Science.gov

Sample records for confocal laser scanning

  1. The relaxed confocal scanning laser ophthalmoscope.

    PubMed

    Van de Velde, F J

    2006-01-01

    The development of the Scanning Laser Ophthalmoscope is reviewed from a historical perspective. Since a flying-spot scanning principle for an electro-optical ophthalmoscope was first disclosed in 1950, enabling milestones have included the introduction of the laser and inversion of the usual Gullstrand's configuration of optical pupils in 1977, and the application of the optical principle of confocality by means of double or de-scanning in 1983. As a result, high resolution and high contrast confocal infra-red ophthalmoscopy with a 790 nm diode laser, at video rates, is a major novel imaging modality when compared to traditional optical techniques. This imaging mode is ideal to provide the necessary fiducial landmarks for microperimetry, therapeutic laser and SD-OCT based optical sectioning of the retina. DPSS or He-Ne lasers emitting at 532, 543, 561 or 575 nm are used for complimentary red-free fundus imaging. The diode 790 nm and DPSS 490 nm lasers are also used for fluorescence excitation.

  2. 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.

  3. 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...

  4. CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE OVARIES

    EPA Science Inventory

    Confocal Laser Scanning Microscopy of Apoptosis in Whole Mouse Ovaries. Robert M. Zucker Susan C. Jeffay and Sally D. Perreault Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle...

  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. 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...

  8. 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

  9. Confocal scanning laser ophthalmoscopy in glaucoma diagnosis and management.

    PubMed

    Alexandrescu, C; Dascalu, A M; Panca, A; Sescioreanu, A; Mitulescu, C; Ciuluvica, R; Voinea, L; Celea, C

    2010-01-01

    The early diagnosis and detection of progression are two key-elements in the actual management of glaucoma. The current opinion in clinical practice is to quantify the structural damage for a better follow-up of the patient and the standardization of the results. The present review is a concise survey of literature covering the period of 1990-2010, documenting the evidence-based role of confocal scanning laser ophthalmoscopy in glaucoma diagnosis and management.

  10. [Application of confocal laser scanning microscope in forensic pathology].

    PubMed

    Zhuo, Luo; Hu, Le-Sheng; Zhou, Lan; Zheng, Na; Liang, Man; Yang, Fan; Liu, Liang

    2009-12-01

    Confocal laser scanning microscopy(CLSM) is a new technique for microscopic imaging, which can collect the transverse section image of the samples and produce three-dimensional reconstruction and present higher spatial resolution than the conventional light microscope. As a precision instrument for the microscopic image, it plays an important role in forensic pathology. The article reviews the recent research achievements from sudden cardiac death, bullet wound and nervous system damage, etc, and explores the potential applications of the forensic pathology research and forensic practice.

  11. The design and construction of a cost-efficient confocal laser scanning microscope

    NASA Astrophysics Data System (ADS)

    Xi, Peng; Rajwa, Bartlomiej; Jones, James T.; Robinson, J. Paul

    2007-03-01

    The optical dissection ability of confocal microscopy makes it a powerful tool for biological materials. However, the cost and complexity of confocal scanning laser microscopy hinders its wide application in education. We describe the construction of a simplified confocal scanning laser microscope and demonstrate three-dimensional projection based on cost-efficient commercial hardware, together with available open source software.

  12. Quantitative single-molecule imaging by confocal laser scanning microscopy.

    PubMed

    Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

    2008-11-25

    A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

  13. Adaptive optics for confocal laser scanning microscopy with adjustable pinhole

    NASA Astrophysics Data System (ADS)

    Yoo, Han Woong; van Royen, Martin E.; van Cappellen, Wiggert A.; Houtsmuller, Adriaan B.; Verhaegen, Michel; Schitter, Georg

    2016-04-01

    The pinhole plays an important role in confocal laser scanning microscopy (CLSM) for adaptive optics (AO) as well as in imaging, where the size of the pinhole denotes a trade-off between out-of-focus rejection and wavefront distortion. This contribution proposes an AO system for a commercial CLSM with an adjustable square pinhole to cope with such a trade-off. The proposed adjustable pinhole enables to calibrate the AO system and to evaluate the imaging performance. Experimental results with fluorescence beads on the coverslip and at a depth of 40 μm in the human hepatocellular carcinoma cell spheroid demonstrate that the proposed AO system can improve the image quality by the proposed calibration method. The proposed pinhole intensity ratio also indicates the image improvement by the AO correction in intensity as well as resolution.

  14. 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

  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. Semiquantitative confocal laser scanning microscopy applied to marine invertebrate ecotoxicology.

    PubMed

    Chandler, G Thomas; Volz, David C

    2004-01-01

    Confocal laser scanning microscopy (CLSM) represents a powerful, but largely unexplored ecotoxicologic tool for rapidly assessing in vivo effects of toxicants on marine invertebrate embryo quality and development. We describe here a new semiquantitative CLSM approach for assessing relative yolk quantity in marine invertebrate embryos (harpacticoid copepods) produced by parents reared from hatching to adult in the polycylic aromatic hydrocarbon chrysene. This method is based on fluorogenic labeling of embryo yolk and subsequent statistical analysis of areal pixel intensities over multiple Z-series using a general linear model (GLM)-nested analysis of variance. The fluorescent yolk-labeling method described here was able to detect statistically significant differences in yolk concentrations in marine copepod (Amphiascus tenuiremis) eggs or embryos from females exposed to ultraviolet light and chrysene-contaminated sediments. Yolk intensities in embryos from females cultured throughout their life cycles in clean sediments were statistically identical with or without UV exposure. In contrast, yolk intensities in embryos of females cultured throughout their life cycle in chrysene-contaminated sediments were significantly higher in the non-UV-exposed treatment with chrysene at 2500 ng/g sediment (65.7% higher) and the UV-exposed treatment with chrysene at 500 ng/g sediment (76.6% higher).

  17. 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.

  18. 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.

  19. Intracellular phthalocyanine localization: confocal laser scanning microscopy studies

    NASA Astrophysics Data System (ADS)

    Chernyaeva, Elena B.; Greve, Jan; de Grooth, Bart G.; Van Leeuwen, A. G.

    1994-02-01

    Phthalocyanines (Pc) are promising second-generation photosensitizers for the photodynamic therapy (PDT) of cancer. We report on the tetrasulfonated aluminum phthalocyanine (AlPcS4) localization in cultured Chinese hamster lung cells studied by means of confocal laser scanning microscopy (CLSM). In these cells AlPcS4 was found in granules surrounding Golgi apparatus and in the peripheral cytoplasmic region. Peripheral Pc-containing granules partially coincided with the acidic cellular compartments. The effect of irradiation with light on Pc intracellular distribution was also studied. In the Pc-free medium disruption of some Pc- containing granules was observed followed by appearance of Pc fluorescence in the cell plasma membrane, the nuclear envelope, and the near-nuclear region. When cells were irradiated in the presence of Pc in external medium a drastic increase of membrane permeability to Pc was observed, followed by Pc binding the cell plasma membrane, nuclear envelope, and some structures in the cytoplasm. Diffusive Pc fluorescence in the nucleus was also observed. The implication of observed Pc redistribution caused by irradiation with light for the PDT protocol is discussed.

  20. CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE AND RAT OVARIES

    EPA Science Inventory

    Confocal Laser Scanning Microscopy of Apoptosis in Whole Mouse and Rat Ovaries. Robert M. Zucker Susan C. Jeffay and Sally D. Perreault Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research ...

  1. 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

  2. Multispectral confocal scanning laser ophthalmoscope for retinal vessel oximetry

    NASA Astrophysics Data System (ADS)

    Lompado, Arthur; Smith, Matthew H.; Hillman, Lloyd W.; Denninghoff, Kurt R.

    2000-03-01

    Scanning laser microscopy is a widely used technique in ophthalmoscopy for providing high-resolution real time images of the retina. We describe a scanning laser ophthalmoscope that acquires retinal images at four wavelengths for the purpose of measuring the oxygen saturation of blood in retinal arteries and veins. Images at all four wavelengths are obtained across a single video frame using a temporal interlacing technique. An extraction procedure then permits analysis of four monochromatic images. A technique for calculating oxygen saturation from a multi-spectral image set is presented, along with preliminary measurements. The choice of wavelengths dramatically affects the oxygen saturation calculation accuracy and we present an optimized wavelength set and the calculated oxygen saturation results. The potential applications for this technology range from the diagnosis of various ophthalmic diseases to the detection of blood loss in trauma victims.

  3. 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.

  4. A confocal scanning laser ophthalmoscope for retinal vessel oximetry

    NASA Astrophysics Data System (ADS)

    Lompado, Arthur

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

  5. 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...

  6. 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...

  7. Laser scanning confocal microscope with programmable amplitude, phase, and polarization of the illumination beam.

    PubMed

    Boruah, B R; Neil, M A A

    2009-01-01

    We describe the design and construction of a laser scanning confocal microscope with programmable beam forming optics. The amplitude, phase, and polarization of the laser beam used in the microscope can be controlled in real time with the help of a liquid crystal spatial light modulator, acting as a computer generated hologram, in conjunction with a polarizing beam splitter and two right angled prisms assembly. Two scan mirrors, comprising an on-axis fast moving scan mirror for line scanning and an off-axis slow moving scan mirror for frame scanning, configured in a way to minimize the movement of the scanned beam over the pupil plane of the microscope objective, form the XY scan unit. The confocal system, that incorporates the programmable beam forming unit and the scan unit, has been implemented to image in both reflected and fluorescence light from the specimen. Efficiency of the system to programmably generate custom defined vector beams has been demonstrated by generating a bottle structured focal volume, which in fact is the overlap of two cross polarized beams, that can simultaneously improve both the lateral and axial resolutions if used as the de-excitation beam in a stimulated emission depletion confocal microscope.

  8. Design and development of multi functional confocal laser scanning microscope with UV / VIS laser source

    NASA Astrophysics Data System (ADS)

    Kanai, Yoshikazu; Kanzaki, Yousuke; Wakaki, Moriaki; Takeyama, Norihide

    2005-08-01

    A high resolution Confocal Laser Scanning Microscope (CLSM) with UV / VIS light sources was developed as the first step of multi-functional microscope. The optical system is designed to optimize for both UV and VIS wavelengths. An UV laser is used to achieve higher resolution, and a VIS is for multi functions. A new objective lens specialized for this application was designed and fabricated. Specification of the lens and the optical system is NA:0.95, EFL:2.5mm, WD:1.5mm, Resolution:160nm and achromatic for two wavelengths of UV 325.0nm / VIS 632.8nm. Several specimens were characterized to check the performance of the system. Some optical materials under study were measured for evaluation, and interesting results could be obtained. Multi-functional measurements are being planed as a next step. This system will help the research of nano-structures, photonic-crystals and biology.

  9. 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.

  10. Confocal scanning laser microscopy and quantitative image analysis: application to cream cheese microstructure investigation.

    PubMed

    Fenoul, F; Le Denmat, M; Hamdi, F; Cuvelier, G; Michon, C

    2008-04-01

    The naked eye observation of cream cheese confocal scanning laser microscopy images only provides qualitative information about its microstructure. Because those products are dense dairy gels, confocal scanning laser microscopy images of 2 different cream cheeses may appear close. Quantitative image analysis is then necessary to compensate for human eye deficiency (e.g., lack of precision, subjectivity). Two kinds of quantitative image analysis were performed in this study: high-order statistical methods and grayscale mathematical morphology. They were applied to study the microstructure of 3 different cream cheeses (same manufacturing process, same dry matter content, but different fat and protein contents). Advantages and drawbacks of both methods are reviewed. The way they may be used to describe cream cheese microstructure is also presented.

  11. Ultrasonic enrichment of microspheres for ultrasensitive biomedical analysis in confocal laser-scanning fluorescence detection

    NASA Astrophysics Data System (ADS)

    Wiklund, M.; Toivonen, J.; Tirri, M.; Hänninen, P.; Hertz, H. M.

    2004-07-01

    An ultrasonic particle concentrator based on a standing-wave hemispherical resonator is combined with confocal laser-scanning fluorescence detection. The goal is to perform ultrasensitive biomedical analysis by concentration of biologically active microspheres. The standing-wave resonator consists of a 4 MHz focusing ultrasonic transducer combined with the optically transparent plastic bottom of a disposable 96-well microplate platform. The ultrasonic particle concentrator collects suspended microspheres into dense, single-layer aggregates at well-defined positions in the sample vessel of the microplate, and the fluorescence from the aggregates is detected by the confocal laser-scanning system. The biochemical properties of the system are investigated using a microsphere-based human thyroid stimulating hormone assay.

  12. 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.

  13. Laser scanning confocal microscopy and laser tweezers based experiments to understand dentine-bacteria interactions

    NASA Astrophysics Data System (ADS)

    Peng, Sum Chee; Mohanty, Samarendra; Gupta, P. K.; Kishen, Anil

    2007-02-01

    Failure of endodontic treatment is commonly due to Enterococcal infection. In this study influence of chemical treatments of type-I collagen membrane by chemical agents commonly used in endodontic treatment on Enterococcus faecalis cell adherence was evaluated. In order to determine the change in number of adhering bacteria after chemical treatment, confocal laser scanning microscopy was used. For this, overnight culture of E faecalis in All Culture broth was applied to chemically treated type-I collagen membrane. It was found that Ca(OH) II treated groups had statistically significant (p value=0.05) increase in population of bacteria adherence. The change in adhesion force between bacteria and collagen was determined by using optical tweezers (1064 nm). For this experiment, Type-I collagen membrane was soaked for 5 mins in a media that contained 50% all culture media and 50% saturated Ca(OH) II . The membrane was spread on the coverslip, on which diluted bacterial suspension was added. The force of laser tweezers on the bacteria was estimated at different trap power levels using viscous drag method and trapping stiffness was calculated using Equipartition theorem method. Presence of Ca(OH) II was found to increase the cell-substrate adherence force from 0.38pN to >2.1pN. Together, these experiments show that it was highly probable that the increase in adherence to collagen was due to a stronger adhesion in the presence of Ca (OH) II.

  14. In vivo confocal imaging of the retina in animal models using scanning laser ophthalmoscopy.

    PubMed

    Seeliger, Mathias W; Beck, Susanne C; Pereyra-Muñoz, Naira; Dangel, Susann; Tsai, Jen-Yue; Luhmann, Ulrich F O; van de Pavert, Serge A; Wijnholds, Jan; Samardzija, Marijana; Wenzel, Andreas; Zrenner, Eberhart; Narfström, Kristina; Fahl, Edda; Tanimoto, Naoyuki; Acar, Niyazi; Tonagel, Felix

    2005-12-01

    Scanning-laser ophthalmoscopy is a technique for confocal imaging of the eye in vivo. The use of lasers of different wavelengths allows to obtain information about specific tissues and layers due to their reflection and transmission characteristics. In addition, fluorescent dyes excitable in the blue and infrared range offer a unique access to the vascular structures associated with each layer. In animal models, a further enhancement in specificity can be obtained by GFP expression under control of tissue-specific promotors. Important fields of application are studies in retinal degenerations and the follow-up of therapeutic intervention.

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

    PubMed

    Wouterlood, Floris G

    2014-04-10

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

  16. 3-D reconstruction of neurons from multichannel confocal laser scanning image series.

