescence

    PubMed Central

    Shi, Yanxiang; Fox, Rodney O.; Olsen, Michael G.

    2014-01-01

    A technique for visualizing and quantifying reactive mixing for laminar and turbulent flow in a microscale chemical reactor using confocal-based microscopic laser induced fluorescence (confocal μ-LIF) was demonstrated in a microscale multi-inlet vortex nanoprecipitation reactor. Unlike passive scalar μ-LIF, the reactive μ-LIF technique is able to visualize and quantify micromixing effects. The confocal imaging results indicated that the flow in the reactor was laminar and steady for inlet Reynolds numbers of 10, 53, and 93. Mixing and reaction were incomplete at each of these Reynolds numbers. The results also suggested that although mixing by diffusion was enhanced near the midplane of the reactor at Rej = 53 and 93 due to very thin bands of acidic and basic fluid forming as the fluid spiraled towards the center of the reactor, near the top, and bottom walls of the reactor, the lower velocities due to fluid friction with the walls hindered the formation of these thin bands, and, thus, resulted in large regions of unmixed and unreacted fluid. At Rej = 240, the flow was turbulent and unsteady. The mixing and reaction processes were still found to be incomplete even at this highest Reynolds number. At the reactor midplane, the flow images at Rej = 240 showed unmixed base fluid near the center of the reactor, suggesting that just as in the Rej = 53 and 93 cases, lower velocities near the top and bottom walls of the reactor hinder the mixing and rection of the acidic and basic streams. Ensemble averages of line-scan profiles for the Rej = 240 were then calculated to provide statistical quantification of the microscale mixing in the reactor. These results further demonstrate that even at this highest Reynolds number investigated, mixing and reaction are incomplete. Visualization and quantification of micromixing using this reactive μ-LIF technique can prove useful in the validation of computational fluid dynamics models of micromixing within

  20. A novel approach to pseudopodia proteomics: excimer laser etching, two-dimensional difference gel electrophoresis, and confocal imaging

    PubMed Central

    Mimae, Takahiro; Ito, Akihiko; Hagiyama, Man; Nakanishi, Jun; Hosokawa, Yoichiroh; Okada, Morihito; Murakami, Yoshinori; Kondo, Tadashi

    2014-01-01

    Pseudopodia are actin-rich ventral cellular protrusions shown to facilitate the migration and metastasis of tumor cells. Here, we present a novel approach to perform pseudopodia proteomics. Tumor cells growing on porous membranes extend pseudopodia into the membrane pores. In our method, cell bodies are removed by horizontal ablation at the basal cell surface with the excimer laser while pseudopodia are left in the membrane pores. For protein expression profiling, whole cell and pseudopodia proteins are extracted with a lysis buffer, labeled with highly sensitive fluorescent dyes, and separated by two-dimensional gel electrophoresis. Proteins with unique expression patterns in pseudopodia are identified by mass spectrometry. The effects of the identified proteins on pseudopodia formation are evaluated by measuring the pseudopodia length in cancer cells with genetically modified expression of target proteins using confocal imaging. This protocol allows global identification of pseudopodia proteins and evaluation of their functional significance in pseudopodia formation within one month. PMID:25309719

  1. Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

    SciTech Connect

    Darvin, M E; Richter, H; Zhu, Y J; Meinke, M C; Knorr, F; Lademann, J; Gonchukov, S A; Koenig, K

    2014-07-31

    Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted. (laser biophotonics)

  2. Detection of fluorescent organic nanoparticles by confocal laser endomicroscopy in a rat model of Barrett’s esophageal adenocarcinoma

    PubMed Central

    Dassie, Elisa; Arcidiacono, Diletta; Wasiak, Iga; Damiano, Nunzio; Dall’Olmo, Luigi; Giacometti, Cinzia; Facchin, Sonia; Cassaro, Mauro; Guido, Ennio; De Lazzari, Franca; Marin, Oriano; Ciach, Tomasz; Fery-Forgues, Suzanne; Alberti, Alfredo; Battaglia, Giorgio; Realdon, Stefano

    2015-01-01

    For many years, novel strategies for cancer detection and treatment using nanoparticles (NPs) have been developed. Esophageal adenocarcinoma is the sixth leading cause of cancer-related deaths in Western countries, and despite recent advances in early detection and treatment, its prognosis is still very poor. This study investigated the use of fluorescent organic NPs as potential diagnostic tool in an experimental in vivo model of Barrett’s esophageal adenocarcinoma. NPs were made of modified polysaccharides loaded with [4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran] (DCM), a well-known fluorescent dye. The NP periphery might or might not be decorated with ASYNYDA peptide that has an affinity for esophageal cancer cells. Non-operated and operated rats in which gastroesophageal reflux was surgically induced received both types of NPs (NP-DCM and NP-DCM-ASYNYDA) by intravenous route. Localization of mucosal NPs was assessed in vivo by confocal laser endomicroscopy, a technique which enables a “real time” and in situ visualization of the tissue at a cellular level. After injection of NP-DCM and NP-DCM-ASYNYDA, fluorescence was observed in rats affected by esophageal cancer, whereas no signal was observed in control non-operated rats, or in rats with simple esophagitis or Barrett’s esophagus mucosa. Fluorescence was observable in vivo 30 minutes after the administration of NPs. Interestingly, NP-DCM-ASYNYDA induced strong fluorescence intensity 24 hours after administration. These observations suggested that NPs could reach the tumor cells, likely by enhanced permeability and retention effect, and the peptide ASYNYDA gave them high specificity for esophageal cancer cells. Thus, the combination of NP platform and confocal laser endomicroscopy could play an important role for highlighting esophageal cancer conditions. This result supports the potential of this strategy as a targeted carrier for photoactive and bioactive molecules in esophageal

  3. Detection of fluorescent organic nanoparticles by confocal laser endomicroscopy in a rat model of Barrett's esophageal adenocarcinoma.

    PubMed

    Dassie, Elisa; Arcidiacono, Diletta; Wasiak, Iga; Damiano, Nunzio; Dall'Olmo, Luigi; Giacometti, Cinzia; Facchin, Sonia; Cassaro, Mauro; Guido, Ennio; De Lazzari, Franca; Marin, Oriano; Ciach, Tomasz; Fery-Forgues, Suzanne; Alberti, Alfredo; Battaglia, Giorgio; Realdon, Stefano

    2015-01-01

    For many years, novel strategies for cancer detection and treatment using nanoparticles (NPs) have been developed. Esophageal adenocarcinoma is the sixth leading cause of cancer-related deaths in Western countries, and despite recent advances in early detection and treatment, its prognosis is still very poor. This study investigated the use of fluorescent organic NPs as potential diagnostic tool in an experimental in vivo model of Barrett's esophageal adenocarcinoma. NPs were made of modified polysaccharides loaded with [4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran] (DCM), a well-known fluorescent dye. The NP periphery might or might not be decorated with ASYNYDA peptide that has an affinity for esophageal cancer cells. Non-operated and operated rats in which gastroesophageal reflux was surgically induced received both types of NPs (NP-DCM and NP-DCM-ASYNYDA) by intravenous route. Localization of mucosal NPs was assessed in vivo by confocal laser endomicroscopy, a technique which enables a "real time" and in situ visualization of the tissue at a cellular level. After injection of NP-DCM and NP-DCM-ASYNYDA, fluorescence was observed in rats affected by esophageal cancer, whereas no signal was observed in control non-operated rats, or in rats with simple esophagitis or Barrett's esophagus mucosa. Fluorescence was observable in vivo 30 minutes after the administration of NPs. Interestingly, NP-DCM-ASYNYDA induced strong fluorescence intensity 24 hours after administration. These observations suggested that NPs could reach the tumor cells, likely by enhanced permeability and retention effect, and the peptide ASYNYDA gave them high specificity for esophageal cancer cells. Thus, the combination of NP platform and confocal laser endomicroscopy could play an important role for highlighting esophageal cancer conditions. This result supports the potential of this strategy as a targeted carrier for photoactive and bioactive molecules in esophageal cancer

  4. Photodynamic therapy with laser scanning mode of tumor irradiation

    NASA Astrophysics Data System (ADS)

    Chepurna, Oksana; Shton, Irina; Kholin, Vladimir; Voytsehovich, Valerii; Popov, Viacheslav; Pavlov, Sergii; Gamaleia, Nikolai; Wójcik, Waldemar; Zhassandykyzy, Maral

    2015-12-01

    In this study we propose a new version of photodynamic therapy performed by laser scanning. The method consists in tumor treatment by a light beam of a small cross section which incrementally moves through the chosen area with a defined delay at each point and repetitively re-scans a zone starting from the initial position. Experimental evaluation of the method in vitro on murine tumor model showed that despite the dose, applied by scanning irradiation mode, was 400 times lower, the tumor inhibition rate conceded to attained with continuous irradiation mode by only 20%.

  5. Visualisation of urban airborne laser scanning data with occlusion images

    NASA Astrophysics Data System (ADS)

    Hinks, Tommy; Carr, Hamish; Gharibi, Hamid; Laefer, Debra F.

    2015-06-01

    Airborne Laser Scanning (ALS) was introduced to provide rapid, high resolution scans of landforms for computational processing. More recently, ALS has been adapted for scanning urban areas. The greater complexity of urban scenes necessitates the development of novel methods to exploit urban ALS to best advantage. This paper presents occlusion images: a novel technique that exploits the geometric complexity of the urban environment to improve visualisation of small details for better feature recognition. The algorithm is based on an inversion of traditional occlusion techniques.

  6. A diffraction-limited scanning system providing broad spectral range for laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Yu, Jiun-Yann; Liao, Chien-Sheng; Zhuo, Zong-Yan; Huang, Chen-Han; Chui, Hsiang-Chen; Chu, Shi-Wei

    2009-11-01

    Diversified research interests in scanning laser microscopy nowadays require broadband capability of the optical system. Although an all-mirror-based optical design with a suitable metallic coating is appropriate for broad-spectrum applications from ultraviolet to terahertz, most researchers prefer lens-based scanning systems despite the drawbacks of a limited spectral range, ghost reflection, and chromatic aberration. One of the main concerns is that the geometrical aberration induced by off-axis incidence on spherical mirrors significantly deteriorates image resolution. Here, we demonstrate a novel geometrical design of a spherical-mirror-based scanning system in which off-axis aberrations, both astigmatism and coma, are compensated to reach diffraction-limited performance. We have numerically simulated and experimentally verified that this scanning system meets the Marechà l condition and provides high Strehl ratio within a 3°×3° scanning area. Moreover, we demonstrate second-harmonic-generation imaging from starch with our new design. A greatly improved resolution compared to the conventional mirror-based system is confirmed. This scanning system will be ideal for high-resolution linear/nonlinear laser scanning microscopy, ophthalmoscopic applications, and precision fabrications.

  7. A first approach to the detection and equalization of distorted latent fingerprints and microtraces on non-planar surfaces with confocal laser microscopy

    NASA Astrophysics Data System (ADS)

    Kirst, Stefan; Clausing, Eric; Dittmann, Jana; Vielhauer, Claus

    2012-10-01

    Fingerprints and microtraces play an important role as evidence within the field of criminalistics. Their conservative acquisition processes, are established, but are altering and impurifying the traces often. In case of microtraces even the integrity of the trace complex is affected. Using contactless methods, the acquisition process becomes non-invasiv and repeatable, but might be distorting on the other hand, when non-planar substrates are in use. Detecting and dealing with distortion in contactless aquired scans of non-planar surfaces is a novel field of research. Nowadays highly distorted fingerprints can only be used, if the substrate can be manually distorted by destroying or deforming it. In this paper we suggest methods for detection and equalization of distortion for use in combination of types of traces. Therefore we define different types of distortion in fingerprints and microtraces. A standardization of types is necessary to develop different solution for equalization. For usage within the field of forensics, each method is evaluated via proper error rates and adaptively used to acquire fingerprints and microtraces. Using our techniques, we are able to detect distortion and equalize fingerprints to support the investigators work. In case of microtraces the presented methods can even be used to equalize mircotraces themselves for better determination of their scale and topology. For all scans the confocal 3D laser microscope "Keyence VK-X110" is used to gather color-, intensity- and topography information in 22 different measurement conditions within 6 different samples consisting of a total of 880 scans. Despite our achievements in the field of distortion detection and equalization there are still challenges, like the non-isometric projection, that need to be focused on. Also, the presented equalization methods may not completely remove any kind of distortion, such as added by deformation. Therefore we suggest and discuss future work for improving the

  8. Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

    PubMed Central

    Zhi, Yanan; Wang, Benquan; Yao, Xincheng

    2016-01-01

    Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact–free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina. PMID:27480461

  9. Confocal Laser Scanning Microscopy Evaluation of an Acellular Dermis Tissue Transplant (Epiflex®)

    PubMed Central

    Hohenberger, Peter

    2012-01-01

    The structure of a biological scaffold is a major determinant of its biological characteristics and its interaction with cells. An acellular dermis tissue transplant must undergo a series of processing steps, to remove cells and genetic material and provide the sterility required for surgical use. During manufacturing and sterilization the structure and composition of tissue transplants may change. The composition of the human cell-free dermis transplant Epiflex® was investigated with specific attention paid to its structure, matrix composition, cellular content and biomechanics. We demonstrated that after processing, the structure of Epiflex remains almost unchanged with an intact collagen network and extracellular matrix (ECM) protein composition providing natural cell interactions. Although the ready to use transplant does contain some cellular and DNA debris, the processing procedure results in a total destruction of cells and active DNA which is a requirement for an immunologically inert and biologically safe substrate. Its biomechanical parameters do not change significantly during the processing. PMID:23056225

  10. Sarcoglycans in human skeletal muscle and human cardiac muscle: a confocal laser scanning microscope study.

    PubMed

    Anastasi, G; Cutroneo, G; Trimarchi, F; Rizzo, G; Bramanti, P; Bruschetta, D; Fugazzotto, D; Cinelli, M P; Soscia, A; Santoro, G; Favaloro, A

    2003-01-01

    Sarcoglycans are a subcomplex of transmembrane proteins which are part of the dystrophin-glycoprotein complex. They are expressed in the skeletal, cardiac and smooth muscle. Although numerous studies have been conducted on the sarcoglycan subcomplex in skeletal and cardiac muscle, the manner of the distribution and localization of these proteins along the nonjunctional sarcolemma is not clear. We therefore carried out an indirect immunofluorescence study on surgical biopsies of normal human skeletal muscle and of healthy human atrial myocardium biopsies of patients affected by valvulopathy. Our results indicate that, in skeletal muscle, sarcoglycans have a costameric distribution and all colocalize with each other. Only in a few cases did the alpha-sarcoglycan not colocalize with other sarcoglycans. In addition, these glycoproteins can be localized in different fibers either in the regions of the sarcolemma over band I or band A. In cardiac muscle, our results show a costameric distribution of all proteins examined and, unlike in skeletal muscle, they show a constant colocalization of all sarcoglycans with each other, along with a consistent localization of these proteins in the region of the sarcolemma over band I. In our opinion, this situation seems to confirm the hypothesis of a correlation between the region of the sarcolemma occupied by costameric proteins and the metabolic type, fast or slow, of the muscular fibers. These data, besides opening a new line of research in understanding interactions between the sarcoglycans and other transmembrane proteins, could also be extended to skeletal and cardiac muscles affected by neuromuscular and cardiovascular pathologies to understand possible structural alterations. PMID:12566627

  11. CONFOCAL LASER SCANNING MICROSCOPY OF WHOLE MOUSE OVARIES: EXCELLENT MORPHOLOGY, APOPTOSIS DETECTION, AND SPECTROSCOPY

    EPA Science Inventory

    Background: Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole...

  12. DETERMINATION OF STRUCTURE OF AGGREGATES BY CONFOCAL SCANNING LASER MICROSCOPY. (R825513C022)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  13. Cellular scanning strategy for selective laser melting: Generating reliable, optimized scanning paths and processing parameters

    NASA Astrophysics Data System (ADS)

    Mohanty, Sankhya; Hattel, Jesper H.

    2015-03-01

    Selective laser melting is yet to become a standardized industrial manufacturing technique. The process continues to suffer from defects such as distortions, residual stresses, localized deformations and warpage caused primarily due to the localized heating, rapid cooling and high temperature gradients that occur during the process. While process monitoring and control of selective laser melting is an active area of research, establishing the reliability and robustness of the process still remains a challenge. In this paper, a methodology for generating reliable, optimized scanning paths and process parameters for selective laser melting of a standard sample is introduced. The processing of the sample is simulated by sequentially coupling a calibrated 3D pseudo-analytical thermal model with a 3D finite element mechanical model. The optimized processing parameters are subjected to a Monte Carlo method based uncertainty and reliability analysis. The reliability of the scanning paths are established using cumulative probability distribution functions for process output criteria such as sample density, thermal homogeneity, etc. A customized genetic algorithm is used along with the simulation model to generate optimized cellular scanning strategies and processing parameters, with an objective of reducing thermal asymmetries and mechanical deformations. The optimized scanning strategies are used for selective laser melting of the standard samples, and experimental and numerical results are compared.

  14. Facial recognition and laser surface scan: a pilot study.

    PubMed

    Lynnerup, Niels; Clausen, Maja-Lisa; Kristoffersen, Agnethe May; Steglich-Arnholm, Henrik

    2009-01-01

    Surface scanning of the face of a suspect is presented as a way to better match the facial features with those of a perpetrator from CCTV footage. We performed a simple pilot study where we obtained facial surface scans of volunteers and then in blind trials tried to match these scans with 2D photographs of the faces of the volunteers. Fifteen male volunteers were surface scanned using a Polhemus FastSCAN Cobra Handheld Laser Scanner. Three photographs were taken of each volunteer's face in full frontal, profile and from above at an angle of 45 degrees and also 45 degrees laterally. Via special software (MIMICS and Photoshop) the surface scans were matched with the photographs in blind trials. The matches were graded as: a good fit; possible fit; and no fit. All the surface scans and photos were matched correctly, although one surface scan could be matched with two angled photographs, meaning that the discriminatory value was 86.7%. We also tested the surface scanner in terms of reliability in establishing point measures on skulls, and compared with physical measurements performed by calipers. The variation was on average 1 mm for five cranial measures. We suggest how surface scanning might be applied in forensic facial identification. PMID:19507073

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

    PubMed Central

    MACRAE, K.; TRAVIS, C.; AMOR, R.; NORRIS, G.; WILSON, S.H.; OPPO, G.‐L.; MCCONNELL, G.