    PubMed

    Wouterlood, Floris G

    2005-08-01

    A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. The operator configures separate channels (laser, filters, detector settings) for each fluorochrome used in a particular experiment. Then, 3-D reconstructions are made from Z-series of confocal images: one series per channel. Channel signal separation is extremely important and measures to avoid bleaching are vital. Post-acquisition deconvolution of the image series is then performed to increase resolution. In the 3-D reconstruction program 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 relationship of 3-D-reconstructed structures with respect to structures seen 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 wherever possible.

  17. In vivo observation of papillae of the human tongue using confocal laser scanning microscopy.

    PubMed

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

    2005-01-01

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

  18. 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.

  19. Nondestructive Sectioning Of Fixed And Living Specimens Using A Confocal Scanning Laser Fluorescence Microscope: Microtomoscopy

    NASA Astrophysics Data System (ADS)

    Stelzer, Ernst H...; Wijnaendts-Van-Resandt, Roelof W.

    1987-08-01

    Modern molecular biologists and in particular cell biologists have a large set of experimental tools at their disposal. Immunocytochemistry, fluorescence labels, and microscopy are only subsets of the entire spectrum of methods. Depending on the fields in which biologists work a lot of results are obtained with classical biochemistry, gel electrophoresis and blotting techniques. Gathering morphological data may not be the least important task, but will in many cases be considered only after all other methods have failed. With the advent of video microscopes and the availability of high speed image processing devices, microscopy can also be used for quantitation. Confocal scanning laser fluorescence microscopy (Ft-CSCM) [Cox 1984] is in fact another technique or method that is entering the rapidly developing field of quantitative microscopy. It is therefore very important to understand the physical properties of the CSCLM in detail and to compare a confocal microscope not only with other confocal microscopes, but also with all the other techniques and methods. The confocal microscope has to find its particular application and it should be understood that it will replace neither conventional microscopy, nor video microscopy, nor electron microscopy. It will not be used for every application and every type of investigation. The CSCM has to find its niche in the laboratories and this paper will present two applications in which it proves its usefulness.

  20. Confocal laser scanning microscopy of porcine skin: implications for human wound healing studies

    PubMed Central

    VARDAXIS, N. J.; BRANS, T. A.; BOON, M. E.; KREIS, R. W.; MARRES, L. M.

    1997-01-01

    The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model. PMID:9183682

  1. Measurement of oxygen saturation in small retinal vessels with adaptive optics confocal scanning laser ophthalmoscope.

    PubMed

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

    2011-11-01

    We have used an adaptive optics confocal scanning laser ophthalmoscope to assess oxygen saturation in small retinal vessels. Images of the vessels with a diameter smaller than 50 μm are recorded at oxygen sensitive and isosbestic wavelengths (680 and 796 nm, respectively). The vessel optical densities (ODs) are determined by a computer algorithm. Then, OD ratios (ODRs), which are inversely proportional to oxygen saturation, are calculated. The results show that arterial ODRs are significantly smaller than venous ODRs, indicating that oxygen saturation in the artery is higher than that in the vein. To the best of our knowledge, this is the first noninvasive measurement of oxygen saturation in small retinal vessels.

  2. Re-description of Craspodema reflectans (Nematoda, Cyatholaimidae) using confocal laser scanning microscopy.

    PubMed

    Semprucci, Federica; Burattini, Sabrina

    2015-06-12

    Craspodema reflectans, erected by Gerlach 1964, is here re-described from some specimens recently found in the Maldivian archipelago and the implication of the new findings for the taxonomy of the Craspodema genus is discussed. Accordingly, an emended diagnosis of Craspodema genus and C. reflectans species are proposed. New data are also provided with the aid of the confocal laser scanning microscopy, using the natural fluorescence of the nematodes. The approach described here lays new foundations for the study of Museum collection material and it may be decisive for capture of new morphological details.

  3. 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.

  4. Characterization of microporous membranes using confocal scanning laser microscopy in fluorescence mode

    NASA Astrophysics Data System (ADS)

    Charcosset, C.; Bernengo, J.-C.

    2000-12-01

    Confocal Scanning Laser Microscopy (CSLM) in fluorescence mode was used to characterize microporous membranes. Two microfiltration membranes were investigated: a mixed ester (cellulose nitrate/cellulose acetate) 1.2 μm-rated membrane and a polycarbonate track-etched membrane with cylindrical pores of 2 μm diameter. Optical sections of the membranes stained with rhodamine and mounted in glycerol were performed at 1 μm intervals, from 0 to 10 μm. CSLM was found useful for microporous membrane characterization, as it gives some insight into bulk membrane morphology.

  5. A study of hydrogenated carbon fibers by scanning electron microscopy and confocal laser scanning microscopy.

    PubMed

    de la Cal, Antonio Madroñero; Aguado-Serrano, Juan; Rojas-Cervantes, Maria Luisa; Adame, Elena V Rosa; Marron, Belen Sarmiento; Rosende, Africa Castro; Nevshupa, Roman

    2009-06-01

    The hydrogen absorption process is studied in carbonaceous fibers produced from a mixture of methane and hydrogen. The absorption of the hydrogen was examined in two types of fibers, in "as-grown" state and after a process of desorption during an annealing to 1.473 K under vacuum. Later to its production process, the fibers withstand an oxidation in air to 973 K. The fibers were examined by means of scanning electron microscopy (SEM) and confocal microscopy by reflection. Differences in the behavior during the oxidation were observed between the fibers in as-grown state and those subjected to a further annealing. It could be verified that the fibers were really constituted by two different phases. In one of the phases, the storage of the hydrogen absorbed took place, whereas in the other phase there was no alteration. The process of annealing prior to the absorption of the hydrogen has an appreciable effect on the desorption rate of the hydrogen.

  6. 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.

  7. Evaluation of conjunctival inflammatory status by confocal scanning laser microscopy and conjunctival brush cytology in patients with atopic keratoconjunctivitis (AKC)

    PubMed Central

    Wakamatsu, Tais Hitomi; Okada, Naoko; Kojima, Takashi; Matsumoto, Yukihiro; Ibrahim, Osama M.A.; Adan, Enrique Sato; Fukagawa, Kazumi; Katakami, Chikako; Tsubota, Kazuo; Shimazaki, Jun; Fujishima, Hiroshi

    2009-01-01

    Purpose To elucidate the status of the conjunctival inflammation in atopic keratoconjunctivitis (AKC) using laser scanning confocal microscopy and compare the relevant findings with conjunctival brush cytology in a prospective controlled study. Methods Twenty eyes from 20 AKC patients as well as 16 eyes from 16 age and sex matched normal subjects were studied. The subjects underwent tear film break-up time (BUT), fluorescein and Rose Bengal staining of the ocular surface, conjunctival confocal microscopy, Schirmer test, and brush cytology. Brush cytology specimens and in vivo confocal microscopy scans underwent evaluation for inflammatory cell densities. Results Brush cytology specimens and in vivo confocal microscopy scans from AKC patients revealed significantly higher numbers of inflammatory cells (p<0.05). Conjunctival inflammatory cell density showed a negative correlation with tear stability and a positive correlation with vital staining scores and conjunctival injection grades. The extent of conjunctival inflammation assessed by in vivo confocal microscopy showed a strong positive linear correlation with the inflammation status evaluated by brush cytology. The corneal inflammatory cell density assessed by in vivo confocal microscopy showed a significant negative correlation with tear stability and a positive linear correlation with corneal fluorescein staining. Conclusions Confocal scanning laser microscopy is an efficient, noninvasive, and a promising tool for the quantitative assessment of conjunctival inflammation, a parameter of this new technology which correlated well with subjective and objective ocular surface clinical findings. PMID:19693288

  8. Concomitant use of Congo red staining and confocal laser scanning microscopy to detect amyloidosis in oral biopsy: A clinicopathological study of 16 patients.

    PubMed

    Scivetti, Michele; Favia, Gianfranco; Fatone, Laura; Maiorano, Eugenio; Crincoli, Vito

    2016-01-01

    Twenty oral biopsies from 16 patients were analyzed both by traditional microscopy and by confocal laser scanning microscopy. Using conventional histopathological techniques, the diagnosis of amyloidosis was confirmed only in 15 biopsies. Using confocal laser scanning microscopy, amyloid deposits were detected in all of the samples. The current study shows that confocal laser scanning analysis helps to identify minimal amyloid deposits that could be overlooked using traditional microscopy, thus raising the sensitivity of oral biopsy up to 100%.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. Confocal laser scanning microscopy detection of chlorophylls and carotenoids in chloroplasts and chromoplasts of tomato fruit.

    PubMed

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

    2014-01-01

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

  14. 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.

  15. 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.

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

    PubMed

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

    2007-12-10

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

  17. Trypan blue as a fluorochrome for confocal laser scanning microscopy of arbuscular mycorrhizae in three mangroves.

    PubMed

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

    2008-06-01

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

  18. 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.

  19. 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.

  20. 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

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

    PubMed

    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.

  2. 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.

  3. 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

  4. High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the mouse.

    PubMed

    Paques, Michel; Simonutti, Manuel; Roux, Michel J; Picaud, Serge; Levavasseur, Etienne; Bellman, Caren; Sahel, José-Alain

    2006-04-01

    We evaluated fundus imaging using a modified confocal scanning laser ophthalmoscope (cSLO) in mice. Examinations were performed in conscious, untrained mice. The largest field of view measured 1,520 x 1,520 mu, with a significant interindividual variability, itself correlated to biometric variability. The composite field of view extended up to the ora serrata. The reflectance imaging associated light reflection from nerve fiber bundles and vessel walls, and absorption by hemoglobin and melanin. Light absorption by the pigment epithelium indeed increased the contrast of the nerve fiber layer, but impaired viewing of the choroid. Due to the confocal mode, fluorescence angiograms with clear separation of retinal and choroidal fluorescence could be obtained even in albino mice. Micrometric-scale transverse resolution and several planes of optical sectioning within the retina were obtained. This permitted for instance tridimensional, subcellular viewing of gfp-expressing retinal microglial cells in CX(3)CR1 mice. We concluded that cSLO is a promising tool for noninvasive, multimodal intravital microscopy of the fundus in the mouse.

  5. Simultaneous Confocal Scanning Laser Ophthalmoscopy Combined with High-Resolution Spectral-Domain Optical Coherence Tomography: A Review

    PubMed Central

    Castro Lima, Verônica; Rodrigues, Eduardo B.; Nunes, Renata P.; Sallum, Juliana F.; Farah, Michel E.; Meyer, Carsten H.

    2011-01-01

    We aimed to evaluate technical aspects and the clinical relevance of a simultaneous confocal scanning laser ophthalmoscope and a high-speed, high-resolution, spectral-domain optical coherence tomography (SDOCT) device for retinal imaging. The principle of confocal scanning laser imaging provides a high resolution of retinal and choroidal vasculature with low light exposure. Enhanced contrast, details, and image sharpness are generated using confocality. The real-time SDOCT provides a new level of accuracy for assessment of the angiographic and morphological correlation. The combined system allows for simultaneous recordings of topographic and tomographic images with accurate correlation between them. Also it can provide simultaneous multimodal imaging of retinal pathologies, such as fluorescein and indocyanine green angiographies, infrared and blue reflectance (red-free) images, fundus autofluorescence images, and OCT scans (Spectralis HRA + OCT; Heidelberg Engineering, Heidelberg, Germany). The combination of various macular diagnostic tools can lead to a better understanding and improved knowledge of macular diseases. PMID:22132313

  6. CD87-positive tumor cells in bone marrow aspirates identified by confocal laser scanning fluorescence microscopy.

    PubMed

    Noack, F; Helmecke, D; Rosenberg, R; Thorban, S; Nekarda, H; Fink, U; Lewald, J; Stich, M; Schutze, K; Harbeck, N; Magdolen, V; Graeff, H; Schmitt, M

    1999-10-01

    Dissemination of single tumor cells to the bone marrow is a common event in cancer. The clinical significance of cytokeratin-positive cells detected in the bone marrow of cancer patients is still a matter of debate. In gastric cancer, overexpression of the receptor (uPAR or CD87) for the serine protease urokinase-type plasminogen activator (uPA) in disseminated cancer cells indicates shorter survival of cancer patients. A new immunofluorescence approach, applying confocal laser scanning microscopy, is introduced to locate CD87 antigen in cytokeratin-positive tumor cells and to quantify the CD87 antigen by consecutive scanning. At first, cytokeratin 8/18/19-positive carcinoma cells are identified at excitation wavelength 488 nm using monoclonal antibody A45B/B3 to the cytokeratins and goat anti-mouse IgG labeled with the fluorochrome Alexa488. Next, CD87 in tumor cells is identified by chicken antibody HU277 to the uPA-receptor and goat anti-chicken IgY labeled with fluorochrome Alexa568 (excitation wavelength 568 nm) and the fluorescence signal quantified on a single cell basis using fluorescently labeled latex beads as the fluorescence reference. From 16 patients with gastric or esophageal carcinoma, bone marrow aspirates were obtained, stained for cytokeratins and CD87 and then subjected to laser scanning fluorescence microscopy. Three of six gastric cancer patients had tumor cells present in the bone marrow of which 2 stained for CD87. Three of ten esophageal carcinoma patients had tumor cells in the bone marrow, all three samples stained for CD87. CD87-positive tumor cells were also dissected from stained bone marrow aspirates by laser microdissection microscope to allow analysis of single cells at the gene level.

  7. Two-dimensional confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  8. Confocal laser scanning microscopy and 3-D reconstructions of neuronal structures in human brain cortex.

    PubMed

    Belichenko, P V; Dahlström, A

    1995-09-01

    Human brain material was studied with Lucifer yellow (LY) microinjections, indirect Texas red immunofluorescence, and confocal laser scanning microscopy (CLSM). The scanned images were transferred to a Silicon Graphics (SG) IRIS computer equipped with software for reconstructing the 3-D architecture of cells. By employing dual channel CLSM (Bio-Rad MRC 600), LY-injected cells and Texas red immunofluorescence could be studied simultaneously. Autopsy material with 2- to 48-h postmortem delays (6 control and 2 Rett's syndrome cases) as well as biopsy material (14 cases with therapy-resistant partial epilepsy--TRPE--undergoing neurosurgery) were used. In each specimen, 100-200 pyramidal and nonpyramidal neurons were visualized by LY microinjection. Single neurons were imaged and 2-D reconstructions of each neuron were made using z-projections of serial optical images; 3-D reconstructions and rotations were computed using the SG workstation, with VoxelView software from Vital Images (UK), and stored in a "neuronal library" on laser or magnetic optical disks. In Ret's syndrome cases and in patients with TRPE various abnormalities in the dendritic geometry of pyramidal and nonpyramidal cells have been found. The combination of LY injections with immunofluorescence allows the investigation of transmitter-related substances around the LY-injected cells. Using antibodies to synaptic vesicle proteins, presynaptic elements docking onto individual spines have been demonstrated. This approach may contribute to the understanding of different neurological and psychiatric disorders and may be useful in the Mapping of the Human Brain project. It may also be integrated with functional imaging by PET scan and with the human genome project.

  9. 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.

  10. 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.