    2015-01-01

    Summary We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic‐scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light‐collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife‐edge method has several advantages over alternative knife‐edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited. PMID:25864964

  16. Airborne laser scanning for high-resolution mapping of Antarctica

    NASA Astrophysics Data System (ADS)

    Csatho, Bea; Schenk, Toni; Krabill, William; Wilson, Terry; Lyons, William; McKenzie, Garry; Hallam, Cheryl; Manizade, Serdar; Paulsen, Timothy

    In order to evaluate the potential of airborne laser scanning for topographic mapping in Antarctica and to establish calibration/validation sites for NASA's Ice, Cloud and land Elevation Satellite (ICESat) altimeter mission, NASA, the U.S. National Science Foundation (NSF), and the U.S. Geological Survey (USGS) joined forces to collect high-resolution airborne laser scanning data.In a two-week campaign during the 2001-2002 austral summer, NASA's Airborne Topographic Mapper (ATM) system was used to collect data over several sites in the McMurdo Sound area of Antarctica (Figure 1a). From the recorded signals, NASA computed laser points and The Ohio State University (OSU) completed the elaborate computation/verification of high-resolution Digital Elevation Models (DEMs) in 2003. This article reports about the DEM generation and some exemplary results from scientists using the geomorphologic information from the DEMs during the 2003-2004 field season.

  17. PARALLEL ION BEAM PROFILE SCAN USING LASER WIRE

    SciTech Connect

    Liu, Yun; Aleksandrov, Alexander V; Huang, Chunning; Long, Cary D; Dickson, Richard W

    2013-01-01

    We report on the world s first experiment of a parallel profile scan of the hydrogen ion (H-) beam using a laser wire system. The system was developed at the superconducting linac of the Spallation Neutron Source (SNS) accelerator complex. The laser wire profile scanner is based on a photo-detachment process and therefore can be conducted on an operational H- beam in a nonintrusive manner. The parallel profile scanning system makes it possible to simultaneously measure profiles of the 1-MW neutron production H- beam at 9 different locations of the linac by using a single light source. This paper describes the design, optical system and software platform development, and measurement results of the parallel profile scanning system.

  18. Non-Contact Measurement Using A Laser Scanning Probe

    NASA Astrophysics Data System (ADS)

    Modjarrad, Amir

    1989-03-01

    Traditional high accuracy touch-trigger probing can now be complemented by high speed, non-contact, profile scanning to give another "dimension" to the three-dimensional Co-ordinate Measuring Machines (CMMs). Some of the features of a specially developed laser scanning probe together with the trade-offs involved in the design of inspection systems that use triangulation are examined. Applications of such a laser probe on CMMs are numerous since high speed scanning allows inspection of many different components and surfaces. For example, car body panels, tyre moulds, aircraft wing skins, turbine blades, wax and clay models, plastics, etc. Other applications include in-process surveillance in manufacturing and food processing, robotics vision and many others. Some of these applications are discussed and practical examples, case studies and experimental results are given with particular reference to use on CMMs. In conclusion, future developments and market trends in high speed non-contact measurement are discussed.

  19. Simultaneous ion beam profile scan using a single laser source

    NASA Astrophysics Data System (ADS)

    Liu, Y.; Long, C.; Huang, C.; Dickson, R.; Aleksandrov, A.

    2013-01-01

    We report on the world’s first experiment of a simultaneous profile scan of the hydrogen ion (H-) beam using a laser wire system. The system was developed and brought to operational level of application at the superconducting linac of the Spallation Neutron Source accelerator complex. The laser wire profile scanner is based on a photodetachment process and therefore can be conducted on a 1-MW neutron production H- beam in a nonintrusive manner. The new simultaneous profile scanning system allows one to simultaneously measure profiles of the H- beam at nine different locations of the linac with high speed and accuracy, and therefore provides a unique tool for accelerator tuning and physics study. This paper describes the design, optical system and software platform developments, and measurement results of the simultaneous profile scanning system.

  20. Confocal endomicroscopy of the larynx

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

    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.

  1. ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

    PubMed Central

    Hng, Keng Imm; Dormann, Dirk

    2013-01-01

    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. PMID:24224017

  2. Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging

    NASA Astrophysics Data System (ADS)

    Mancuso, James J.; Larson, Adam M.; Wensel, Theodore G.; Saggau, Peter

    2009-05-01

    The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.

  3. Micro-scanning mirrors for high-power laser applications in laser surgery

    NASA Astrophysics Data System (ADS)

    Sandner, Thilo; Kimme, Simon; Grasshoff, Thomas; Todt, Ulrich; Graf, Alexander; Tulea, Cristian; Lenenbach, Achim; Schenk, Harald

    2014-03-01

    We present two novel micro scanning mirrors with large aperture and HR dielectric coatings suitable for high power laser applications in a miniaturized laser-surgical instrument for neurosurgery to cut skull tissue. An electrostatic driven 2D-raster scanning mirror with 5x7.1mm aperture is used for dynamic steering of a ps-laser beam of the laser cutting process. A second magnetic 2D-beam steering mirror enables a static beam correction of a hand guided laser instrument. Optimizations of a magnetic gimbal micro mirror with 6 mm x 8 mm mirror plate are presented; here static deflections of 3° were reached. Both MEMS devices were successfully tested with a high power ps-laser at 532nm up to 20W average laser power.

  4. Registration Procedures for Terrestrial Laser Scanning in Geomorphologic Studies

    NASA Astrophysics Data System (ADS)

    Collins, B. D.; Kayen, R.; Minasian, D.

    2006-12-01

    Terrestrial based laser scanning, from either vehicle or tripod mounts allows the collection of geomorphologic data at previously unprecedented detail and volume. However, despite the ease of collecting this data in many settings, post-processing datasets collected without laser-visible reflectors within individual scans can lead to difficulties in both registration and georeferencing procedures. We have been actively involved in gathering data sets from a number of different environments and have been developing various techniques to post-process the data using surface registration methods. These methods use the point cloud or model surface to find a best-fit of the three-dimensional terrain. Recently, we have collected laser scan data of levee breaches in New Orleans following Hurricane Katrina, a glacial cirque basin in the Canadian Rockies, a deep-seated landslide mass in Ventura County, California, rapidly evolving coastal bluffs in Central California, and sand bars and archeological sites in Grand Canyon National Park, Arizona. In each of these projects, setting up accurately surveyed reflectors was impractical due to the locations dynamic and fairly inaccessible setting. Robust surface registration procedures were therefore needed to provide accurate terrain models. We have used laser scanning results from these projects to assess the efficiency of the various post- processing methodologies for obtaining final registered and georeferenced point clouds and surface models. We compared registration results obtained both with and without accurate GPS coordinates for the laser scanner origin (Ventura and coastal landslides), use of a supporting total station unit (Grand Canyon), and collection of DGPS data on targets imaged in the LIDAR data after the scanning process (Katrina Levees). In many of these settings, the model fit improved by four times, from a root mean square error of 20 cm to 5cm when accurately surveyed coordinates were utilized for the laser scan

  5. In-situ investigation of thermal instabilities and solid state dewetting in polycrystalline platinum thin films via confocal laser microscopy

    SciTech Connect

    Jahangir, S.; Cheng, Xuan; Huang, H. H.; Nagarajan, V.; Ihlefeld, J.

    2014-10-28

    Solid state dewetting and the subsequent morphological changes for platinum thin films grown on zinc oxide (ZnO) buffered (001) silicon substrates (Pt/ZnO/SiO{sub 2}/(001)Si system) is investigated under vacuum conditions via a custom-designed confocal laser microscope coupled with a laser heating system. Live imaging of thin film dewetting under a range of heating and quenching vacuum ambients reveals events including hillock formation, hole formation, and hole growth that lead to formation of a network of Pt ligaments, break up of Pt ligaments to individual islands and subsequent Pt islands shape reformation, in chronological fashion. These findings are corroborated by ex-situ materials characterization and quantitative electron microscopy analysis. A secondary hole formation via blistering before film rupture is revealed to be the critical stage, after which a rapid dewetting catastrophe occurs. This process is instantaneous and cannot be captured by ex-situ methods. Finally, an intermetallic phase forms at 900 °C and alters the morphology of Pt islands, suggesting a practical limit to the thermal environments that may be used for these platinized silicon wafers in vacuum conditions.

  6. Skeletal remodeling dynamics: New approaches with imaging instrumentation. [Laser confocal microscopy:a2

    SciTech Connect

    Parks, N.J.; Pinkerton, K.E.; Seibert, J.A.; Pool, R.R.

    1991-01-01

    This report of progress and future objectives timetable is based on an included schematic of goals and objectives and the project abstract which is included as Appendix 1. Five matters are summarized in the order of (1) novel methods of calcified bone confocal microscopy and reconstruction image analysis of decalcified beagle and human cortical bone serial sections, (2) macroscopic cross-correlation of beagle and human cortical and cancellous bone fractions with CT analysis, (3) guidance to the most radiobiologically important skeletal regions of interest with the just completed {sup 90}Sr bone tumor map from life time beagle studies, (4) deposition patterns of radioactive agents that participate in apatite crystal nucleation processes in bone and leave radiation-excited electrons trapped in bone mineral, and (5) the budget period timetable. The discovery that beta particles from {sup 166}Ho (T{sub {1/2}} =26 hr, {beta}{sub max} = 1.8 MeV) phosphonic acid bone agents leave detectable, long-lived, electron paramagnetic resonance signals in bone is included in Appendix 2 as a joint report.