  11. Combined molecular ecological and confocal laser scanning microscopic analysis of peat bog methanogen populations.

    PubMed

    Upton, M; Hill, B; Edwards, C; Saunders, J R; Ritchie, D A; Lloyd, D

    2000-12-15

    Confocal laser scanning microscopy, using fluorescently labelled oligonucleotide probes targeting the 16S rRNA of different physiological groups of methanogens, was used to identify which methanogenic genera were present and to describe their in situ spatial locations in samples taken at different depths from blanket peat bog cores. Total bacterial DNA was also extracted and purified from the samples and used as template for amplification of 16S rRNA and regions of methyl CoM reductase-encoding genes using the polymerase chain reaction, as well as for oligonucleotide hybridisation experiments. These techniques, used in concert, demonstrated that methanogens of several physiological groups were present in highest numbers in the mid regions of 25 cm deep peat cores. Some discrepancies were apparent in the findings of the microscopic and molecular methods, though these may be partially accounted for by the different sensitivities of the techniques employed. The combined approaches used in this study gave an insight into the diversity and distribution of methanogens in peat environments not possible using molecular ecological methods alone.

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

    PubMed Central

    MacDonald, Glen H.; Rubel, Edwin W

    2008-01-01

    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 5 parts methyl salicylate and 3 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

  13. Reflective confocal laser scanning microscopy and nonlinear microscopy of cross-linked rabbit cornea

    NASA Astrophysics Data System (ADS)

    Krueger, Alexander; Hovakimyan, Marina; Ramirez, Diego F.; Stachs, Oliver; Guthoff, Rudolf F.; Heisterkamp, Alexander

    2009-07-01

    Cross-linking of the cornea with application of Ribovlavin and UV-A light is an evolving clinical treatment of the eye disease keratoconus. Despite the positive clinical track record of corneal cross-linking, the complex wound healing process after the treatment is still under investigation. In this study an animal model was used to clarify the state of wound healing 5 weeks after treatment. Cross-linked rabbit corneae were imaged with reflective confocal laser scanning and nonlinear microscopy, namely second harmonic imaging microscopy (SHIM) and two-photon excited autofluorescence. First results show that the NAD(P) H-autofluorescence of the corneal keratocytes and their scattering signal still show a signature of the treatment five weeks after the cross-linking procedure. The SHIM signals show the structural morphology of the fibrous collagen sheets in the stroma of the cornea. SHIM detected in the forward direction differs substantially from backward SHIM, but no signature of treatment was found in both detection channels of the SHIM signal.

  14. 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.

  15. The investigation of the dynamic morphology of block copolymer solutions by laser scanning confocal microscopy (LSCM)

    NASA Astrophysics Data System (ADS)

    Lee, Hyunjung

    2005-03-01

    Recently we applied laser scanning confocal microscopy (LSCM) for the study of block copolymer 3D morphology. Besides static measurement of microstructures (direct 3-D imaging of block copolymer morphology), LSCM also enables the tracking of the fast dynamic process which has been impossible by conventional microscopic techniques such as TEM (transmission electron microscopy) or AFM (atomic force microscopy). In this study, in-situ LSCM investigation of the morphology of confined photonic BCP solution was performed in conjunction with spectroscopic measurement for the first time. When a lamellar forming polystyrene-b-isoprene (480k-360k, PS/PI) in cumene was placed between cover glasses, the continuous evaporation of the solvent induced a shear field along the radial direction (evaporation direction). As a result, the photonic lamellar BCP solution over the whole area developed a series of concentric ring pattern covering entire visible colors (blue to red). Comparison of the experimental result with theoretical calculation (transfer matrix method) revealed that this phenomenon mainly comes from the change of the orientation of BCP lamella based on the reflectivity at each region along the radius..

  16. 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.

  17. Presynaptic structure of Aplysia single live neuron by atomic force and confocal laser scanning microscope.

    PubMed

    Park, Aee-Young; Chae, Yeon-Su; Lee, Seung-Hee; Kaang, Bong-Kiun; Lee, Seonghoon

    2013-05-02

    The structural and functional plasticity of Aplysia mechanosensory presynaptic neurons has been studied in relation with the mechanism underlying learning and memory. Long-term facilitation (LTF), which is a well-known cellular model for long-term memory in Aplysia, is accompanied by new synaptic structural growth or change. We developed a combined atomic force microscope and confocal laser scanning microscope (AFM-CLSM) system integrated with a MATLAB routine for image processing to concurrently obtain high-resolution 3-dimensional (3D) outer-surface morphological images and 3D interior fluorescence images. With our combined AFM-CLSM system, volumetric changes in the presynaptic structures (varicosities) of Aplysia live sensory-motor neuron cocultures were observed. The spatial distribution of synaptic vesicle molecules in the preexisting varicosities was monitored together with a volumetric change in the varicosities. Our combined AFM-CLSM system is successfully adapted for measuring learning-related structural changes and the movement of synaptic molecules in the single live neuron through interaction force and fluorescence imaging.

  18. Visualization of ultradeformable liposomes penetration pathways and their skin interaction by confocal laser scanning microscopy.

    PubMed

    Subongkot, Thirapit; Wonglertnirant, Nanthida; Songprakhon, Pucharee; Rojanarata, Theerasak; Opanasopit, Praneet; Ngawhirunpat, Tanasait

    2013-01-30

    The objective of this study was to elucidate the skin penetration pathway of the generated ultradeformable liposomes (ULs) with terpenes for transdermal drug delivery of fluorescein sodium (NaFl). ULs with d-limonene were selected to investigate the penetration pathways and vesicle-skin interaction in terms of release and attachment processes via Confocal Laser Scanning Microscopy (CLSM). A co-localization technique was employed to visualize the skin penetration behavior of UL-labeled red fluorescence (Rh-PE) and fluorescence-entrapped drug (NaFl) through porcine skin. Our results suggested that ULs with entrapped drug might attach to any part of the skin before releasing the entrapped drug into the skin. Most ULs and entrapped drug penetrated through hair follicles more than through the nonfollicular region. In summary, the transfollicular pathway was the major penetration pathway of ULs with d-limonene for transdermal drug delivery of NaFl, whereas the intercellular and transcellular pathways were the minor penetration pathways.

  19. 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.

  20. Three-dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy.

    PubMed

    Buda, Gregory J; Isaacson, Tal; Matas, Antonio J; Paolillo, Dominick J; Rose, Jocelyn K C

    2009-10-01

    Full appreciation of the roles of the plant cuticle in numerous aspects of physiology and development requires a comprehensive understanding of its biosynthesis and deposition; however, much is still not known about cuticle structure, trafficking and assembly. To date, assessment of cuticle organization has been dominated by 2D imaging, using histochemical stains in conjunction with light and fluorescence microscopy. This strategy, while providing valuable information, has limitations because it attempts to describe a complex 3D structure in 2D. An imaging technique that could accurately resolve 3D architecture would provide valuable additions to the growing body of information on cuticle molecular biology and biochemistry. We present a novel application of 3D confocal scanning laser microscopy for visualizing the architecture, deposition patterns and micro-structure of plant cuticles, using the fluorescent stain auramine O. We demonstrate the utility of this technique by contrasting the fruit cuticle of wild-type tomato (Solanum lycopersicum cv. M82) with those of cutin-deficient mutants. We also introduce 3D cuticle modeling based on reconstruction of serial optical sections, and describe its use in identification of several previously unreported features of the tomato fruit cuticle.

  1. Laser scanning confocal microscopy for in situ monitoring of alkali-silica reaction.

    PubMed

    Collins, C L; Ideker, J H; Kurtis, K E

    2004-02-01

    Alkali-silica reaction (ASR) occurs in concrete between reactive siliceous components in the aggregate and the strongly alkaline pore solution, resulting in the formation of a potentially expansive gel product. Lithium additives have been shown to reduce expansion associated with ASR, but the mechanism(s) by which lithium reduces expansion have not been understood. Therefore, development of an in situ method to observe reactions associated with ASR is highly desirable, as it will allow for non-destructive observation of the reaction product formation and damage evolution over time, as the reaction progresses. A technique to image into mortar through glass aggregate by laser scanning confocal microscopy (LSCM), producing three-dimensional representations of the sample was developed. This LSCM technique was utilized to monitor the progress of alkali-silica reaction in mortar samples prepared with alkali-reactive glass aggregate both in the presence and in the absence of lithium additives: LiNO3, LiCl or LiOH. The method proved to be effective in qualitatively monitoring crack formation and growth and product formation, within cracks and at the paste/aggregate interface. In particular, dendritic products were observed at the paste/aggregate interface only in those samples containing lithium, suggesting that these products may play a role in ASR mitigation.

  2. 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.

  3. Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling.

    PubMed

    Sonomura, Takahiro; Furuta, Takahiro; Nakatani, Ikuko; Yamamoto, Yo; Unzai, Tomo; Matsuda, Wakoto; Iwai, Haruki; Yamanaka, Atsushi; Uemura, Masanori; Kaneko, Takeshi

    2013-01-01

    Recently, three-dimensional reconstruction of ultrastructure of the brain has been realized with minimal effort by using scanning electron microscopy (SEM) combined with focused ion beam (FIB) milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy (EM) provides a great advantage in that molecules of interest are specifically localized in ultrastructures. Thus, we applied immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in confocal laser-scanning microcopy (CF-LSM). Dendrites of medium-sized spiny neurons in the rat neostriatum were visualized using a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively. In contrast-inverted FIB-SEM images, silver precipitations and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were as easily recognizable as those in the transmission electron microscopy (TEM) images. Furthermore, in the sites of interest, some appositions displayed synaptic specializations of an asymmetric type. Thus, the present method was useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connections in the central neural circuit.

  4. Development of a high speed laser scanning confocal microscope with an acquisition rate up to 200 frames per second.

    PubMed

    Choi, S; Kim, P; Boutilier, R; Kim, M Y; Lee, Y J; Lee, H

    2013-10-07

    There has been an increasing interest for observing fast biological phenomena such as cell movements in circulations and action potentials. The laser scanning confocal microscopy offers a good spatial resolution and optical sectioning ability to observe various in vivo animal models. We developed a high speed laser scanning confocal microscope capable of acquiring 512 by 512 pixel images at 200 fps (frames per second). We have incorporated a fast rotating polygonal scanning mirror with 128 facets for the X-axis scanner. In order to increase the throughput of the Y-axis scanner, we applied a bi-directional scanning method for vertical scanning. This made it possible to scan along the Y-axis two times during each scanner motion cycle. For the image acquisition, we used a custom photomultiplier tube amplifier with a broad frequency band. In addition, custom imaging software was written for the new microscope. In order to verify the acquisition speed of the developed confocal microscope, a resolution target moving at a series of constant speeds and a sedated mouse with slight movements due to heartbeats were observed. By comparing successive frames, the frame acquisition speeds were calculated.

  5. Application of Confocal and Spectrally Resolved Techniques to Scanning Laser Photoluminescence Microscopy.

    NASA Astrophysics Data System (ADS)

    Bowron, John William

    Both confocal microscopes and photoluminescence wafer mapping systems are well-developed technologies, however the application of confocal techniques to photoluminescence microscopy is not common in the literature. While developing this microscope a novel design for a spectrally-resolved detection arm was implemented. The microscope shows full confocal capabilities in reflected light operation, good spectral sensitivity in the visible region and a range of possible spectral resolutions between 10 nm and 0.1 nm, however the axial response in photoluminescence operation was found to be broader than expected by a factor of two. Calculations were performed to model and understand the new microscope. Simulations of the axial-response of an infinity-connected microscope in reflected light agreed well with experimental data. A new prediction showed that under certain circumstances the maximum signal is not always obtained at best focus. This prediction was confirmed later by experiment. These calculations were extended to understand the broadening observed in photoluminescence imaging. Three factors were considered: absorption in the material, diffusion of photo-excited carriers and the high refractive index of the material. The utility of the microscope was demonstrated by using it to image several different samples. Near-infrared fluorescence imaging was demonstrated for a stained biological specimen. Auto-fluorescence imaging was demonstrated using an ultra-violet laser and spectrally-resolved images were used to distinguish between various materials in the specimen. Confocal image stacking was demonstrated in photoluminescence on a CuO sample. Confocal photoluminescence images were shown to have higher spatial resolution than non-confocal images. Quantitative information was obtained for a SiC sample containing several polytypes. The optical measurements were then correlated with X-ray diffraction measurements in order to arrive at a polytype identification scheme

  6. Local intracellular ion measurements with luminescent indicators using confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Opitz, N.; Merten, E.; Acker, H.

    1995-09-01

    Ion sensitive fluoroprobes such as BCECF (pH) and FURA-II (Ca2+) are frequently used indicators for determination of ion activities in single cells and subcellular compartments, e.g. by video enhanced or video intensified microscopy. Moreover, using confocal laser scanning microscopy (CLSM) with its inherent potential for noninvasive optical sectioning of cells and tissues and subsequent 3D image reconstruction, intracellular ion topographies can be monitored via pseudocolor encoded ratio imaging from pixel to pixel enabling in vivo measurements of dynamic intracellular processes. Regardless of the degree of spatial resolution, reliable qualtitative determinations essentially depend on accurate calibration of the intracellularly entrapped fluoroprobe. Calibration is either established on the basis of a whole cell or within a more or less extended subcellular compartment and the characteristics are displayed as concentration encoded pseudocolor bar within the image frame. This calibration is assumed to be valid for other cellular compartments and, in case of ion imaging, it is even thought to be valid for every single pixel of the complete pixel field. However, the assumption of a topographically invariant intracellular calibration requires a reliable behavior of the intracellularly applied indicator. This intracellular integrity of the dyes often does not seem to exist since intracellular calibration curves considerably deviate from in vitro calibration characteristics. Deviations may be due to intracellular interactions of indicator molecules with cytoplasmic macromolecules, e.g. proteins, resulting in spectral distortions and/or sensitivity deficits as demonstrated by the indicators BCECF and FURA-RED (a FURA-II analogue) or to intracellular redistribution of the indicator as exemplified by pH measurements using carboxy-SNARF-1. Consequences of these investigations as well as further potential interferences are discussed with special respect to ion imaging

  7. 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.

  8. Fast intracellular motion in the living cell by video rate reflection confocal laser scanning microscopy

    PubMed Central

    VESELY, PAVEL; BOYDE, ALAN

    2001-01-01

    Fast intracellular motion (FIM) was first revealed by back scattered light (BSL) imaging in video rate confocal scanning laser microscopy (VRCSLM), beyond the limits of spatial and temporal resolution obtainable with conventional optical microscopy. BSL imaging enabled visualisation of intra and extracellular motion with resolution in space down to 0.2 μm and in time to 1/25th of a second. Mapping the cell space at 0.2 μm×0.2 μm (XY = in instantaneous best focal plane)×0.5 μm (Z = height/depth, optic axis direction) volume steps revealed a communication layer above the known contact layer and an integrated dynamic spatial network (IDSN) towards the cell centre. FIM was originally observed as localised quasichaotic dancing (dithering) or reflecting patches/spots in the cell centre, faster in the darker nuclear space. Later, a second type of FIM was recognised which differed by the presence of a varied proportion of centrifugal and centripetal directional movements and/or jumping of patches/spots in the cell centre and outside the nuclear space. The first type is characteristic for cells in slightly adverse conditions while the second type has so far only been found in eutrophic cells. Temporal speeding up and coarsening of FIM, followed by slowing and eventually cessation at cell death, was found on exposure to strong stressors. It was concluded that the state of FIM provides instantaneous information about individual cell reactions to actual treatment and about cell survival. A putative switch between the first and second type FIM could be considered as an indicator of timing of cellular processes. The significance of FIM for the biology of the cell is seen in the rapid assessment of the condition of an individual live cell investigated by combination of various methods. Requirements for further development of this approach are outlined. PMID:11465857

  9. Laser scanning confocal microscopy characterization of water repellent distribution in a sandstone pore network.

    PubMed

    Zoghlami, Karima; Gómez-Gras, David; Corbella, Mercè; Darragi, Fadila

    2008-11-01

    In the present work, we propose the use of the Laser Scanning Confocal Microscopy (LSCM) to determine the effect of water repellents on rock's pore-network configuration and interconnection. The rocks studied are sandstones of Miocene age, a building material that is commonly found in the architectural heritage of Tunisia. The porosity quantitative data of treated and untreated samples, obtained by mercury porosimetry tests, were compared. The results show a slight decrease in total porosity with the water repellent treatment, which reduced both microporosity and macroporosity. This reduction produced a modification in pore size distribution and a shift of the pore access size mode interval toward smaller pore diameters (from the 30-40 microm to the 20-30 microm intervals). The water repellent was observed in SEM images as a continuous film coating grain surfaces; moreover, it was easily visualized in LSCM, by staining the water repellent with Epodye fluorochrome, and the coating thickness was straightforwardly measured (1.5-2 microm). In fact, the combination of mercury intrusion porosimetry data and LSCM observations suggests that the porosity reduction and the shift of the pore diameter mode were mainly due to the general reduction of pore diameters, but also to the plugging of the smallest pores (less than 3-4 microm in diameter) by the water repellent film. Finally, the LSCM technique enabled the reconstruction of 3D views of the water repellent coating film in the pore network, indicating that its distribution was uniform and continuous over the 100 microm thick sample. The LSCM imaging facilitates the integration and interpretation of mercury porosimetry and SEM data.

  10. 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.

  11. 3-D confocal laser scanning microscopy used in morphometric analysis of rat Purkinje cell dendritic spines after chronic ethanol consumption.

    PubMed

    Wenisch, S; Fortmann, B; Steinmetz, T; Kriete, A; Leiser, R; Bitsch, I

    1998-12-01

    A confocal laser scanning microscope (with a 543 nm laser) was used for imaging rat Purkinje cell dendritic spines at high 3-D resolution. In a nutritionally controlled study of the rat, 5 months of ethanol consumption was demonstrated to alter the spines of Purkinje cell dendrites in rat cerebellum. Intact spines showed significant elongation after ethanol exposure, whereas this neuromorphological alteration could not be detected in controls. Spine elongation could be regarded as compensative growth of spines in search of new synaptic contacts due to alcohol induced cell loss.

  12. A confocal video-rate laser-beam scanning reflected-light microscope with no moving parts.

    PubMed

    Goldstein, S R; Hubin, T; Rosenthal, S; Washburn, C

    1990-01-01

    A no-moving-parts, 30 frames/s, laser-beam scanning confocal reflected-light microscope has been developed. In principle, the technique can be extended to fluorescence and transmission light microscopy. Acousto-optic beam deflectors controlled by digital electronics move a laser beam in a 512-line interlaced 8.5 x 8.5-mm raster. The light passes through a beam splitter, enters an inverted microscope through the side camera port, and is imaged at the object by the microscope objective. Reflected light returns through the objective, exits the camera port, is reflected off the beam splitter, and is imaged on to the photocathode of an image dissector tube (IDT). Confocality is provided by raster scanning the IDT aperture coincident with the congruent image of the laser beam incident on the object. Real-time jitter-free reflected light images of a variety of biological objects have been produced. Computer-controlled alignment of the laser scan and IDT is performed in several seconds.

  13. In-vivo confocal scanning laser ophthalmoscopic characterization of retinal pathology in a small-eye-animal model

    NASA Astrophysics Data System (ADS)

    Zwick, Harry; Elliot, Rowe; Schuschereba, Steven T.; Lund, David J.; Stuck, Bruce E.

    1997-05-01

    We have used confocal scanning laser ophthalmoscopy (CSLO) to evaluate acute laser retinal injury in a small eye animal model. THe snake eye is optically unique, combining a high numerical aperture with a clear ocular media and a cornea covered with a hard dry spectacle. These optical qualities allow detailed resolution of photoreceptors, retinal nerve fiber, and retinal capillary blood cells in an intact vertebrate eye. We demonstrated that acute laser exposures capable of damaging the photoreceptor matrix may also alter blood flow at more anterior levels of the retina. Changes in photoreceptor density and orientation were indicated in the early post exposure seconds at high dose acute Argon laser exposures. An increase in photoreceptor reflectivity was observed in surviving photoreceptors and was enhanced with a near IR CSLO imaging source. Q-switched exposure failed to show this enhancement, possibly because of greater subchoroidal involvement associated with acoustic damage processes.

  14. 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)

  15. 3D imaging of cement-based materials at submicron resolution by combining laser scanning confocal microscopy with serial sectioning.

    PubMed

    Yio, M H N; Mac, M J; Wong, H S; Buenfeld, N R

    2015-05-01

    In this paper, we present a new method to reconstruct large volumes of nontransparent porous materials at submicron resolution. The proposed method combines fluorescence laser scanning confocal microscopy with serial sectioning to produce a series of overlapping confocal z-stacks, which are then aligned and stitched based on phase correlation. The method can be extended in the XY plane to further increase the overall image volume. Resolution of the reconstructed image volume does not degrade with increase in sample size. We have used the method to image cementitious materials, hardened cement paste and concrete and the results obtained show that the method is reliable. Possible applications of the method such as three-dimensional characterization of the pores and microcracks in hardened concrete, three-dimensional particle shape characterization of cementitious materials and three-dimensional characterization of other porous materials such as rocks and bioceramics are discussed.

  16. 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.

  17. A simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope.

    PubMed

    Rumio, C; Morini, M; Miani, A; Barajon, I; Castano, P

    1995-01-01

    A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips ('sandwich' style) and the optical tomography was performed from both sides by turning the 'sandwich' upside-down. The samples were impregnated using standard Golgi-Cox, 'rapid Golgi' or other silver methods. The ability to turn the 'sandwich' upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e., near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.

  18. SarConfoCal: simultaneous sarcomere length and cytoplasmic calcium measurements for laser scanning confocal microscopy images.

    PubMed

    Pasqualin, Côme; Gannier, François; Yu, Angèle; Malécot, Claire O; Bredeloux, Pierre; Maupoil, Véronique

    2016-12-22

    Simultaneous recordings of myocytes contractility and their cytoplasmic calcium concentration allow powerful studies, particularly on heart failure and other cardiac dysfunctions. Such studies require dedicated and expensive experimental devices that are difficult to use. Thus we propose SarConfoCal, the first and only software to simultaneously analyse both cytoplasmic calcium variations (from fluorescence signal) and myocytes contractility (from sarcomere length measurement) on laser scanning confocal microscopy images. SarConfoCal is easy to set up and use, especially by people without programming skills.

  19. 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.

  20. Short-pulsed diode lasers as an excitation source for time-resolved fluorescence applications and confocal laser scanning microscopy in PDT

    NASA Astrophysics Data System (ADS)

    Kress, Matthias; Meier, Thomas H.; El-Tayeb, Tarek A. A.; Kemkemer, Ralf; Steiner, Rudolf W.; Rueck, Angelika C.

    2001-11-01

    This article describes a setup for subcellular time-resolved fluorescence spectroscopy and fluorescence lifetime measurements using a confocal laser scanning microscope in combination with a short pulsed diode laser for fluorescence excitation and specimen illumination. The diode laser emits pulses at 398 nm wavelength with 70 ps full width at half maximum (FWHM) duration. The diode laser can be run at a pulse repetition rate of 40 MHz down to single shot mode. For time resolved spectroscopy a spectrometer setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Subcellular fluorescence lifetime measurements were achieved using a time-correlated single photon counting (TCSPC) module instead of the spectrometer setup. The capability of the short pulsed diode laser for fluorescence imaging, fluorescence lifetime measurements and time-resolved spectroscopy in combination with laser scanning microscopy is demonstrated by fluorescence analysis of several photosensitizers on a single cell level.

  1. 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.

  2. Application of confocal laser scanning microscopy for the diagnosis of amyloidosis.

    PubMed

    Castellani, Chiara; Fedrigo, Marny; Frigo, Anna Chiara; Barbera, Mila Della; Thiene, Gaetano; Valente, Marialuisa; Adami, Fausto; Angelini, Annalisa

    2017-02-20

    We analysed specificity and sensitivity of confocal laser microscopy (CLSM) on tissue sections for a diagnosis of amyloidosis, in an attempt to reduce technical errors and better standardise pathological diagnosis. We first set up a protocol for the use of CLSM on this type of specimen, using a group of 20 amyloid negative and 20 positive samples. Of all specimens, 2, 4 and 8-μm sections were cut. Sections were stained with Congo red (CR) and thioflavin-T (ThT) and observed by cross-polarised light microscopy (CR-PL), epifluorescence microscopy (CRF-epiFM and ThT-epiFM) and CLSM (CRF-CLSM and ThT-CLSM). To validate the method in a diagnostic setting, we examined tissue samples from 116 consecutive patients with clinical suspicion of amyloidosis, selected from the period 2005 to 2014 from the database of the Pathology Unit of the University of Padua. The results were compared with those of transmission electron microscopy (TEM), which we consider as reference. We found that with CRF-CLSM, the false negative rate was reduced from 17 to 5%, while the sensitivity of detection increased to 12%. The results were in complete agreement with those of TEM ThT-CLSM; both sensitivity and specificity were 100%. Finally, ThT-CLSM results did not vary with section thickness, and small amounts of amyloid could even be detected in 2-μm sections. In conclusion, we found ThT-CLSM to be more sensitive as a screening method for amyloidosis than CR and ThT epifluorescence optical imaging. The method was easier to standardise, provided images with better resolution and resulted in more consistent pathologist diagnoses.

  3. The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy.

    PubMed

    Cheng, Y; Huo, Q; Lu, J; Li, R; Wang, K

    1999-08-01

    A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol. From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and extracellular concentration. For a given concentration, the transport time of [Ln(cit)2]3- is much shorter than that of Ln3+, since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4'-diisothiocyanato-2,2'-stilbenendisulfonate on the transport of [Ln(cit)2]3-. On the other hand, the transport of free Ln3+ might be attributed to the enhanced permeability of erythrocytes owing to Ln3+ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes.

  4. 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 ...

  5. Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method.

    PubMed

    Gutierre, R C; Vannucci Campos, D; Mortara, R A; Coppi, A A; Arida, R M

    2017-01-05

    Confocal laser-scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser-scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China ink-injected specimens by the Spalteholz method. This method may be suitable for global, three-dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis.

  6. 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.

  7. Extracting the ridge set as a graph for actin filament length estimation from confocal laser scanning microscopic images

    NASA Astrophysics Data System (ADS)

    Birkholz, Harald

    2012-04-01

    The progress in image acquisition techniques provides life sciences with an abundance of data. Image analysis facilitates the assessment. The actin cytoskeleton plays a crucial role in understanding the behavior of osteoblastic cells on biomaterials. In the flat basal part of the cells, it can be visualized by confocal laser scanning microscopy. In the microscopic images, the stained cytoskeleton appears as a dense network of bright ridges which is so far only qualitatively assessed. For its quantification, there is a need for ridge detection techniques that provide a geometrical description of this graph feature. The state of the art methods do not cope with the systematical degradation by noise, unspecific luminance, and uneven dye uptake. This work presents the key part of a ridge-tracking technique, which makes more efficient use of context information, and evaluate it by its length measurement accuracy. Two random models illustrate the performance against ground truth. Representative microscopic images confirm the applicability.

  8. 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.

  9. 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.

  10. Imaging of oxidative stress at subcellular level by confocal laser scanning microscopy after fluorescent derivatization of cellular carbonyls.

    PubMed Central

    Pompella, A.; Comporti, M.

    1993-01-01

    Confocal laser scanning fluorescence microscopy plus image videoanalysis was used to visualize the tissue areas and the subcellular sites first involved by oxidative stress and lipid peroxidation, in the well-established experimental model of lipid peroxidation induced by haloalkane intoxication in the liver cell. The fluorescent reagent 3-hydroxy-2-naphthoic acid hydrazide was employed to derivativize the carbonyl functions originating from the lipoperoxidative process in situ, in liver cryostat sections from in vivo intoxicated rats, as well as in isolated hepatocytes exposed in vitro to the pro-oxidant action of haloalkanes. The results obtained indicate that: 1) the detection of fluorescent derivatives of carbonyls indeed offers a gain in sensitivity, 2) haloalkane-induced lipid peroxidation in hepatocytes primarily involves the perinuclear endoplasmic reticulum, whereas the plasma membrane and the nuclear compartment are unaffected, and 3) lipid peroxidation also induces an increase of liver autofluorescence. Images Figure 2 Figure 4 PMID:8494040

  11. Confocal scanning laser tomography of the optic nerve head on the patients with Alzheimer's disease compared to glaucoma and control.

    PubMed

    Kurna, Sevda Aydin; Akar, Gokcen; Altun, Ahmet; Agirman, Yasemin; Gozke, Eren; Sengor, Tomris

    2014-12-01

    The purpose of this study was to evaluate optic nerve head (ONH) differences of the patients with Alzheimer's disease (AD) measured by confocal scanning laser tomography [Heidelberg Retina Tomograph (HRT) III] and compare with glaucoma and control subjects. Eighty-four patients were enrolled into the study: 44 eyes of 24 patients with mild to moderate AD (Group 1), 68 eyes of 35 patients with glaucoma (Group 2), and 49 eyes of 25 heathy volunteers as a control (Group 3). A complete ophthalmologic examination as well as a confocal scanning laser ophthalmoscopic assessment with HRT III were performed on all patients. Mean values of the ONH topographic parameters such as rim area (RA), rim volume (RV), height variation contour, linear cup/disc ratio, cup shape measure, and retinal nerve fiber layer (RNFL) were recorded. Mean values of RNFL thickness was 0.23 ± 0.07 in AD, 0.22 ± 0.09 in glaucoma and 0.24 ± 0.07 in the control group (p = 0.323). RA and RV were significantly lower, and linear C/D ratio was significantly higher in the glaucoma group when compared to AD and control (p < 0.05). There was no statistically significant difference between AD and control for the optic disc parameters tested (p > 0.05). We observed a negative correlation of the age with RNFL in all of the groups (p < 0.005). Age was the most important parameter affecting RNFL. Our results suggest that HRT does not demonstrate ONH differences between AD and control group, while it successfully differentiates glaucoma from AD and control cases of older age.

  12. Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara.

    PubMed

    Borges, J N; Costa, V S; Mantovani, C; Barros, E; Santos, E G N; Mafra, C L; Santos, C P

    2017-02-01

    Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish-eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P. macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox-1) are presented. Phylogeny reconstructions linked P. macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin-fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P. macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High-resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes.

  13. 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.

  14. 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.

  15. Confocal laser scanning microscopy elucidation of the micromorphology of the leaf cuticle and analysis of its chemical composition.