  7. 3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  8. Automated house internal geometric quality inspection using laser scanning

    NASA Astrophysics Data System (ADS)

    Wang, Yuchen; Zhang, Zhichao; Qiu, Zhouyan

    2015-12-01

    Taking a terrestrial laser scanner to scan the room of a house, the scanned data can be used to inspect the geometric quality of the room. Taking advantage of the scan line feature, we can quickly calculate normal of the scanned points. Afterwards, we develop a fast plane segmentation approach to recognize the walls of the room according to the semantic constraints of a common room. With geometric and semantic constraints, we can exclude points that don't belong to the inspecting room. With the segmented results, we can accurately do global search of max and min height, width and length of a room, and the flatness of the wall as well. Experiment shows the robustness of this geometric inspecting approach. This approach has the ability to measure some important indicators that cannot be done by manual work.

  9. Image inpainting for the differential confocal microscope

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  10. Segment based shape matching in terrestrial laser scanning point clouds

    NASA Astrophysics Data System (ADS)

    Bremer, Magnus; Rutzinger, Martin; Wichmann, Volker

    2013-04-01

    Change detection of dynamic surface elements is an important application in geomorphological analysis. In order to be able to investigate such changes, the high spatial resolution and accuracy of the laser scanning technology is exploited. Dealing with laser scanning data, most change detection approaches are aiming at the assessment of volumetric changes due to erosion and deposition by geomorphologic processes. In these cases the areas of erosion and deposition are spatially separated and can be investigated in a cut-and-fill analysis. Where slow changes are controlled by interior deformation of material mixtures due to gravity, surface changes are mostly due to slight movements of objects and not to absolute material losses and gains. In complex terrain an object-based approach for the reconstruction of 3D change vectors is required. Depending on the level of scale, terrain can be subdivided into a large number of small planar patches. Using 3D point cloud data from terrestrial laser scanning, this can be done by a planar segmentation procedure grouping laser points of flat surfaces. Rotating each point cloud segment into its best fit plane, its 2D footprint shows specific local surface characteristics. Thus, each surface patch has a unique fingerprint that can be described by a variety of segment features. In an experimental framework we test the capability of shape based matching for the derivation of change vectors on dynamic surfaces. To consider different data characteristics such as varying point densities and scan perspectives, terrestrial laser scans of a rock glacier are acquired from three positions with an Optech ILRIS3D terrestrial laser scanner. Additionally, the point density is manipulated in order to simulate three different levels of point density. For the matching of surface patches, we test various non-metric shape features such as roundness, concavity and elongation. Besides, we use metric shape features such as patch area, perimeter and the

  11. Effects of infestation of Rhyzopertha dominica F. on sorghum endosperm: A laser scanning confocal microscopy and differential scanning calorimetery study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infestations of Rhyzopertha dominica (F.), the lesser grain borer, can cause loss of biomass and decrease grain quality through feeding damage or contamination with insect fragments and uric acid. R. dominica can change dough properties of wheat and negatively affect bread quality. However, few pub...

  12. Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

    NASA Astrophysics Data System (ADS)

    Darvin, M. E.; Richter, H.; Zhu, Y. J.; Meinke, M. C.; Knorr, F.; Gonchukov, S. A.; Koenig, K.; Lademann, J.

    2014-07-01

    Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.

  13. Binocular eye tracking with the Tracking Scanning Laser Ophthalmoscope.

    PubMed

    Stevenson, S B; Sheehy, C K; Roorda, A

    2016-01-01

    The development of high magnification retinal imaging has brought with it the ability to track eye motion with a precision of less than an arc minute. Previously these systems have provided only monocular records. Here we describe a modification to the Tracking Scanning Laser Ophthalmoscope (Sheehy et al., 2012) that splits the optical path in a way that slows the left and right retinas to be scanned almost simultaneously by a single system. A mirror placed at a retinal conjugate point redirects half of each horizontal scan line to the fellow eye. The collected video is a split image with left and right retinas appearing side by side in each frame. Analysis of the retinal motion in the recorded video provides an eye movement trace with very high temporal and spatial resolution. Results are presented from scans of subjects with normal ocular motility that fixated steadily on a green laser dot. The retinas were scanned at 4° eccentricity with a 2° square field. Eye position was extracted offline from recorded videos with an FFT based image analysis program written in Matlab. The noise level of the tracking was estimated to range from 0.25 to 0.5arcmin SD for three subjects. In the binocular recordings, the left eye/right eye difference was 1-2arcmin SD for vertical motion and 10-15arcmin SD for horizontal motion, in agreement with published values from other tracking techniques. PMID:25676884

  14. A setup for combined multiphoton laser scanning microscopic and multi-electrode patch clamp experiments on brain slices

    NASA Astrophysics Data System (ADS)

    Helm, P. Johannes; Reppen, Trond; Heggelund, Paul

    2009-02-01

    Multi Photon Laser Scanning Microscopy (MPLSM) appears today as one of the most powerful experimental tools in cellular neurophysiology, notably in studies of the functional dynamics of signal processing in single neurons. Simultaneous recording of fluorescence signals at high spatial and temporal resolution and electric signals by means of multi electrode patch clamp techniques have provided new paths for the systematic investigation of neuronal mechanisms. In particular, this approach has opened for direct studies of dendritic signal processing in neurons. We report about a setup optimized for simultaneous electrophysiological multi electrode patch clamp and multi photon laser scanning fluorescence microscopic experiments on brain slices. The microscopic system is based on a modified commercially available confocal scanning laser microscope (CLSM). From a technical and operational point of view, two developments are important: Firstly, in order to reduce the workload for the experimentalist, who in general is forced to concentrate on controlling the electrophysiological parameters during the recordings, a system of shutters has been installed together with dedicated electronic modules protecting the photo detectors against destructive light levels caused by erroneous opening or closing of microscopic light paths by the experimentalist. Secondly, the standard detection unit has been improved by installing the photomultiplier tubes (PMT) in a Peltier cooled thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields. The electrophysiological system is based on an industrial standard multi patch clamp unit ergonomically arranged around the microscope stage. The electrophysiological and scanning processes can be time coordinated by standard trigger electronics.

  15. Laser scanning system for inspecting large underwater hydroelectric structures

    NASA Astrophysics Data System (ADS)

    Mirallès, François; Beaudry, Julien; Blain, Michel; de Santis, Romano M.; Houde, Régis; Hurteau, Richard; Robert, André; Sarraillon, Serge; Soucy, Nathalie

    2010-04-01

    A novel robotic laser scanning system for the inspection of large underwater hydroelectric structures is proposed. This system has been developed at the Hydro Quebec Research Institute and consists of a laser camera mounted on a 2-D Cartesian manipulator. Mechanical, electronic, and software design aspects; overall operational modalities; and proof of concept results are presented. We evaluated the performances of the system in the course of laboratory experiments and inspection trials carried out under normal operating conditions at the site of three of Hydro Quebec's hydroelectric dams.

  16. Practical Enhancement of Terrestrial Laser Scanning for Fluvial Geomorphology Surveys

    NASA Astrophysics Data System (ADS)

    Hwang, K.; Chandler, D. G.

    2014-12-01

    Accurate measurement of microtopography plays an important role in fluvial geomorphology. Whereof the surface is obscured by vegetation or landform, airborne remote sensing can be impractical and ground-based surveys using terrestrial laser scanning (TLS) show promise. TLS provides high resolution observations of the land surface for relatively low cost and with simple setup. However, the scanning range is effectively limited to less than 100 m, requiring individual scenes to be merged in software to represent larger landforms. For studies requiring several scenes, an efficient scanning strategy should be established in advance to optimize for time, resolution and spatial coverage. This requires careful consideration of scanner placement to merge scenes. We address problems encountered with blind spots. TLS is generally conducted on a 2-m (or shorter) tripod and the low scanning angle to the land surface at long distance inevitably causes blind spots in rugose or complex terrain. Similarly, the distance between TLS placement points is limited by the ability to resolve matching targets from sequential surveys. Here we present a simple geometry-based scanning plan regardless of the type and range of the instrument, with modification of the survey instrument platform. The half of a minimum range is used to make at least 18% of a superposed area with the next scan. Since scanning height barely affects the scanning range, the tripod was substituted to a 3-m stepladder and the platform of the scanner was modified to level and adjust the device easily with one hand. The results show that the new scanning plan performs well regardless of the topography and figure of the area of interest, with sufficient superposed area for combination with other adjacent scans. The modification of the platform also turned out to be more efficient to secure the observing angle and improve usability. The physical enhancement for TLS will provide valuable opportunity to conduct a standardized

  17. Monitoring stream bluff erosion using repeat terrestrial laser scanning

    NASA Astrophysics Data System (ADS)

    Neitzel, G.; Gran, K. B.