    PubMed

    Nadiminti, Pavani P; Rookes, James E; Boyd, Ben J; Cahill, David M

    2015-11-01

    Electron microscopy techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been invaluable tools for the study of the micromorphology of plant cuticles. However, for electron microscopy, the preparation techniques required may invariably introduce artefacts in cuticle preservation. Further, there are a limited number of methods available for quantifying the image data obtained through electron microscopy. Therefore, in this study, optical microscopy techniques were coupled with staining procedures and, along with SEM were used to qualitatively and quantitatively assess the ultrastructure of plant leaf cuticles. Leaf cryosections of Triticum aestivum (wheat), Zea mays (maize), and Lupinus angustifolius (lupin) were stained with either fat-soluble azo stain Sudan IV or fluorescent, diarylmethane Auramine O and were observed under confocal laser scanning microscope (CLSM). For all the plant species tested, the cuticle on the leaf surfaces could be clearly resolved in many cases into cuticular proper (CP), external cuticular layer (ECL), and internal cuticular layer (ICL). Novel image data analysis procedures for quantifying the epicuticular wax micromorphology were developed, and epicuticular waxes of L. angustifolius were described here for the first time. Together, application of a multifaceted approach involving the use of a range of techniques to study the plant cuticle has led to a better understanding of cuticular structure and provides new insights into leaf surface architecture.

  16. An Automated Approach for Localizing Retinal Blood Vessels in Confocal Scanning Laser Ophthalmoscopy Fundus Images.

    PubMed

    Kromer, Robert; Shafin, Rahman; Boelefahr, Sebastian; Klemm, Maren

    In this work, we present a rules-based method for localizing retinal blood vessels in confocal scanning laser ophthalmoscopy (cSLO) images and evaluate its feasibility. A total of 31 healthy participants (17 female; mean age: 64.0 ± 8.2 years) were studied using manual and automatic segmentation. High-resolution peripapillary scan acquisition cSLO images were acquired. The automated segmentation method consisted of image pre-processing for gray-level homogenization and blood vessel enhancement (morphological opening operation, Gaussian filter, morphological Top-Hat transformation), binary thresholding (entropy-based thresholding operation), and removal of falsely detected isolated vessel pixels. The proposed algorithm was first tested on the publically available dataset DRIVE, which contains color fundus photographs, and compared to performance results from the literature. Good results were obtained. Monochromatic cSLO images segmented using the proposed method were compared to those manually segmented by two independent observers. For the algorithm, a sensitivity of 0.7542, specificity of 0.8607, and accuracy of 0.8508 were obtained. For the two independent observers, a sensitivity of 0.6579, specificity of 0.9699, and accuracy of 0.9401 were obtained. The results demonstrate that it is possible to localize vessels in monochromatic cSLO images of the retina using a rules-based approach. The performance results are inferior to those obtained using fundus photography, which could be due to the nature of the technology.

  17. 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.

  18. 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

  19. Three-dimensional volume reconstruction of extracellular matrix proteins in uveal melanoma from fluorescent confocal laser scanning microscope images

    PubMed Central

    BAJCSY, P.; LEE, S-C.; LIN, A.; FOLBERG, R.

    2006-01-01

    Summary The distribution of looping patterns of laminin in uveal melanomas and other tumours has been associated with adverse outcome. Moreover, these patterns are generated by highly invasive tumour cells through the process of vasculogenic mimicry and are not therefore blood vessels. Nevertheless, these extravascular matrix patterns conduct plasma. The three-dimensional (3D) configuration of these laminin-rich patterns compared with blood vessels has been the subject of speculation and intensive investigation. We have developed a method for the 3D reconstruction of volume for these extravascular matrix proteins from serial paraffin sections cut at 4 μm thicknesses and stained with a fluorescently labelled antibody to laminin (Maniotis et al., 2002). Each section was examined via confocal laser-scanning focal microscopy (CLSM) and 13 images were recorded in the Z-dimension for each slide. The input CLSM imagery is composed of a set of 3D subvolumes (stacks of 2D images) acquired at multiple confocal depths, from a sequence of consecutive slides. Steps for automated reconstruction included (1) unsupervised methods for selecting an image frame from a subvolume based on entropy and contrast criteria, (2) a fully automated registration technique for image alignment and (3) an improved histogram equalization method that compensates for spatially varying image intensities in CLSM imagery due to photo-bleaching. We compared image alignment accuracy of a fully automated method with registration accuracy achieved by human subjects using a manual method. Automated 3D volume reconstruction was found to provide significant improvement in accuracy, consistency of results and performance time for CLSM images acquired from serial paraffin sections. PMID:16438687

  20. 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.

  1. 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.

  2. Effect of paeoniflorin on the calcium ion concentration in salivary gland cells using confocal laser scanning microscopy

    PubMed Central

    Qian, Xian; Shi, Xiaolu; Wang, Hongyi

    2016-01-01

    Objective: To investigate the effects of paeoniflorin, the main monomer component of Jinxueyuan granules, on the Ca2+ concentrations in salivary gland cells to further explore the salivation-promoting mechanism and effective monomer components of Jinxueyuan granules. Methods: The salivary gland cells of suckling rats were cultured in vitro and loaded with a Fluo-3AM fluorescent probe, and changes in the intracellular Ca2+ concentrations were observed using a confocal laser scanning microscope. Results: No significant changes in the intracellular Ca2+ concentrations were demonstrated (P>0.05) in the paeoniflorin-free Hank’s media treatment group or in the higher-dose paeoniflorin (10-2 mol/L) Hank’s media treatment group; however, a significant increase in the intracellular Ca2+ concentration in the lower-dose paeoniflorin (10-4 mol/L) treatment group was observed (P=0.001). Further study showed that treatment with the calcium channel blocker verapamil hydrochloride or with Ca2+-free D-Hank’s media did not block the paeoniflorin-induced (10-4 mol/L) increase in intracellular Ca2+ (P<0.05). Conclusion: Paeoniflorin promotes the release of endogenous calcium to upregulate the intracellular Ca2+ concentration. Further studies should be performed to investigate the association between paeoniflorin and the Ca2+ concentration in salivary gland cells and to elucidate the corresponding functional pathways. PMID:27725850

  3. 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

  4. 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

  5. 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.

  6. Video rate confocal laser scanning reflection microscopy in the investigation of normal and neoplastic living cell dynamics.

    PubMed

    Vesely, P; Boyde, A

    1996-01-01

    The introduction of video rate confocal laser scanning microscopes (VRCLSM) used in reflection mode with high magnification, high aperture objective lenses and with further magnification by a zoom facility allowed the first detailed observations of the activity of living cytoplasm and offered a new tool for investigation of the structural transition from the living state to the specimen fixed for electron microscopy (EM). We used a Noran Odyssey VRCLSM in reflection (backscattered) mode. A greater degree of oversampling and more comfortable viewing of the liver or taped video image was achieved at zoom factor 5, giving a display monitor field width of 10 microns. A series of mesenchyme derived cell lines--from normal cells to sarcoma cells of different malignancy--was used to compare behaviour of the observed intracellular structures and results of fixation. We contrasted the dynamic behaviour of fine features in the cytoplasm of normal and neoplastic living cells and changes induced by various treatments. The tubulomembraneous 3D structure of cytoplasm in living cells is dynamic with motion observable at the new limits of resolution provided by VRCLSM. All organelles appear integrated into one functional compartment supporting the continuous 3D trafficking of small particles (vesicles). This integrated dynamic spatial network (IDSN) was found to be largest in neoplastic cells.

  7. Distribution and quantification of polyethylenimine oligodeoxynucleotide complexes in human skin after iontophoretic delivery using confocal scanning laser microscopy.

    PubMed

    Brus, Carola; Santi, Patrizia; Colombo, Paolo; Kissel, Thomas

    2002-12-05

    Iontophoresis may be a potentially useful technique for the delivery of oligonucleotides into the skin. To enhance intracellular uptake during iontophoresis, we investigated the dermal delivery of oligodeoxynucleotides (ODN) as a polyelectrolyte complex with polyethylenimine (PEI). Perpendicular cross-sectioning was performed to visualize and quantify the penetration properties of double labeled PEI/ODN complexes across full thickness human skin. Due to the net positive charge of the complexes, anodal iontophoresis was expected to enhance skin delivery by electrorepulsion compared to passive diffusion. Confocal laser scanning microscopy demonstrated that non-complexed ODN could penetrate the skin after 1 h of cathodal iontophoresis but not by passive diffusion or anodal iontophoresis. However, extensive degradation occurred as documented by a dramatic decrease of fluorescence intensity within viable skin tissue after 10 h. Anodal iontophoresis of the complexes led to a deep penetration of both the TAMRA-labeled ODN and the Oregon Green-labeled PEI. A constant increase in fluorescence indicated a protective effect of the polymer against nuclease degradation. Co-localization of red and green fluorescence was noted within numerous nuclei of epidermal keratinocytes. In contrast, passive diffusion of the complexes did not lead to successful uptake into keratinocytes and was limited to the stratum corneum. Complexation of ODN by PEI, therefore, seems to be a promising method to enhance both the transport of charged complexes into the skin and to facilitate intracellular uptake, which may potentially be useful for the local treatment of skin diseases using ODN.

  8. Vessel Labeling in Combined Confocal Scanning Laser Ophthalmoscopy and Optical Coherence Tomography Images: Criteria for Blood Vessel Discrimination

    PubMed Central

    Motte, Jeremias; Alten, Florian; Ewering, Carina; Osada, Nani; Kadas, Ella M.; Brandt, Alexander U.; Oberwahrenbrock, Timm; Clemens, Christoph R.; Eter, Nicole; Paul, Friedemann; Marziniak, Martin

    2014-01-01

    Introduction The diagnostic potential of optical coherence tomography (OCT) in neurological diseases is intensively discussed. Besides the sectional view of the retina, modern OCT scanners produce a simultaneous top-view confocal scanning laser ophthalmoscopy (cSLO) image including the option to evaluate retinal vessels. A correct discrimination between arteries and veins (labeling) is vital for detecting vascular differences between healthy subjects and patients. Up to now, criteria for labeling (cSLO) images generated by OCT scanners do not exist. Objective This study reviewed labeling criteria originally developed for color fundus photography (CFP) images. Methods The criteria were modified to reflect the cSLO technique, followed by development of a protocol for labeling blood vessels. These criteria were based on main aspects such as central light reflex, brightness, and vessel thickness, as well as on some additional criteria such as vascular crossing patterns and the context of the vessel tree. Results and Conclusion They demonstrated excellent inter-rater agreement and validity, which seems to indicate that labeling of images might no longer require more than one rater. This algorithm extends the diagnostic possibilities offered by OCT investigations. PMID:25203135

  9. 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.

  10. 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.

  11. Evaluation of Enterococcus faecalis adhesion, penetration, and method to prevent the penetration of Enterococcus faecalis into root cementum: Confocal laser scanning microscope and scanning electron microscope analysis

    PubMed Central

    Halkai, Rahul S.; Hegde, Mithra N.; Halkai, Kiran R.

    2016-01-01

    Aim: To ascertain the role of Enterococcus faecalis in persistent infection and a possible method to prevent the penetration of E. faecalis into root cementum. Methodology: One hundred and twenty human single-rooted extracted teeth divided into five groups. Group I (control): intact teeth, Group II: no apical treatment done, Group III divided into two subgroups. In Groups IIIa and IIIb, root apex treated with lactic acid of acidic and neutral pH, respectively. Group IV: apical root cementum exposed to lactic acid and roughened to mimic the apical resorption. Group V: apical treatment done same as Group IV and root-end filling done using mineral trioxide aggregate (MTA). Apical one-third of all samples immersed in E. faecalis broth for 8 weeks followed by bone morphogenetic protein and obturation and again immersed into broth for 8 weeks. Teeth split into two halves and observed under confocal laser scanning microscope and scanning electron microscope, organism identified by culture and polymerase chain reaction techniques. Results: Adhesion and penetration was observed in Group IIIa and Group IV. Only adhesion in Group II and IIIB and no adhesion and penetration in Group I and V. Conclusion: Adhesion and penetration of E. faecalis into root cementum providing a long-term nidus for subsequent infection are the possible reason for persistent infection and root-end filling with MTA prevents the adhesion and penetration. PMID:27994316

  12. Dynamic behavior of binary component ion-exchange displacement chromatography of proteins visualized by confocal laser scanning microscopy.

    PubMed

    Shi, Qing-Hong; Shi, Zhi-Cong; Sun, Yan

    2012-09-28

    Confocal laser scanning microscopy (CLSM) was introduced to visualize particle-scale binary component protein displacement behavior in Q Sepharose HP column. To this end, displacement chromatography of two intrinsic fluorescent proteins, enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP), were developed using sodium saccharin (NaSac) as a displacer. The results indicated that RFP as well as eGFP could be effectively displaced in the single-component experiments by 50 mmol/L NaSac at 120 and 140 mmol/L NaCl whereas a fully developed displacement train with eGFP and RFP was only observed at 120 mmol/L NaCl in binary component displacement. At 140 mmol/L NaCl, there was a serious overlapping of the zones of the two proteins, indicating the importance of induced-salt effect on the formation of an isotachic displacement train. CLSM provided particle-scale evidence that induced-salt effect occurred likewise in the interior of an adsorbent and was synchronous to the introduction of the displacer. CLSM results at 140 mmol/L NaCl also demonstrated that both the proteins had the same fading rate at 50 mmol/L NaSac in the initial stage, suggesting the same displacement ability of NaSac to both the proteins. In the final stage, the fading rate of RFP in the adsorbent became slow, particularly at lower displacer concentrations. In the binary component displacement, the two proteins exhibited distinct fading rates as compared to the single component displacement and the remarkable lagging of the fading rate was observed in protein displacements. It suggested that the co-adsorbed proteins had significant influence on the formation of an isotachic train and the displacement chromatography of the proteins. Therefore, this research provided particle-scale insight into the dynamic behavior and complexity in the displacement of proteins.

  13. 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

  14. The simplicity of males: dwarf males of four species of Osedax (Siboglinidae; Annelida) investigated by confocal laser scanning microscopy.

    PubMed

    Worsaae, Katrine; Rouse, Greg W

    2010-02-01

    Dwarf males of the bone-eating worms Osedax (Siboglinidae, Annelida) have been proposed to develop from larvae that settle on females rather than on bone. The apparent arrest in somatic development and resemblance of the males to trochophore larvae has been posited as an example of paedomorphosis. Here, we present the first investigation of the entire muscle and nervous system in dwarf males of Osedax frankpressi, O. roseus, O. rubiplumus, and O. "spiral" analyzed by multistaining and confocal laser scanning microscopy. Sperm shape and spermiogenesis, the sperm duct and internal and external ciliary patterns were likewise visualized. The males of all four species possess morphological traits typical of newly settled siboglinid larvae: a prostomium, a peristomium with a prototroch, one elongate segment and a second shorter segment. Each segment has a ring of eight long-handled hooked chaetae. The longitudinal muscles are distributed as evenly spaced strands forming a grid with the thin outer circular muscles. Oblique protractor and retractor muscles are associated with each of the chaetal sacs. The nervous system comprises a cerebral ganglion, a prototroch nerve ring, paired dorsolateral longitudinal nerves, five ventral longitudinal nerves with paired, posterior ganglia and a terminal commissure, as well as a net of fine peripheral transverse plexuses surrounding the first segment. Internal ciliation occurs as paired ventrolateral bands along the first segment. The bands appear to lead the free mature sperm to a ciliated duct and seminal vesicle lying just behind the prototroch region. A duct then runs from the seminal vesicle into the dorsal part of the prostomium. The similarity of Osedax males to the larvae of Osedax and other siboglinid annelids as well as similarities shown here to the neuromuscular organization seen in other annelid larvae supports the hypothesis of paedomorphosis in males of Osedax.