    2012-12-01

    Terrestrial laser scanning (TLS) technology provides high-resolution topographic data that can be used to detect geomorphic change in fluvial environments. In this study, we utilize successive terrestrial laser scans to investigate the relationship between peak flow rates and stream bluff erosion in the Amity Creek watershed in Duluth, Minnesota. We also combine TLS scan results with bluff inventories from airborne lidar to estimate the volume of sediment erosion from bluffs in the watershed, which is an important source of fine sediment contributing to the creek's turbidity impairment. We selected nine study bluffs to conduct terrestrial laser scans on after all significant flood events over a two-year time period. The study employs a Faro Focus 3D phase-shift laser to collect data. Post-processing of the TLS-point cloud data sets involves: (1) removal of vegetation and objects other than the erosional surface of interest; (2) decimation of the point cloud in PC Tools and extraction of zmin values to produce a data set manageable in GIS; (3) creation of a bare earth digital elevation model (DEM) for each set of scans using ArcMap; and (4) utilization of Geomorphic Change Detection (GCD) software to generate DEMs of Difference (DODs) from subsequent terrestrial laser scans. Preliminary results from three flooding events indicate significant erosional activity at all field sites. Slumps were observed at two bluffs following spring melt and freeze/thaw cycling. Two major precipitation events in late spring and early summer provided a unique opportunity to observe the impact of extreme high flow events on bluff erosion throughout the watershed using TLS technology. 4.75 inches of intermittent rain over a six-day period in late May 2012 (May 23-28) resulted in slumping at many bluffs and one major failure. The ≥100-year flood that occurred on June 19-20 (7.25 inches), 2012 was powerful enough to induce considerable channel change. Slumps occurred at six study sites

  18. Laser-Scan Ultrasonic/Thermographic Inspection System

    NASA Technical Reports Server (NTRS)

    Bar-Cohen, Yoseph; Marzwell, Neville; Chung, Tom; Carman, Greg

    1996-01-01

    Laser ultrasonic/thermographic inspection system is conceptual instrument used in nondestructive inspection of large areas of structures for subsurface defects. For example, operated in field to examine aircraft and pressure vessels for hidden cracks. System would not require contact with inspected structure or use of ultrasonic-coupling medium. Instrument obtains data in ultrasonic and thermographic C-scans over structural surfaces with contours characterized by surface normal vectors varying by angles as large as 45 degrees.

  19. Selective retinal therapy with a continuous line scanning laser

    NASA Astrophysics Data System (ADS)

    Paulus, Yannis M.; Jain, ATul; Gariano, Ray F.; Nomoto, Hiroyuki; Schuele, Georg; Sramek, Christopher; Charalel, Resmi; Palanker, Daniel

    2010-02-01

    This study evaluates the effects of exposure duration, beam diameter, and power on the safety, selectivity, and healing of retinal lesions created using a continuous line scanning laser. A 532 nm laser (PASCALTM) with retinal beam diameters of 40 and 66 μm was applied to 60 eyes of 30 Dutch-Belted rabbits. Retinal exposure duration varied from 15 to 60 μs. Lesions were acutely assessed by ophthalmoscopy and fluorescein angiography (FA). RPE flatmounts were evaluated with live-dead fluorescent assay (LD). Histological analysis was performed at 1 hour, 1 and 3 days, 1 and 2 weeks, and 1 and 2 months following laser treatment. Ophthalmoscopic visibility (OV) of the lesions corresponded to photoreceptor damage on histological analysis at 1 hour. In subvisible lesions, FA and LD yielded similar thresholds of RPE damage. The ratios of the threshold of rupture and of OV to FA visibility (measures of safety and selectivity) increased with decreasing duration and beam diameter. Above the threshold of OV, histology showed focal RPE damage and photoreceptor loss at one day without inner retinal effects. By one week, continuity of photoreceptor and RPE layers was restored. By 1 month, photoreceptors appeared normal while hypertrophy and hyperpigmentation of the RPE persisted. Retinal therapy with a fast scanning continuous laser achieves selective targeting of the RPE and, at higher power, of the photoreceptors. The damage zone in the photoreceptor layer is quickly filled-in, likely due to photoreceptor migration from adjacent zones. Continuous scanning laser can treat large retinal areas within standard eye fixation time.

  20. Wedge-filtering of geomorphologic terrestrial laser scan data.

    PubMed

    Panholzer, Helmut; Prokop, Alexander

    2013-01-01

    Terrestrial laser scanning is of increasing importance for surveying and hazard assessments. Digital terrain models are generated using the resultant data to analyze surface processes. In order to determine the terrain surface as precisely as possible, it is often necessary to filter out points that do not represent the terrain surface. Examples are vegetation, vehicles, and animals. Filtering in mountainous terrain is more difficult than in other topography types. Here, existing automatic filtering solutions are not acceptable, because they are usually designed for airborne scan data. The present article describes a method specifically suitable for filtering terrestrial laser scanning data. This method is based on the direct line of sight between the scanner and the measured point and the assumption that no other surface point can be located in the area above this connection line. This assumption is only true for terrestrial laser data, but not for airborne data. We present a comparison of the wedge filtering to a modified inverse distance filtering method (IDWMO) filtered point cloud data. Both methods use manually filtered surfaces as reference. The comparison shows that the mean error and root-mean-square-error (RSME) between the results and the manually filtered surface of the two methods are similar. A significantly higher number of points of the terrain surface could be preserved, however, using the wedge-filtering approach. Therefore, we suggest that wedge-filtering should be integrated as a further parameter into already existing filtering processes, but is not suited as a standalone solution so far. PMID:23429548

  1. Supraresolution imaging in brain slices using stimulated-emission depletion 2-photon laser scanning microscopy

    PubMed Central

    Ding, Jun; Takasaki, Kevin T.; Sabatini, Bernardo L.

    2009-01-01

    SUMMARY Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescent imaging of neuronal structure and function within neural tissue. However, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here we demonstrate supraresolution 2PLSM within brain slices. Imaging beyond the diffraction limit is accomplished by using near-infrared (NIR) lasers for both pulsed 2-photon excitation and continuous wave stimulation emission depletion (STED). Furthermore, we demonstrate that Alexa Fluor-594, a bright fluorophore commonly used for both live cell and fixed tissue fluorescence imaging, is suitable for STED 2PLSM. STED 2PLSM supraresolution microscopy achieves approximately 3 fold improvement in resolution in the radial direction over conventional 2PLSM, revealing greater detail in the structure of dendritic spines located ~100 microns below the surface of brain slices. Further improvements in resolution are theoretically achievable, suggesting that STED 2PLSM will permit nanoscale imaging of neuronal structures located in relatively intact brain tissue. PMID:19709626

  2. Calibration technology in application of robot-laser scanning system

    NASA Astrophysics Data System (ADS)

    Ren, YongJie; Yin, ShiBin; Zhu, JiGui

    2012-11-01

    A system composed of laser sensor and 6-DOF industrial robot is proposed to obtain complete three-dimensional (3-D) information of the object surface. Suitable for the different combining ways of laser sensor and robot, a new method to calibrate the position and pose between sensor and robot is presented. By using a standard sphere with known radius as a reference tool, the rotation and translation matrices between the laser sensor and robot are computed, respectively in two steps, so that many unstable factors introduced in conventional optimization methods can be avoided. The experimental results show that the accuracy of the proposed calibration method can be achieved up to 0.062 mm. The calibration method is also implemented into the automated robot scanning system to reconstruct a car door panel.

  3. Ta Keo Temple Reconstruction Based on Terrestrial Laser Scanning Technology

    NASA Astrophysics Data System (ADS)

    Xi, X.; Wang, C.; Wan, Y. P.; Khuon, K. N.

    2015-08-01

    Ta Keo temple is one of the very famous temple complex of Angkor Wat in northwestern Cambodia. It has been suffering massive collapse and other serious damages in recent years. Nowadays, Terrestrial Laser Scanning(TLS) technology is considered as a wellestablished resource for heritage documentation and protection (Lerma et al, 2008; Reshetyuk, 2009). This paper used TLS to reconstruct Ta Keo Temple. Firstly, we acquired 71 scanning stations of points cloud data with high density and high accuracy, and over one thousand images with high spatial resolution about the temple. Secondly, the raw points cloud data were denoised, reduced and managed efficiently, and registrated using an adjusted ICP algorithm. Thirdly, a triangulation method was used to model most objects. At last, we mapped the texture data into the digital model and a 3-D model of Ta Keo with high accuracy was achieved. The authors focus on large object reconstruction by TLS technology, and pay much attention to the scanning design, multi-station data and the whole project's data registration, and texture mapping and so on. The research result will be useful for Ta Keo restoration, reconstruction and protection. Also, it is a good reference source for large complex buildings reconstruction when using terrestrial laser scanning technology.

  4. Point-to-plane registration of terrestrial laser scans

    NASA Astrophysics Data System (ADS)

    Grant, Darion; Bethel, James; Crawford, Melba

    2012-08-01

    The registration of pairs of Terrestrial Laser Scanning data (TLS) is an integral precursor to 3D data analysis. Of specific interest in this research work is the class of approaches that is considered to be fine registration and which does not require any targets or tie points. This paper presents a pairwise fine registration approach called P2P that is formulated using the General Least Squares adjustment model. Given some initial registration parameters, the proposed P2P approach utilizes the scanned points and estimated planar features of both scans, along with their stochastic properties. These quantities are used to determine the optimum registration parameters in the least squares sense. The proposed P2P approach was tested on both simulated and real TLS data, and experimental results showed it to be four times more accurate than the registration approach of Chen and Medioni (1991).