  15. 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

  16. Distribution of biomolecules in porous nitrocellulose membrane pads using confocal laser scanning microscopy and high-speed cameras.

    PubMed

    Mujawar, Liyakat Hamid; Maan, Abid Aslam; Khan, Muhammad Kashif Iqbal; Norde, Willem; van Amerongen, Aart

    2013-04-02

    The main focus of our research was to study the distribution of inkjet printed biomolecules in porous nitrocellulose membrane pads of different brands. We produced microarrays of fluorophore-labeled IgG and bovine serum albumin (BSA) on FAST, Unisart, and Oncyte-Avid slides and compared the spot morphology of the inkjet printed biomolecules. The distribution of these biomolecules within the spot embedded in the nitrocellulose membrane was analyzed by confocal laser scanning microscopy in the "Z" stack mode. By applying a "concentric ring" format, the distribution profile of the fluorescence intensity in each horizontal slice was measured and represented in a graphical color-coded way. Furthermore, a one-step diagnostic antibody assay was performed with a primary antibody, double-labeled amplicons, and fluorophore-labeled streptavidin in order to study the functionality and distribution of the immune complex in the nitrocellulose membrane slides. Under the conditions applied, the spot morphology and distribution of the primary labeled biomolecules was nonhomogenous and doughnut-like on the FAST and Unisart nitrocellulose slides, whereas a better spot morphology with more homogeneously distributed biomolecules was observed on the Oncyte-Avid slide. Similar morphologies and distribution patterns were observed when the diagnostic one-step nucleic acid microarray immunoassay was performed on these nitrocellulose slides. We also investigated possible reasons for the differences in the observed spot morphology by monitoring the dynamic behavior of a liquid droplet on and in these nitrocellulose slides. Using high speed cameras, we analyzed the wettability and fluid flow dynamics of a droplet on the various nitrocellulose substrates. The spreading of the liquid droplet was comparable for the FAST and Unisart slides but different, i.e., slower, for the Oncyte-Avid slide. The results of the spreading of the droplet and the penetration behavior of the liquid in the

  17. 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

  18. Confocal laser scanning, scanning electron, and transmission electron microscopy investigation of Enterococcus faecalis biofilm degradation using passive and active sodium hypochlorite irrigation within a simulated root canal model.

    PubMed

    Mohmmed, Saifalarab A; Vianna, Morgana E; Penny, Matthew R; Hilton, Stephen T; Mordan, Nicola; Knowles, Jonathan C

    2017-02-28

    Root canal irrigation is an important adjunct to control microbial infection. The aim of this study was to investigate the effect of 2.5% (wt/vol) sodium hypochlorite (NaOCl) agitation on the removal, killing, and degradation of Enterococcus faecalis biofilm. A total of 45 root canal models were manufactured using 3D printing with each model comprising an 18 mm length simulated root canal of apical size 30 and taper 0.06. E. faecalis biofilms were grown on the apical 3 mm of the models for 10 days. A total of 60 s of 9 ml of 2.5% NaOCl irrigation using syringe and needle was performed, the irrigant was either left stagnant in the canal or agitated using manual (Gutta-percha), sonic, and ultrasonic methods for 30 s. Following irrigation, the residual biofilms were observed using confocal laser scanning, scanning electron, and transmission electron microscopy. The data were analyzed using one-way ANOVA with Dunnett post hoc tests at a level of significance p ≤ .05. Consequence of root canal irrigation indicate that the reduction in the amount of biofilm achieved with the active irrigation groups (manual, sonic, and ultrasonic) was significantly greater when compared with the passive and untreated groups (p < .05). Collectively, finding indicate that passive irrigation exhibited more residual biofilm on the model surface than irrigant agitated by manual or automated (sonic, ultrasonic) methods. Total biofilm degradation and nonviable cells were associated with the ultrasonic group.

  19. 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.

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

    PubMed Central

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

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required. PMID:24298422

  1. Characterization of a subwavelength-scale 3D void structure using the FDTD-based confocal laser scanning microscopic image mapping technique.

    PubMed

    Choi, Kyongsik; Chon, James W; Gu, Min; Lee, Byoungho

    2007-08-20

    In this paper, a simple confocal laser scanning microscopic (CLSM) image mapping technique based on the finite-difference time domain (FDTD) calculation has been proposed and evaluated for characterization of a subwavelength-scale three-dimensional (3D) void structure fabricated inside polymer matrix. The FDTD simulation method adopts a focused Gaussian beam incident wave, Berenger's perfectly matched layer absorbing boundary condition, and the angular spectrum analysis method. Through the well matched simulation and experimental results of the xz-scanned 3D void structure, we first characterize the exact position and the topological shape factor of the subwavelength-scale void structure, which was fabricated by a tightly focused ultrashort pulse laser. The proposed CLSM image mapping technique based on the FDTD can be widely applied from the 3D near-field microscopic imaging, optical trapping, and evanescent wave phenomenon to the state-of-the-art bio- and nanophotonics.

  2. Confocal laser scanning microscopic observation on adult Schistosoma japonicum harbored in mice following treatment with single-dose mefloquine.

    PubMed

    Xiao, Shu-Hua; Sun, Jun; Xue, Jian

    2012-06-01

    The aim of the present study is to assess the mefloquine-induced alteration of adult Schistosoma japonicum using confocal laser scanning microscopy (CLSM). Eight out of ten mice infected with 60-80 S. japonicum cercariae for 35 days were treated orally with mefloquine at a single dose of 400 mg/kg. Four groups of two mice were killed at 24 h and 3, 7, and 14 days post-treatment, and schistosomes were collected by perfusion from the liver and mesenteric veins, fixed in 70% alcohol, stained with acid carmine, and examined by CLSM. Worms obtained from untreated mice served as controls. Twenty-four hours post-treatment, focal tegument of adult male and female worms, which composed of fine and short villus-like materials, became thicker and longer, or disorder arrangement, while the musculatures beneath the tegument revealed in focal and irregular swelling with various degrees. In the gut of male and female schistosomes, severe dilatation accompanied by swelling, collapse, and peeling of gut mucosa was universal. In the reproductive organs, no apparent alteration in the testis structure of male worms was seen, while in female worms, slight damage to the ovary included loose arrangement of mature ovary cells accompanied by some of them degenerated and collapsed. As to vitelline glands, severe damage, such as swelling, indistinction, fusion or collapse of vitelline cells, and apparent swelling of parenchymal tissues in vitelline gland lobules, was seen. Meanwhile, abnormal ova emerged in the uterus at this time point. Three to 7 days post-treatment, the damage to the worms aggravated either in extent or in severity along with time. In some focally swollen worm body, the parenchymal tissues revealed in severe swelling. In addition, a large piece of degenerated and necrotic parenchymal tissues emerged closed to the severe destructed oral or ventral sucker. In the gut of male and female worms, the major alterations manifested by focal collapse or peeling of mucosa, and

  3. Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

    PubMed Central

    Auty, M. A. E.; Gardiner, G. E.; McBrearty, S. J.; O'Sullivan, E. O.; Mulvihill, D. M.; Collins, J. K.; Fitzgerald, G. F.; Stanton, C.; Ross, R. P.

    2001-01-01

    The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ∼108 bacteria/ml (equivalent to ∼107 CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent. PMID:11133474

  4. 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

  5. Relationship between gustatory function and average number of taste buds per fungiform papilla measured by confocal laser scanning microscopy in humans.

    PubMed

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

    2017-02-01

    The aim of this study was to elucidate the relationship between the gustatory function and average number of taste buds per fungiform papilla (FP) in humans. Systemically healthy volunteers (n = 211), pre-operative patients with chronic otitis media (n = 79), and postoperative patients, with or without a chorda tympani nerve (CTN) severed during middle ear surgery (n = 63), were included. Confocal laser scanning microscopy was employed to observe fungiform taste buds because it allows many FP to be observed non-invasively in a short period of time. Taste buds in an average of 10 FP in the midlateral region of the tongue were counted. In total, 3,849 FP were observed in 353 subjects. The gustatory function was measured by electrogustometry (EGM). An inverse relationship was found between the gustatory function and average number of fungiform taste buds per papilla. The healthy volunteers showed a lower EGM threshold (better gustatory function) and had more taste buds than did the patients with otitis media, and the patients with otitis media showed a lower EGM threshold and had more taste buds than did postoperative patients, reflecting the severity of damage to the CTN. It was concluded that the confocal laser scanning microscope is a very useful tool for using to observe a large number of taste buds non-invasively.

  6. High-resolution three-dimensional images from confocal scanning laser microscopy. Quantitative study and mathematical correction of the effects from bleaching and fluorescence attenuation in depth.

    PubMed

    Rigaut, J P; Vassy, J

    1991-08-01

    Three-dimensional images can be assembled by piling up consecutive confocal fluorescent images obtained by confocal scanning laser microscopy. The present work was based on three-dimensional (50-microns-deep) images at high (x, y) resolution obtained with an MRC-500 after en bloc staining of thick slices of rat liver by chromomycin A3 for nuclear DNA. The results of studies on bleaching, fluorescence excitation and emission intensities at various depths of histologic preparations are described. These effects could be evaluated separately by acquiring piled-up ("brick-stepping") and non-piled-up ("side-stepping") (x, y) images at consecutive depths and also (x, z) images. Empirical equations allowed the fitting of experimental plots of bleaching versus time, at different laser intensities and at different depths, and of fluorescence emission intensity versus depth. The main conclusions were that under our experimental conditions: (1) there was no attenuation by depth of the fluorochrome penetration, (2) there was no attenuation of the exciting beam intensity up to at least 50 microns deep, (3) there was an attenuation of the fluorescence emission intensity by depth, (4) bleaching happened equally on all planes above and below any confocal plane being studied, and (5) the fluorescence bleaching half-life was independent of depth. A mathematical correction scheme designed to compensate for bleaching and for attenuation of fluorescence emission in depth is presented. This correction is required for obtaining three-dimensional images of better quality, for optimal three-dimensional image segmentation and for any quantitative analysis based upon voxel-discretized emission intensities (gray levels)--e.g., estimating, by confocal image cytometry, textural chromatin parameters and nuclear DNA amounts.

  7. Simple high-speed confocal line-scanning microscope.

    PubMed

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

    2005-06-27

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

  8. Mean cell size and collagen orientation from 2D Fourier analysis on confocal laser scanning microscopy and two-photon fluorescence microscopy on human skin in vivo

    NASA Astrophysics Data System (ADS)

    Lucassen, Gerald W.; Bakker, Bernard L.; Neerken, Sieglinde; Hendriks, Rob F. M.

    2003-07-01

    We present results from 2D Fourier analysis on 3D stacks of images obtained by confocal laser scanning reflectance microscopy (CLSM) and two-photon fluorescence microscopy (2PM) on human skin in vivo. CLSM images were obtained with a modified commercial system (Vivascope1000, Lucid Inc, excitation wavelength 830 nm) equipped with a piezo-focusing element (350 μm range) for depth positioning of the objective lens. 2PM was performed with a specially designed set-up with excitation wavelength 730 nm. Mean cell size in the epidermal layer and structural orientation in the dermal layer have been determined as a function of depth by 2D Fourier analysis. Fourier analysis on microscopic images enables automatic non-invasive quantitative structural analysis (mean cell size and orientation) of living human skin.

  9. Data on characterization of nano- and micro-structures resulting from glycine betaine surfactant/kappa-carrageenan interactions by Laser Scanning Confocal Microscopy and Transmission Electron Microscopy.

    PubMed

    Gaillard, Cédric; Wang, Yunhui; Covis, Rudy; Vives, Thomas; Benoit, Maud; Benvegnu, Thierry

    2016-12-01

    This article contains data on the Laser Scanning Confocal Microscopy (LSCM) and Transmission Electron Microscopy (TEM) images related to multi-scaled self-assemblies resulting from 'green' cationic glycine betaine surfactant/anionic kappa-carrageenan interactions. These data gave clear evidence of the evolution of the micron-, nano-sized structures obtained at two surfactant/polymer molar ratios (3.5 and 0.8) and after the dilution of the aqueous dispersions with factors of 5 and 10 times. This data article is related to the research article entitled, "Monitoring the architecture of anionic ĸ-carrageenan/cationic glycine betaine amide surfactant assemblies by dilution: A multiscale approach" (Gaillard et al., 2017) [1].

  10. 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

  11. Confocal laser scanning microscopy coupled to a spectrofluorometric detector as a rapid tool for determining the in vivo effect of metals on phototrophic bacteria.

    PubMed

    Burnat, Mireia; Diestra, Elia; Esteve, Isabel; Solé, Antonio

    2010-01-01

    In this paper, we determine for the first time the in vivo effect of heavy metals in a phototrophic bacterium. We used Confocal Laser Scanning Microscopy coupled to a spectrofluorometric detector as a rapid technique to measure pigment response to heavy-metal exposure. To this end, we selected lead and copper (toxic and essential metals) and Microcoleus sp. as the phototrophic bacterium because it would be feasible to see this cyanobacterium as a good biomarker, since it covers large extensions of coastal sediments. The results obtained demonstrate that, while cells are still viable, pigment peak decreases whereas metal concentration increases (from 0.1 to 1 mM Pb). Pigments are totally degraded when cultures were polluted with lead and copper at the maximum doses used (25 mM Pb(NO(3))(2) and 10 mM CuSO(4)). The aim of this study was also to identify the place of metal accumulation in Microcoleus cells. Element analysis of this cyanobacterium in the above mentioned conditions determined by Energy Dispersive X-ray microanalysis (EDX) coupled to Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), shows that Pb (but not Cu) accumulates externally and internally in cells.

  12. Confocal Laser Scanning and Atomic-Force Microscopy in Estimation of Elastic Properties of Organic-Rich Rocks, Bazhenov Formation, Russia.

    NASA Astrophysics Data System (ADS)

    Ahmadov, R.; Vanorio, T.; Mavko, G.

    2008-12-01

    We estimate the indentation modulus (related to Young's modulus via Poisson's ratio) of organic-rich shale samples using a nano-indentation technique based on atomic-force microscopy, coupled with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Our approach is based on elaboration of data from two types of microscopy (SEM and CLSM to separate organic-rich (kerogen containing) regions from the mineral matrix of oil shales, with subsequent nanoscale probing via AFM. First, the microtexture of shales is characterized by SEM. Possible regions of interest are selected, and CLSM imaging is performed to confirm the presence of organic matter. Then, an AFM-based nano- indentation probe is employed to test the hardness of the previously identified region. Finally, nano- indentation modulus values are determined for individual mineral and organic-rich phases. This allows mapping of the absolute value of the modulus, providing spatial variation of elasticity, which then can be correlated with the initial mineralogy of the sample.

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

    PubMed

    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.

  14. Modular Scanning Confocal Microscope with Digital Image Processing.

    PubMed

    Ye, Xianjun; McCluskey, Matthew D

    2016-01-01

    In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength.

  15. Modular Scanning Confocal Microscope with Digital Image Processing

    PubMed Central

    McCluskey, Matthew D.

    2016-01-01

    In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength. PMID:27829052

  16. 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

  17. Investigation of the cutaneous penetration behavior of dexamethasone loaded to nano-sized lipid particles by EPR spectroscopy, and confocal Raman and laser scanning microscopy.

    PubMed

    Lohan, Silke B; Saeidpour, Siavash; Solik, Agnieszka; Schanzer, Sabine; Richter, Heike; Dong, Pin; Darvin, Maxim E; Bodmeier, Roland; Patzelt, Alexa; Zoubari, Gaith; Unbehauen, Michael; Haag, Rainer; Lademann, Jürgen; Teutloff, Christian; Bittl, Robert; Meinke, Martina C

    2016-12-30

    An improvement of the penetration efficiency combined with the controlled release of actives in the skin can facilitate the medical treatment of skin diseases immensely. Dexamethasone (Dx), a synthetic glucocorticoid, is frequently used for the treatment of inflammatory skin diseases. To investigate the penetration of nano-sized lipid particles (NLP) loaded with Dx in comparison to a commercially available base cream, different techniques were applied. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the penetration of Dx, which was covalently labeled with the spin probe 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA). The penetration into hair follicles was studied using confocal laser scanning microscopy (CLSM) with curcumin-loaded NLP. The penetration of the vehicle was followed by confocal Raman microscopy (CRM). Penetration studies using excised porcine skin revealed a more than twofold higher penetration efficiency for DxPCA into the stratum corneum (SC) after 24h incubation compared to 4h incubation when loaded to the NLP, whereas when applied in the base cream, almost no further penetration was observed beyond 4h. The distribution of DxPCA within the SC was investigated by consecutive tape stripping. The release of DxPCA from the base cream after 24h in deeper SC layers and the viable epidermis was shown by EPR. For NLP, no release from the carrier was observed, although DxPCA was detectable in the skin after the complete SC was removed. This phenomenon can be explained by the penetration of the NLP into the hair follicles. However, penetration profiles measured by CRM indicate that NLP did not penetrate as deeply into the SC as the base cream formulation. In conclusion, NLP can improve the accumulation of Dx in the skin and provide a reservoir within the SC and in the follicular infundibula.

  18. 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.

  19. Direct observation of intraparticle equilibration and the rate-limiting step in adsorption of proteins in chromatographic adsorbents with confocal laser scanning microscopy.

    PubMed

    Kasche, Volker; de Boer, Michael; Lazo, Cesar; Gad, Moustafa

    2003-06-25

    The adsorption of different proteins in a single biospecific and hydrophobic adsorbent particle for preparative protein chromatography has been observed directly by confocal laser scanning microscopy as a function of time at a constant bulk concentration c(b). The bulk concentration was in the non-linear part of the adsorption isotherm. At all times the concentration of free protein at the particle surface was almost equal to the bulk content indicating that external mass transfer resistance is not rate limiting for the adsorption under these conditions. Inside the particles a distinct maximum in adsorbed and free protein concentration that moved inside to a distance of approximately 0.2 R (R particle radius) from the particle surface, was observed. This is due to a decreasing solid-phase density and adsorptive capacity in the particle between 0.8 R and R indicating that the fraction of macropores (or void space) is larger in the outer than in the inner part of the adsorbent particles. By increasing the bulk concentration by a factor of 10 the equilibration time was reduced by about the same magnitude. This is in agreement with the concentration dependence of the effective pore diffusion coefficient D(p,eff)=D(p)/[epsilon(p)[1+nK/(K +c)(2)

  20. Ultrasensitive and selective gold film-based detection of mercury (II) in tap water using a laser scanning confocal imaging-surface plasmon resonance system in real time.

    PubMed

    Zhang, Hongyan; Yang, Liquan; Zhou, Bingjiang; Liu, Weimin; Ge, Jiechao; Wu, Jiasheng; Wang, Ying; Wang, Pengfei

    2013-09-15

    An ultrasensitive and selective detection of mercury (II) was investigated using a laser scanning confocal imaging-surface plasmon resonance system (LSCI-SPR). The detection limit was as low as 0.01ng/ml for Hg(2+) ions in ultrapure and tap water based on a T-rich, single-stranded DNA (ssDNA)-modified gold film, which can be individually manipulated using specific T-Hg(2+)-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 Hg(2+) ion concentration, which is unaffected by the presence of other metal ions. The coefficients obtained for ultrapure and tap water were 0.99902 and 0.99512, respectively, for the linear part over a range of 0.01-100ng/ml. The results show that the double-effect sensor has potential for practical applications with ultra sensitivity and selectivity, especially in online or real-time monitoring of Hg(2+) ions pollution in tap water with the further improvement of portable LSCI-SPR instrument.

  1. Comparison of fundus autofluorescence images acquired by the confocal scanning laser ophthalmoscope (488 nm excitation) and the modified Topcon fundus camera (580 nm excitation).

    PubMed

    Deli, A; Moetteli, L; Ambresin, A; Mantel, I

    2013-12-01

    To compare autofluorescence (AF) images obtained with the confocal scanning laser ophthalmoscope (using the Heidelberg retina angiograph; HRA) and the modified Topcon fundus camera, in a routine clinical setting. A prospective comparative study conducted at the Jules-Gonin Eye Hospital. Fifty-six patients from the medical retina clinic. All patients had complete ophthalmic slit-lamp and fundus examinations, colour and red-free fundus photography, AF imaging with both instruments, and fluorescein angiography. Cataract and fixation were graded clinically. AF patterns were analyzed for healthy and pathological features. Differences of image noise were analyzed by cataract grading and fixation. A total of 105 eyes were included. AF patterns discovered by the retina angiograph and the fundus camera images, respectively, were a dark optic disc in 72 % versus 15 %, a dark fovea in 92 % versus 4 %, sub- and intraretinal fluid visible as hyperautofluorescence on HRA images only, lipid exudates visible as hypoautofluorescence on HRA images only. The same autofluorescent pattern was found on both images for geographic atrophy, retinal pigment changes, drusen and haemorrhage. Image noise was significantly associated with the degree of cataract and/or poor fixation, favouring the fundus camera. Images acquired by the fundus camera before and after fluorescein angiography were identical. Fundus AF images differ according to the technical differences of the instruments used. Knowledge of these differences is important not only for correctly interpreting images, but also for selecting the most appropriate instrument for the clinical situation.

  2. 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

    Hao, Likai; Li, Jianli; Kappler, Andreas; Obst, Martin

    2013-11-01

    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 Fe(3+), Cu(2+), Zn(2+), and Hg(2+), 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.

  3. 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.

  4. 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.

  5. Bone natural autofluorescence and confocal laser scanning microscopy: Preliminary results of a novel useful tool to distinguish between forensic and ancient human skeletal remains.

    PubMed

    Capasso, Luigi; D'Anastasio, Ruggero; Guarnieri, Simone; Viciano, Joan; Mariggiò, Maria

    2017-03-01

    The fast, high-throughput distinction between palaeoanthropological/archaeological remains and recent forensic/clinical bone samples is of vital importance in the field of medico-legal science. In this paper, a novel dating method was developed using the autofluorescence of human bones and the confocal laser scanning microscope as the means to distinguish between archaeological and forensic anthropological skeletal findings. Human bones exhibit fluorescence, typically induced by natural antibiotics that are absorbed by collagen, and provide secondary, exogenous fluorophores. However, primary natural fluorescence (or autofluorescence) caused by enigmatic endogenous fluorophores is also present as a micro-phenomenon, whose nature is still obscure. Here, we show that the endogenous fluorophores are mucopolysaccharides of the Rouget-Neumann sheath and, more relevant, that the intensity of the natural fluorescence in human bone decreases in a relationship to the antiquity of the samples. These results suggest that the autofluorescence of bone is a promising technique for the assessment of skeletal remains that may be potentially of medico-legal interest. A larger study is proposed to confirm these findings and to create a predictive model between the autofluorescence intensity and the time since death.

  6. Novel application of confocal laser scanning microscopy and 3D volume rendering toward improving the resolution of the fossil record of charcoal.

    PubMed

    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.

  7. 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.

  8. A Dual Laser Scanning Confocal and Transmission Electron Microscopy Analysis of the Intracellular Localization, Aggregation and Particle Formation of African Horse Sickness Virus Major Core Protein VP7.

    PubMed

    Wall, Gayle V; Rutkowska, Daria A; Mizrachi, Eshchar; Huismans, Henk; van Staden, Vida

    2017-02-01

    The bulk of the major core protein VP7 in African horse sickness virus (AHSV) self-assembles into flat, hexagonal crystalline particles in a process appearing unrelated to viral replication. Why this unique characteristic of AHSV VP7 is genetically conserved, and whether VP7 aggregation and particle formation have an effect on cellular biology or the viral life cycle, is unknown. Here we investigated how different small peptide and enhanced green fluorescent protein (eGFP) insertions into the VP7 top domain affected VP7 localization, aggregation, and particle formation. This was done using a dual laser scanning confocal and transmission electron microscopy approach in conjunction with analyses of the solubility, aggregation, and fluorescence profiles of the proteins. VP7 top domain modifications did not prevent trimerization, or intracellular trafficking, to one or two discrete sites in the cell. However, modifications that resulted in a misfolded and insoluble VP7-eGFP component blocked trafficking, and precluded protein accumulation at a single cellular site, perhaps by interfering with normal trimer-trimer interactions. Furthermore, the modifications disrupted the stable layering of the trimers into characteristic AHSV VP7 crystalline particles. It was concluded that VP7 trafficking is driven by a balance between VP7 solubility, trimer forming ability, and trimer-trimer interactions.

  9. Studies in Confocal Scanning Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Corle, Timothy Richard

    Optical microscopes have been used as measurement tools in many areas of science of the past 300 years. Despite their maturity, there is still active research in the field. In particular the development of confocal scanning optical microscopes (CSOMs) in the 1970's has extended the usefulness of optical microscopes by giving them depth imaging capabilities. In a CSOM a defocused image disappears rather than blurring as it does with a standard microscope. The shallow depth of focus allows structures with a height difference smaller than one wavelength to be imaged independently, and thus quantitative measurements of height can be made. The design and construction of two CSOMs is discussed. The first is a mechanically scanned single pinhole microscope. This instrument was developed as a test bed on which to try out ideas relating to phase contrast imaging. The second is a Nipkow disk based real-time confocal scanning optical microscope (RSOM). These two microscopes were used to investigate the transverse and depth resolution of CSOMs. It is demonstrated that although they do not intrinsically have any better transverse resolution than a standard optical microscope, CSOMs produce a visually sharper image with increased contrast. The depth response of the CSOM is also investigated. A vector theory for the depth response is derived and compared with experimental results. It is shown that previously unexplained asymmetries in the sidelobe structure of this response can be accounted for by aberrations in the microscope objective. Phase contrast images can be generated by periodically defocusing the microscope, either mechanically or electro -optically and detecting a signal at the modulation frequency. A new electro-optic phase contrast microscope is described. The microscope is used to quantitatively measure both the height and width of thin film gratings. The depth response and point spread function of this microscope are also derived. It is shown that the sidelobe

  10. In situ observation of the growth of biofouling layer in osmotic membrane bioreactors by multiple fluorescence labeling and confocal laser scanning microscopy.

    PubMed

    Yuan, Bo; Wang, Xinhua; Tang, Chuyang; Li, Xiufen; Yu, Guanghui

    2015-05-15

    Since the concept of the osmotic membrane bioreactor (OMBR) was introduced in 2008, it has attracted growing interests for its potential applications in wastewater treatment and reclamation; however, the fouling mechanisms of forward osmosis (FO) membrane especially the development of biofouling layer in the OMBR are not yet clear. Here, the fouled FO membranes were obtained from the OMBRs on days 3, 8 and 25 in sequence, and then the structure and growing rule of the biofouling layer formed on the FO membrane samples were in-situ characterized by multiple fluorescence labeling and confocal laser scanning microscopy (CLSM). CLSM images indicated that the variations in abundance and distribution of polysaccharides, proteins and microorganisms in the biofouling layer during the operation of OMBRs were significantly different. Before the 8th day, their biovolume dramatically increased. Subsequently, the biovolumes of β-d-glucopyranose polysaccharides and proteins continued increasing and leveled off after 8 days, respectively, while the biovolumes of α-d-glucopyranose polysaccharides and microorganisms decreased. Extracellular polymeric substances (EPS) played a significant role in the formation and growth of biofouling layer, while the microorganisms were seldom detected on the upper fouling layer after 3 days. Based on the results obtained in this study, the growth of biofouling layer on the FO membrane surface in the OMBR could be divided into three stages. Initially, EPS was firstly deposited on the FO membrane surface, and then microorganisms associated with EPS located in the initial depositing layer to form clusters. After that, the dramatic increase of the clusters of EPS and microorganisms resulted in the quick growth of biofouling layer during the flux decline of the OMBR. However, when the water flux became stable in the OMBR, some microorganisms and EPS would be detached from the FO membrane surface.

  11. Combination of synchrotron radiation-based Fourier transforms infrared microspectroscopy and confocal laser scanning microscopy to understand spatial heterogeneity in aquatic multispecies biofilms.

    PubMed

    Reuben, Sheela; Banas, Krzysztof; Banas, Agnieszka; Swarup, Sanjay

    2014-11-01

    Understanding the spatial heterogeneity within environmental biofilms can provide an insight into compartmentalization of different functions in biofilm communities. We used a non-destructive and label-free method by combining Synchrotron Radiation-based Fourier Transform Infrared Microspectroscopy (SR-FTIR) with Confocal Laser Scanning Microscopy (CLSM) to distinguish the spatial chemical changes within multispecies biofilms grown from natural storm waters in flow cells. Among the different surfaces tested for biofilm growth and optimal imaging, mylar membranes were most suited and it enabled successful spatial infrared imaging of natural biofilms for obtaining reliable and interpretable FTIR spectra. Time series analysis of biofilm growth showed that influx of water during biofilm growth, results in significant changes in biofilm formation. Early biofilms showed active nutrient acquisition and desiccation tolerance mechanisms corresponding with accumulation of secreted proteins. Statistical approach used for the evaluation of chemical spectra allowed for clustering and classification of various regions of the biofilm. Microheterogeneity was observed in the polymeric components of the biofilm matrix, including cellulose, glycocalyx and dextran-like molecules. Fructan and glycan-rich regions were distinguishable and glycocalyx was abundant in the strongly adhering peripheral regions of biofilms. Inner core showed coexistence of oxygen dimers and ferrihydrite that will likely support growth of Fe (II)-oxidising bacteria. The combined SR-FTIR microspectroscopy and CSLM approach for complex natural biofilms described here will be useful both in understanding heterogeneity of matrix components and in correlating functions of juxtaposed microbial species in complex natural biofilms with physicochemical microenvironment to which they are exposed.

  12. Laser scanning confocal microscopy and quantitative microscopy with a charge coupled device camera improve detection of human papillomavirus DNA revealed by fluorescence in situ hybridization.