  5. Composition analysis by scanning femtosecond laser ultraprobing (CASFLU).

    DOEpatents

    Ishikawa, Muriel Y.; Wood, Lowell L.; Campbell, E. Michael; Stuart, Brent C.; Perry, Michael D.

    2002-01-01

    The composition analysis by scanning femtosecond ultraprobing (CASFLU) technology scans a focused train of extremely short-duration, very intense laser pulses across a sample. The partially-ionized plasma ablated by each pulse is spectrometrically analyzed in real time, determining the ablated material's composition. The steering of the scanned beam thus is computer directed to either continue ablative material-removal at the same site or to successively remove nearby material for the same type of composition analysis. This invention has utility in high-speed chemical-elemental, molecular-fragment and isotopic analyses of the microstructure composition of complex objects, e.g., the oxygen isotopic compositions of large populations of single osteons in bone.

  6. Dental scanning in CAD/CAM technologies: laser beams

    NASA Astrophysics Data System (ADS)

    Sinescu, Cosmin; Negrutiu, Meda; Faur, Nicolae; Negru, Radu; Romînu, Mihai; Cozarov, Dalibor

    2008-02-01

    Scanning, also called digitizing, is the process of gathering the requisite data from an object. Many different technologies are used to collect three dimensional data. They range from mechanical and very slow, to radiation-based and highly-automated. Each technology has its advantages and disadvantages, and their applications and specifications overlap. The aims of this study are represented by establishing a viable method of digitally representing artifacts of dental casts, proposing a suitable scanner and post-processing software and obtaining 3D Models for the dental applications. The method is represented by the scanning procedure made by different scanners as the implicated materials. Scanners are the medium of data capture. 3D scanners aim to measure and record the relative distance between the object's surface and a known point in space. This geometric data is represented in the form of point cloud data. The contact and no contact scanners were presented. The results show that contact scanning procedures uses a touch probe to record the relative position of points on the objects' surface. This procedure is commonly used in Reverse engineering applications. Its merits are represented by efficiency for objects with low geometric surface detail. Disadvantages are represented by time consuming, this procedure being impractical for artifacts digitization. The non contact scanning procedure implies laser scanning (laser triangulation technology) and photogrammetry. As a conclusion it can be drawn that different types of dental structure needs different types of scanning procedures in order to obtain a competitive complex 3D virtual model that can be used in CAD/CAM technologies.

  7. The effect of copper on different phototrophic microorganisms determined in vivo and at cellular level by confocal laser microscopy.

    PubMed

    Seder-Colomina, M; Burgos, A; Maldonado, J; Solé, A; Esteve, I

    2013-01-01

    Microbial mats are coastal ecosystems that consist mainly of cyanobacteria, primary producers in these habitats that play an important role in stabilising delta sediments. However, these ecosystems are subject to various kinds of pollution, including metal contamination, placing their survival at risk. Among heavy metals, copper is an essential metal at low doses and toxic at high doses. This metal is present in different pesticides used in rice production, a thriving agro-industry in the Ebro Delta (Spain). For several years, our group has been studying the Ebro Delta microbial mats and has developed a method for determining the effect that metals cause on cyanobacteria populations. This method is based on confocal laser microscopy coupled to a spectrofluorometer, which rapidly provides simultaneous three-dimensional information on photosynthetic microorganisms and their fluorescence spectra profiles. The current study determines the copper effect on different photosynthetic microorganisms from culture collection (Chroococcus sp. PCC 9106 and Spirulina sp. PCC 6313) and isolated from the environment (Microcoleus-like and the microalga DE2009). Comparing all results obtained it can be observed that the minimum dose of Cu that is capable of significantly altering chlorophyll a (chl a) fluorescence intensity were 1 × 10(-7) M in Chroococcus sp. PCC 9106; 1 × 10(-7) M in Spirulina sp. PCC 6313; 3 × 10(-7) M in Microcoleus and 5 × 10(-6) M in the microalga DE2009. Moreover, the sensitivity of the technique used was 1 × 10(-7) M. PMID:23138333

  8. Multicolor probe-based confocal laser endomicroscopy: a new world for in vivo and real-time cellular imaging

    NASA Astrophysics Data System (ADS)

    Vercauteren, Tom; Doussoux, François; Cazaux, Matthieu; Schmid, Guillaume; Linard, Nicolas; Durin, Marie-Amélie; Gharbi, Hédi; Lacombe, François

    2013-03-01

    Since its inception in the field of in vivo imaging, endomicroscopy through optical fiber bundles, or probe-based Confocal Laser Endomicroscopy (pCLE), has extensively proven the benefit of in situ and real-time examination of living tissues at the microscopic scale. By continuously increasing image quality, reducing invasiveness and improving system ergonomics, Mauna Kea Technologies has turned pCLE not only into an irreplaceable research instrument for small animal imaging, but also into an accurate clinical decision making tool with applications as diverse as gastrointestinal endoscopy, pulmonology and urology. The current implementation of pCLE relies on a single fluorescence spectral band making different sources of in vivo information challenging to distinguish. Extending the pCLE approach to multi-color endomicroscopy therefore appears as a natural plan. Coupling simultaneous multi-laser excitation with minimally invasive, microscopic resolution, thin and flexible optics, allows the fusion of complementary and valuable biological information, thus paving the way to a combination of morphological and functional imaging. This paper will detail the architecture of a new system, Cellvizio Dual Band, capable of video rate in vivo and in situ multi-spectral fluorescence imaging with a microscopic resolution. In its standard configuration, the system simultaneously operates at 488 and 660 nm, where it automatically performs the necessary spectral, photometric and geometric calibrations to provide unambiguously co-registered images in real-time. The main hardware and software features, including calibration procedures and sub-micron registration algorithms, will be presented as well as a panorama of its current applications, illustrated with recent results in the field of pre-clinical imaging.

  9. The case of diagnostics of invasive pulmonary aspergillosis by in vivo probe-based confocal laser endomicroscopy of central and distal airways

    PubMed Central

    Danilevskaya, Olesya; Averyanov, Alexander; Klimko, Nikolay; Lesnyak, Viktor; Sorokina, Anastasia; Sazonov, Dmitry; Zabozlaev, Fedor

    2014-01-01

    We present a case of 41-year-old patient with invasive pulmonary aspergillosis (IPA) in which probe-based confocal laser endomicroscopy (pCLE) imaging of central and distal airways was first performed in vivo. pCLE imaging showed the signs of complete or partial destruction of elastin network of alveolar wall with fibrillar branching fluorescent structures in the zone with typical IPA changes on HRCT. PMID:25180153

  10. Globally consistent registration of terrestrial laser scans via graph optimization

    NASA Astrophysics Data System (ADS)

    Theiler, Pascal Willy; Wegner, Jan Dirk; Schindler, Konrad

    2015-11-01

    In this paper we present a framework for the automatic registration of multiple terrestrial laser scans. The proposed method can handle arbitrary point clouds with reasonable pairwise overlap, without knowledge about their initial orientation and without the need for artificial markers or other specific objects. The framework is divided into a coarse and a fine registration part, which each start with pairwise registration and then enforce consistent global alignment across all scans. While we put forward a complete, functional registration system, the novel contribution of the paper lies in the coarse global alignment step. Merging multiple scans into a consistent network creates loops along which the relative transformations must add up. We pose the task of finding a global alignment as picking the best candidates from a set of putative pairwise registrations, such that they satisfy the loop constraints. This yields a discrete optimization problem that can be solved efficiently with modern combinatorial methods. Having found a coarse global alignment in this way, the framework proceeds by pairwise refinement with standard ICP, followed by global refinement to evenly spread the residual errors. The framework was tested on six challenging, real-world datasets. The discrete global alignment step effectively detects, removes and corrects failures of the pairwise registration procedure, finally producing a globally consistent coarse scan network which can be used as initial guess for the highly non-convex refinement. Our overall system reaches success rates close to 100% at acceptable runtimes < 1 h, even in challenging conditions such as scanning in the forest.

  11. Application of laser scanning microscopy for the analysis of oral biofilm dissolution by different endodontic irrigants

    PubMed Central

    del Carpio-Perochena, Aldo; Bramante, Clovis Monteiro; Hungaro Duarte, Marco Antonio; de Andrade, Flaviana Bombarda; Cavenago, Bruno Cavalini; Villas-Bôas, Marcelo Haas; Ordinola-Zapata, Ronald; Amoroso-Silva, Pablo

    2014-01-01

    Background: Multi-specie biofilms are highly resistant to antimicrobials due to cellular interactions found in them. The purpose of this study was to evaluate, by confocal laser scanning microscopy, the biofilm dissolution effectiveness of different irrigant solutions on biofilms developed on infected dentin in situ. Materials and Methods: A total of 120 bovine dentin specimens infected intraorally (30/group) were treated by the following solutions: 2% of chlorhexidine digluconate, 1%, 2.5% and 5.25% of sodium hypochlorite (NaOCl). The solutions were utilized for 5, 15 and 30 min with 2 experimental volumes 500 μL and 1 mL. All the samples were stained using an acridine orange and the biofilm thickness before (control group) and after the experiments were evaluated, utilizing a confocal microscope at ×40. The Mann-Whitney U and the nom-parametric Kruskal-Wallis Dunns tests were utilized to determine the influence of the volume and to perform the comparisons among the groups respectively. The significance level was set at P < 0.05. Results: Statistical differences were not found among the control and the 2% chlorhexidine digluconate groups at any experimental period (P > 0.05). The biofilm dissolution treated with 1% NaOCl was directly proportional to the exposure time (P < 0.05). The higher values of biofilm dissolution were found in 2.5% and 5.25% NaOCl groups (P > 0.05). Conclusion: The higher exposure times and concentrations of NaOCl were not sufficient to dissolve 100% of the biofilm. However, all NaOCl solutions were more effective than 2% chlorhexidine digluconate to dissolve organic matter. PMID:25225556

  12. A new pulsed laser deposition technique: scanning multi-component pulsed laser deposition method.

    PubMed

    Fischer, D; de la Fuente, G F; Jansen, M

    2012-04-01

    The scanning multi-component pulsed laser deposition (PLD) method realizes uniform depositions of desired coatings by a modified pulsed laser deposition process, preferably with a femto-second laser-system. Multi-component coatings (single or multilayered) are thus deposited onto substrates via laser induced ablation of segmented targets. This is achieved via horizontal line-scanning of a focused laser beam over a uniformly moving target's surface. This process allows to deposit the desired composition of the coating simultaneously, starting from the different segments of the target and adjusting the scan line as a function of target geometry. The sequence and thickness of multilayers can easily be adjusted by target architecture and motion, enabling inter/intra layer concentration gradients and thus functional gradient coatings. This new, simple PLD method enables the achievement of uniform, large-area coatings. Case studies were performed with segmented targets containing aluminum, titanium, and niobium. Under the laser irradiation conditions applied, all three metals were uniformly ablated. The elemental composition within the rough coatings obtained was fixed by the scanned area to Ti-Al-Nb = 1:1:1. Crystalline aluminum, titanium, and niobium were found to coexist side by side at room temperature within the substrate, without alloy formation up to 600 °C. PMID:22559543

  13. A new pulsed laser deposition technique: Scanning multi-component pulsed laser deposition method

    SciTech Connect

    Fischer, D.; Jansen, M.; Fuente, G. F. de la

    2012-04-15

    The scanning multi-component pulsed laser deposition (PLD) method realizes uniform depositions of desired coatings by a modified pulsed laser deposition process, preferably with a femto-second laser-system. Multi-component coatings (single or multilayered) are thus deposited onto substrates via laser induced ablation of segmented targets. This is achieved via horizontal line-scanning of a focused laser beam over a uniformly moving target's surface. This process allows to deposit the desired composition of the coating simultaneously, starting from the different segments of the target and adjusting the scan line as a function of target geometry. The sequence and thickness of multilayers can easily be adjusted by target architecture and motion, enabling inter/intra layer concentration gradients and thus functional gradient coatings. This new, simple PLD method enables the achievement of uniform, large-area coatings. Case studies were performed with segmented targets containing aluminum, titanium, and niobium. Under the laser irradiation conditions applied, all three metals were uniformly ablated. The elemental composition within the rough coatings obtained was fixed by the scanned area to Ti-Al-Nb = 1:1:1. Crystalline aluminum, titanium, and niobium were found to coexist side by side at room temperature within the substrate, without alloy formation up to 600 deg. C.

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

    PubMed Central

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

    2014-01-01

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

  15. Efficient terrestrial laser scan segmentation exploiting data structure

    NASA Astrophysics Data System (ADS)

    Mahmoudabadi, Hamid; Olsen, Michael J.; Todorovic, Sinisa

    2016-09-01

    New technologies such as lidar enable the rapid collection of massive datasets to model a 3D scene as a point cloud. However, while hardware technology continues to advance, processing 3D point clouds into informative models remains complex and time consuming. A common approach to increase processing efficiently is to segment the point cloud into smaller sections. This paper proposes a novel approach for point cloud segmentation using computer vision algorithms to analyze panoramic representations of individual laser scans. These panoramas can be quickly created using an inherent neighborhood structure that is established during the scanning process, which scans at fixed angular increments in a cylindrical or spherical coordinate system. In the proposed approach, a selected image segmentation algorithm is applied on several input layers exploiting this angular structure including laser intensity, range, normal vectors, and color information. These segments are then mapped back to the 3D point cloud so that modeling can be completed more efficiently. This approach does not depend on pre-defined mathematical models and consequently setting parameters for them. Unlike common geometrical point cloud segmentation methods, the proposed method employs the colorimetric and intensity data as another source of information. The proposed algorithm is demonstrated on several datasets encompassing variety of scenes and objects. Results show a very high perceptual (visual) level of segmentation and thereby the feasibility of the proposed algorithm. The proposed method is also more efficient compared to Random Sample Consensus (RANSAC), which is a common approach for point cloud segmentation.

  16. Microanalysis of dental caries using laser-scanned fluorescence

    NASA Astrophysics Data System (ADS)

    Barron, Joseph R.; Paton, Barry E.; Zakariasen, Kenneth L.

    1992-06-01

    It is well known that enamel and dentin fluoresce when illuminated by short-wavelength optical radiation. Fluorescence emission from carious and non-carious regions of teeth have been studied using a new experimental scanning technique for fluorescence analysis of dental sections. Scanning in 2 dimensions will allow surface maps of dental caries to be created. These surface images are then enhanced using the conventional and newer image processing techniques. Carious regions can be readily identified and contour maps can be used to graphically display the degree of damage on both surfaces and transverse sections. Numerous studies have shown that carious fluorescence is significantly different than non-carious regions. The scanning laser fluorescence spectrometer focuses light from a 25 mW He-Cd laser at 442 nm through an objective lens onto a cross-section area as small as 3 micrometers in diameter. Microtome prepared dental samples 100 micrometers thick are laid flat onto an optical bench perpendicular to the incident beam. The sample is moved under computer control in X & Y with an absolute precision of 0.1 micrometers . The backscattered light is both spatial and wavelength filtered before being measured on a long wavelength sensitized photomultiplier tube. High precision analysis of dental samples allow detailed maps of carious regions to be determined. Successive images allow time studies of caries growth and even the potential for remineralization studies of decalcified regions.

  17. Laser cutting of irregular shape object based on stereo vision laser galvanometric scanning system

    NASA Astrophysics Data System (ADS)

    Qi, Li; Zhang, Yixin; Wang, Shun; Tang, Zhiqiang; Yang, Huan; Zhang, Xuping

    2015-05-01

    Irregular shape objects with different 3-dimensional (3D) appearances are difficult to be shaped into customized uniform pattern by current laser machining approaches. A laser galvanometric scanning system (LGS) could be a potential candidate since it can easily achieve path-adjustable laser shaping. However, without knowing the actual 3D topography of the object, the processing result may still suffer from 3D shape distortion. It is desirable to have a versatile auxiliary tool that is capable of generating 3D-adjusted laser processing path by measuring the 3D geometry of those irregular shape objects. This paper proposed the stereo vision laser galvanometric scanning system (SLGS), which takes the advantages of both the stereo vision solution and conventional LGS system. The 3D geometry of the object obtained by the stereo cameras is used to guide the scanning galvanometers for 3D-shape-adjusted laser processing. In order to achieve precise visual-servoed laser fabrication, these two independent components are integrated through a system calibration method using plastic thin film target. The flexibility of SLGS has been experimentally demonstrated by cutting duck feathers for badminton shuttle manufacture.

  18. Confocal Imaging of porous media

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  19. Scanning Laser Optical Tomography Resolves Structural Plasticity during Regeneration in an Insect Brain

    PubMed Central

    Eickhoff, René; Lorbeer, Raoul-Amadeus; Scheiblich, Hannah; Heisterkamp, Alexander; Meyer, Heiko; Stern, Michael; Bicker, Gerd

    2012-01-01

    Background Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system. Methodology We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx. Conclusions This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations. PMID:22829931

  20. Laser light scan analysis of the “anticonvulsant face”

    PubMed Central

    Orup, H. Ivan; Deutsch, Curtis K.; Holmes, Lewis B.

    2014-01-01

    BACKGROUND The “anticonvulsant face”, comprised of a short nose, low nasal bridge, epicanthal folds, and wide mouth, was suggested in the 1970s to indicate teratogenesis caused by the anticonvulsant drugs phenytoin and phenobarbital. However, these were based on subjective clinical observations. In the present study we have applied objective and reliable quantitative measures to the operational definitions of craniofacial features in anticonvulant-exposed cases. We have adopted anthropometric analysis based on image analysis of laser light scans. Using morphometric methods, we established the positions of physical features and objectively determined the changes in the size and shape of affected soft tissues of the faces of children exposed to those anticonvulsant drugs during pregnancy. METHODS Thirteen individuals, exposed throughout pregnancy to phenytoin as either monotherapy or polytherapy, were identified in a previous analysis as having significant changes in their craniofacial features based on measurements of cephalometric radiographs, changes associated with “the anticonvulsant face”.. The soft tissues of their faces were imaged by 3D laser (structured light) scanning. RESULTS The notable changes in soft tissues identified by laser light scans were a wide philtrum (between the left and right cristae philtri), narrow mouth (between the left and right cheilions), short nasal bridge (between nasale and pronasale), short nose height (between the nasale and subnasale), and flat orbits (based on the orbital protrusion index). CONCLUSIONS This analysis of phenytoin-exposed individuals is the first anthropometric analysis of the craniofacial surface, designed to render the identification of abnormal features both objective and realiable. These analyses demonstrated that there were several significant changes in the soft tissue of the face, corroborating earlier studies of alterations of the craniofacial skeleton in the anticonvulsant face. Two of the

  1. Road Orthophoto/dtm Generation from Mobile Laser Scanning

    NASA Astrophysics Data System (ADS)

    Vallet, B.; Papelard, J.-P.

    2015-08-01

    This paper proposes a pipeline to produce road orthophoto and DTM from Mobile Laser Scanning (MLS). For the ortho, modern laser scanners provide a reflectance information allowing for high quality grayscale images, at a much finer resolution than aerial photography can offer. For DTM, MLS offers a much higher accuracy and density than aerial products. This increased precision and resolution leverages new applications for both ortho and DEM. The first task is to filter ground vs non ground, then an interpolation is conducted to build image tiles from the filtered points. Finally, multiple layers are registered and blended to allow for seamless fusion. Our proposed approach achieves high quality products and scaling up is demonstrated.

  2. Hot Slab Surface Inspection By Laser Scanning Method

    NASA Astrophysics Data System (ADS)

    Matsubara, Toshiro; Toyota, Toshio; Fujiyama, Akihiro

    1986-10-01

    An optical flaw detector with laser as the external light source, which is called LST ( laser scanning tester ), has been developed. This equipment automatically inspects the entire surface of hot slabs. The results are used to examine the suitability of those slabs for hot charge rolling. The characteristics of LST are its high optical resolving power and the signal processing method with which two-dimensional information on the type of the flaw is processed. For the opening width of O.4mm and over, the detection ratio is nearly 100%. This equipment started commercial operation in January 1983 in Nippon Steel's Yawata Works and its application has increased the hot charge rolling ratio.

  3. Scanning laser ophthalmoscopy: optimized testing strategies for psychophysics

    NASA Astrophysics Data System (ADS)

    Van de Velde, Frans J.

    1996-12-01

    Retinal function can be evaluated with the scanning laser ophthalmoscope (SLO). the main advantage is a precise localization of the psychophysical stimulus on the retina. Four alternative forced choice (4AFC) and parameter estimation by sequential testing (PEST) are classic adaptive algorithms that have been optimized for use with the SLO, and combined with strategies to correct for small eye movements. Efficient calibration procedures are essential for quantitative microperimetry. These techniques measure precisely visual acuity and retinal sensitivity at distinct locations on the retina. A combined 632 nm and IR Maxwellian view illumination provides a maximal transmittance through the ocular media and has a animal interference with xanthophyll or hemoglobin. Future modifications of the instrument include the possibility of binocular evaluation, Maxwellian view control, fundus tracking using normalized gray-scale correlation, and microphotocoagulation. The techniques are useful in low vision rehabilitation and the application of laser to the retina.

  4. Thermal expansion of scanning tunneling microscopy tips under laser illumination

    NASA Astrophysics Data System (ADS)

    Grafström, S.; Schuller, P.; Kowalski, J.; Neumann, R.

    1998-04-01

    The periodic thermal expansion of scanning tunneling microscopy (STM) tips arising under irradiation with power-modulated laser light has been investigated. The expansion was determined by comparison with a calibrated piezomotion measured in an STM, which was operated in the constant-current mode, and instrumental effects were corrected for. The experimental data concerning the frequency response of the thermal expansion for various geometries of the tip and for different positions of the laser focus are compared with theoretical results which were derived from a numerical solution of the equation of heat conduction. A very good agreement is found. The results are also interpreted in terms of simplified analytical expressions. Furthermore, the theoretical data are used to derive the response of the tip to fast transients of the light power as in the case of pulsed irradiation.

  5. Automatic Railway Power Line Extraction Using Mobile Laser Scanning Data

    NASA Astrophysics Data System (ADS)

    Zhang, Shanxin; Wang, Cheng; Yang, Zhuang; Chen, Yiping; Li, Jonathan

    2016-06-01

    Research on power line extraction technology using mobile laser point clouds has important practical significance on railway power lines patrol work. In this paper, we presents a new method for automatic extracting railway power line from MLS (Mobile Laser Scanning) data. Firstly, according to the spatial structure characteristics of power-line and trajectory, the significant data is segmented piecewise. Then, use the self-adaptive space region growing method to extract power lines parallel with rails. Finally use PCA (Principal Components Analysis) combine with information entropy theory method to judge a section of the power line whether is junction or not and which type of junction it belongs to. The least squares fitting algorithm is introduced to model the power line. An evaluation of the proposed method over a complicated railway point clouds acquired by a RIEGL VMX450 MLS system shows that the proposed method is promising.

  6. Galvanometer beam-scanning system for laser fiber drawing.

    PubMed

    Oehrle, R C

    1979-02-15

    A major difficulty in using a laser to draw optical fibers from a glass preform has been uniformally distributing the laser's energy around the melt zone. Several systems have evolved in recent years, but to date the most successful technique has been the off-axis rotating lens system (RLS). The inability of this device to structure efficiently and dynamically the heat zone longitudinally along the preform has restricted its use to preform of less than 8-mm diameter. A new technique reported here employs two orthogonal mounted mirrors, driven by galvanometers to distribute the laser energy around the preform. This system can be retrofitted into the RLS to replace the rotating lens element. The new system, the galvanometer scanning system (GSS), operates at ten times the rotational speed of the RLS and can instantaneously modify the melt zone. The ability of the GSS to enlarge the melt zone reduces the vaporization rate at the surface of the preform permitting efficient use of higher laser power. Experiments i dicate that fibers can be drawn from significantly larger preforms by using the expanded heat zone provided by the GSS. PMID:20208750

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

    PubMed Central

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

    2015-01-01

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

  8. Membrane Vibration Studies Using a Scanning Laser Vibrometer

    NASA Technical Reports Server (NTRS)

    Gaspar, James L.; Solter, Micah J.; Pappa, Richard S.

    2001-01-01

    This paper summarizes on-going experimental work at NASA Langley Research Center to measure the dynamics of a 1.016 m (40 in.) square polyimide film Kapton membrane. A fixed fully automated impact hammer and Polytec PSV-300-H scanning laser vibrometer were used for non-contact modal testing of the membrane with zero-mass-loading. The paper discusses the results obtained by testing the membrane at various tension levels and at various excitation locations. Results obtained by direct shaker excitation to the membrane are also discussed.

  9. Membrane Vibration Studies Using a Scanning Laser Vibrometer

    NASA Astrophysics Data System (ADS)

    Gaspar, James L.; Solter, Micah J.; Pappa, Richard S.

    2001-02-01

    This paper summarizes on-going experimental work at NASA Langley Research Center to measure the dynamics of a 1.016 m (40 in.) square polyimide film Kapton membrane. A fixed fully automated impact hammer and Polytec PSV-300-H scanning laser vibrometer were used for non-contact modal testing of the membrane with zero-mass-loading. The paper discusses the results obtained by testing the membrane at various tension levels and at various excitation locations. Results obtained by direct shaker excitation to the membrane are also discussed.

  10. Precision targeting with a tracking adaptive optics scanning laser ophthalmoscope

    NASA Astrophysics Data System (ADS)

    Hammer, Daniel X.; Ferguson, R. Daniel; Bigelow, Chad E.; Iftimia, Nicusor V.; Ustun, Teoman E.; Noojin, Gary D.; Stolarski, David J.; Hodnett, Harvey M.; Imholte, Michelle L.; Kumru, Semih S.; McCall, Michelle N.; Toth, Cynthia A.; Rockwell, Benjamin A.

    2006-02-01

    Precise targeting of retinal structures including retinal pigment epithelial cells, feeder vessels, ganglion cells, photoreceptors, and other cells important for light transduction may enable earlier disease intervention with laser therapies and advanced methods for vision studies. A novel imaging system based upon scanning laser ophthalmoscopy (SLO) with adaptive optics (AO) and active image stabilization was designed, developed, and tested in humans and animals. An additional port allows delivery of aberration-corrected therapeutic/stimulus laser sources. The system design includes simultaneous presentation of non-AO, wide-field (~40 deg) and AO, high-magnification (1-2 deg) retinal scans easily positioned anywhere on the retina in a drag-and-drop manner. The AO optical design achieves an error of <0.45 waves (at 800 nm) over +/-6 deg on the retina. A MEMS-based deformable mirror (Boston Micromachines Inc.) is used for wave-front correction. The third generation retinal tracking system achieves a bandwidth of greater than 1 kHz allowing acquisition of stabilized AO images with an accuracy of ~10 μm. Normal adult human volunteers and animals with previously-placed lesions (cynomolgus monkeys) were tested to optimize the tracking instrumentation and to characterize AO imaging performance. Ultrafast laser pulses were delivered to monkeys to characterize the ability to precisely place lesions and stimulus beams. Other advanced features such as real-time image averaging, automatic highresolution mosaic generation, and automatic blink detection and tracking re-lock were also tested. The system has the potential to become an important tool to clinicians and researchers for early detection and treatment of retinal diseases.