    PubMed

    Lizard, G; Chignol, M C; Souchier, C; Schmitt, D; Chardonnet, Y

    1994-04-01

    Epithelial cervical CaSki, SiHa and HeLa cells containing respectively 600 copies of human papillomavirus (HPV) DNA type 16, 1-2 copies of HPV DNA type 16 and 10-50 copies of HPV DNA type 18 were used as model to detect different quantities of integrated HPV genome. The HPV DNA was identified on cell deposits with specific biotinylated DNA probes either by enzymatic in situ hybridization (EISH) or fluorescence in situ hybridization (FISH) involving successively a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and streptavidin-alkaline phosphatase complex or streptavidin-fluorescein isothiocyanate complex. With brightfield microscopy and EISH, hybridization spots were observed in CaSki and HeLa cells but hardly any in SiHa cells. With fluorescence microscopy and FISH, hybridization spots were clearly seen only on CaSki cell nuclei. In an attempt to improve the detection of low quantities of HPV DNA signals revealed by FISH, laser scanning confocal microscopy (LSCM) and quantitative microscopy with an intensified charge coupled device (CCD) camera were used. With both LSCM and quantitative microscopy, as few as 1-2 copies of HPV DNA were detected and found to be confined to cell nuclei counterstained with propidium iodide. Under Nomarski phase contrast, a good preservation of the cell structure was observed. With quantitative microscopy, differences in the number, size, total area and integrated fluorescence intensity of hybridization spots per nucleus were revealed between CaSki, SiHa and HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

    2014-05-06

    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.

  18. High-speed line scanning confocal microscope for biological imaging

    NASA Astrophysics Data System (ADS)

    Jung, Seung-Hwan; Kim, Chang-Keun; Ju, Sung-Bin; Cho, Yong-Jin; Jeong, Hyun-Woo; Kim, Beop-Min

    2007-02-01

    We constructed a high-speed laser line-scanning confocal microscope (LSCM) using He-Ne laser (633 nm), a line CCD camera, and an acousto-optic deflector (AOD). The line scanner consists of an AOD and a cylindrical lens, which create a line focus sweeping over the sample. The line scanner generates two-dimensional confocal images (512× 512 pixel image) up to 191 frames per second with no mechanically-moving parts. This system is configured as an inverted microscope for imaging biological organisms or tissues. Images of various biological samples were obtained including rabbit cornea, onion cells, mouse melanoma tumor cells (B16BL6), and human breast tumor cells (BT-20). The frame rate may be further improved up to over 700 frames per second when the image size is reduced (512×128 pixel image). This system may be useful for analyzing fast phenomena during biological and chemical interactions and for imaging 3D structures rapidly.

  19. 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

  20. 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.

  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. 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.

  3. Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity

    NASA Astrophysics Data System (ADS)

    De Luca, Giulia; Breedijk, Ronald; Hoebe, Ron; Stallinga, Sjoerd; Manders, Erik

    2017-03-01

    Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial resolution and signal-to-noise ratio, are characterized and compared with properties of standard confocal microscopy. The results show that the lateral resolution of RCM is ~170 nm compared to ~240 nm of confocal microscopy for 488 nm excitation and 1.49 NA. As the theory predicts, this improved lateral resolution is independent of the pinhole diameter. In standard confocal microscopy, the same lateral resolution can only be achieved with an almost closed pinhole and, consequently, with a major loss of signal. We show that the sectioning capabilities of the standard confocal microscope are preserved in RCM and that the axial resolution of RCM is slightly better (~15%) than the standard confocal microscope. Furthermore, the signal-to-noise ratio in RCM is a factor of 2 higher than in standard confocal microscopy, also due to the use of highly sensitive modern cameras. In case the pinhole of a confocal microscope is adjusted in such way that the lateral resolution is comparable to that of RCM, the signal-to-noise ratio in RCM is 4 times higher than standard confocal microscopy. Therefore, RCM offers a good alternative to standard confocal microscopy for higher lateral resolution with the main advantage of strongly improved sensitivity.

  4. 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

  5. Scanning laser polarimetry - a review.

    PubMed

    Da Pozzo, Stefano; Marchesan, Roberta; Ravalico, Giuseppe

    2009-01-01

    Glaucoma is a leading cause of irreversible blindness worldwide. Retinal ganglion cells and their axons represent the selective target of the disease. When visual function is still intact on standard automated perimetry and optic disc appearance is suspicious, an early diagnosis may be supported by the identification of a retinal nerve fibre layer (RNFL) defect in the peripapillary area. At present days, computer-based, real-time imaging of the peripapillary RNFL is available through instruments of easy use and with high levels of accuracy and reproducibility. Scanning laser polarimetry is performed by a confocal scanning laser ophthalmoscope with an integrated polarimeter (GDx-VCC). There is a considerable amount of scientific evidence about the role of this imaging technique for glaucoma diagnosis. The aim of this review is to describe the principles of operation, the examination procedure, the clinical role, the results of main diagnostic studies and the future development of the software for the scanning laser polarimetry.

  6. 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.

  7. 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.

  8. Digital adaptive optics line-scanning confocal imaging system

    PubMed Central

    Liu, Changgeng; Kim, Myung K.

    2015-01-01

    Abstract. 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

  9. 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.

  10. 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.

  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. 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.

  13. Improvements in visualisation and localisation of human papillomavirus DNA in CaSki cells by fluorescence in situ hybridization, laser scanning confocal microscopy and three-dimensional image reconstruction.

    PubMed

    Lizard, G; Usson, Y; Chignol, M C; Chardonnet, Y

    1994-07-01

    The visual interpretation and localisation of specific DNA sequences in three dimensions in cell nuclei was investigated by fluorescence in situ hybridization (FISH) and laser scanning confocal microscopy (LSCM) using CaSki cells containing 600 copies per cell of human papillomavirus (HPV) DNA type 16 integrated in cellular DNA. Biotinylated DNA probes were used and DNA-DNA hybrids were revealed by a three-step reaction involving a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. The DNA from cell nuclei was counterstained with propidium iodide. With standard fluorescence microscopy, some dense fluorescent spots were seen in the cell nuclei. Similarly, with LSCM, some hybridization spots were observed in the cell nuclei but they were at different levels of the nuclei as shown by successive nuclear sections taken along the z axis. The visualisation of multiple hybridization spots confirmed the presence of multiple integration sites of HPV 16 DNA in CaSki cells. Association of LSCM with three-dimensional reconstructions lead to spatial images of hybridization spots obtained by stacking (x,y) images from consecutive confocal planes. Rotation of the reconstructed cell nuclei around the y axis makes it possible to distinguish closely adjacent spots. The combination of these techniques improves the detection of hybridization spots and may be of interest to further determine whether the HPV DNA is episomal or integrated in infected cells.

  14. 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.

  15. 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.

  16. 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.

  17. Laser confocal microscope with wavelet-profiled point spread function

    NASA Astrophysics Data System (ADS)

    Romero, Mary Jacquiline; Bautista, Godofredo; Daria, Vincent Ricardo; Saloma, Caesar

    2010-04-01

    We report a laser-scanning confocal reflectance microscope with a wavelet-profiled point spread function (PSF) for rapid multi-resolution extraction and analysis of microscopic object features. The PSF is generated via holography by encoding a π-phase shifting disk unto a collimated laser beam via a phase-only spatial light modulator (SLM) that is positioned at the pupil plane of the focusing objective lens. Scaling of the transverse PSF distribution is achieved by selecting a suitable ratio of the π-phase shifting disk radius and the pupil aperture radius. With one and the same objective lens and one SLM to control the phase profile of the pupil function, we produce amplitude PSF distributions that are accurate scaled representations of the circularly-symmetric Mexican hat mother wavelet.

  18. 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

  19. 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

  20. Optical axial scanning in confocal microscopy using an electrically tunable lens.

    PubMed

    Jabbour, Joey M; Malik, Bilal H; Olsovsky, Cory; Cuenca, Rodrigo; Cheng, Shuna; Jo, Javier A; Cheng, Yi-Shing Lisa; Wright, John M; Maitland, Kristen C

    2014-02-01

    This paper presents the use and characterization of an electrically focus tunable lens to perform axial scanning in a confocal microscope. Lateral and axial resolution are characterized over a >250 µm axial scan range. Confocal microscopy using optical axial scanning is demonstrated in epithelial tissue and compared to traditional stage scanning. By enabling rapid axial scanning, minimizing motion artifacts, and reducing mechanical complexity, this technique has potential to enhance in vivo three-dimensional imaging in confocal endomicroscopy.

  1. 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

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

    PubMed

    Chiu, L-D; Su, L; Reichelt, S; Amos, W B

    2012-05-01

    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.

  3. Colonization of potato rhizosphere by GFP-tagged Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44 shown on large sections of roots using enrichment sample preparation and confocal laser scanning microscopy.

    PubMed

    Krzyzanowska, Dorota; Obuchowski, Michal; Bikowski, Mariusz; Rychlowski, Michal; Jafra, Sylwia

    2012-12-18

    The ability to colonize the host plants' rhizospheres is a crucial feature to study in the case of Plant Growth Promoting Rhizobacteria (PGPRs) with potential agricultural applications. In this work, we have created GFP-tagged derivatives of three candidate PGPRs: Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44. The presence of these strains in the rhizosphere of soil-grown potato (Solanum tuberosum L.) was detected with a classical fluorescence microscope and a confocal laser scanning microscope (CLSM). In this work, we have used a broad-field-of-view CLMS device, dedicated to in vivo analysis of macroscopic objects, equipped with an automated optical zoom system and tunable excitation and detection spectra. We show that features of this type of CLSM microscopes make them particularly well suited to study root colonization by microorganisms. To facilitate the detection of small and scattered bacterial populations, we have developed a fast and user-friendly enrichment method for root sample preparation. The described method, thanks to the in situ formation of mini-colonies, enables visualization of bacterial colonization sites on large root fragments. This approach can be easily modified to study colonization patterns of other fluorescently tagged strains. Additionally, dilution plating of the root extracts was performed to estimate the cell number of MB73/2, P482 and A44 in the rhizosphere of the inoculated plants.

  4. Comparison of line-scanned and point-scanned dual-axis confocal microscope performance.

    PubMed

    Wang, D; Chen, Y; Wang, Y; Liu, J T C

    2013-12-15

    The point-scanned dual-axis confocal (PS-DAC) microscope has been shown to exhibit superior capability to reject out-of-focus and multiply scattered light in comparison to its conventional single-axis counterpart. However, the slow frame rate (typically <5 Hz) resulting from point-by-point data collection makes these systems vulnerable to motion artifacts. While video-rate point-scanned confocal microscopy is possible, a line-scanned dual-axis confocal (LS-DAC) microscope provides a simpler means of achieving high-speed imaging through line-by-line data collection, but sacrifices contrast due to loss of confocality along one dimension. Here we evaluate the performance trade-offs between an LS-DAC and PS-DAC microscope with identical spatial resolutions. Characterization experiments of the LS-DAC and PS-DAC microscopes with tissue phantoms, in reflectance mode, are shown to match results from Monte Carlo scattering simulations of the systems. Fluorescence images of mouse brain vasculature, obtained using resolution-matched LS-DAC and PS-DAC microscopes, demonstrate the comparable performance of LS-DAC and PS-DAC microscopy at shallow depths.

  5. Improving Resolution of Confocal Laser Induced Fluorescence in Argon Helicon Plasma

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

    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 dichroic mirror is used to separate the injected laser light from the collected emission. The measurement location is scanned radially by manually adjusting the final focusing lens position. In the initial version of the confocal optical system, measurements were poorly resolved radially because they were integrated over a fairly large path length (~4 cm) centered at the focal point. Here we present collected data from a modified configuration that significantly improves the special resolution of confocal measurements. The confocal measurements are compared to traditional, two-port, LIF measurements over the same radial range.

  6. Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes.

    PubMed

    Eissing, Nathalie; Heger, Lukas; Baranska, Anna; Cesnjevar, Robert; Büttner-Herold, Maike; Söder, Stephan; Hartmann, Arndt; Heidkamp, Gordon F; Dudziak, Diana

    2014-09-01

    Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line.

  7. Comparison of methods for fluorescent detection of viable, dead, and total Escherichia coli O157:H7 cells in suspensions and on apples using confocal scanning laser microscopy following treatment with sanitizers.

    PubMed

    Burnett, Scott L; Beuchat, Larry R

    2002-03-25

    The influence of treating Escherichia coli O157:H7 cells labeled with an enhanced green fluorescent protein (EGFP) plasmid with 20 microg/ml active chlorine, 100 mg/ml hydrogen peroxide, and 80 mg/ml acetic acid on fluorescence intensity was determined. In addition, fluorescent staining methods to differentiate viable and dead E. coli O157:H7 cells on the cuticle of Red Delicious cv. apples following treatment with water or 200 microg/ml active chlorine were evaluated. Suspensions of E. coli O157:H7 EGFP+ cells were exposed to chemical treatment solutions for 0, 30, 60, 120, or 300 s before populations (log10 cfu/ml) were determined by surface plating, and fluorescence intensities of suspensions and individual cells were measured using spectrofluorometry and confocal scanning laser microscopy (CSLM), respectively. The relative fluorescence intensity of suspensions and individual cells changed upon exposure to various treatments. Results indicate that the use of EGFP to tag E. coli O157:H7 may not be appropriate for investigations seeking to microscopically differentiate viable and dead cells on produce following surface treatment with sanitizers. SYTOX Orange and SYTOX Green nucleic acid stains fluorescently labeled dead E. coli O157:H7 cells attached to apple cuticles more intensely than did propidium iodide. A cross-signal occurred between CSLM photomultipliers when examining tissues treated with SYTOX Orange to detect dead cells and antibody labeled with Alexa Fluor 488 to detect total (dead and viable) cells. Because of the possibility of cross-signal resulting in an overestimation of the number of dead cells on apples and, perhaps, other produce treated with these stains, SYTOX Green is preferred to detect dead cells and antibody labeled with Alexa Fluor 594 is preferred to detect the total number of cells on apple surfaces following treatment with sanitizers. The performance of SYTOX Green in combination with Alexa Fluor 594 to detect dead and total cells of

  8. Evaluating the toxicity/fixation balance for corneal cross-linking with sodium hydroxymethylglycinate (SMG) and riboflavin-UVA (CXL) in an ex vivo rabbit model using confocal laser scanning fluorescence microscopy

    PubMed Central

    Kim, Su-Young; Babar, Natasha; Takaoka, Anna; Zyablitskaya, Mariya; Nagasaki, Takayuki; Trokel, Stephen L.; Paik, David C.

    2015-01-01

    Purpose To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA (CXL) for therapeutic tissue cross-linking of the cornea. Methods Eleven (11) fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. Following epithelial debridement, the tissue was exposed to 1/4 Max (9.765mM) or 1/3Max (13.02mM) SMG at pH 8.5 for 30min or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Post-exposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts following a standardized algorithm. Results The ΔTm following CXL, 1/3 SMG, and 1/4 SMG was 2.19±0.91°C, 1.33±0.49 °C, and 1.10 ±0.46 °C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high powered field. The values were 2.95±1.74% (control) and 8.86±11.10% (CXL) [p=0.390]; 0.98±0.20% (control) and 19.53±32.22% (1/3max SMG) [p=0.426]; and 2.70±2.37% (control) and 2.84±2.24% (1/4 max SMG) [p=0.938];. The values for endothelial toxicity were then indexed over the shift in Tm in order to yield a toxicity/fixation index. The values were as follows: 2.70 for CXL, 13.95 for 1/3 max, and 0.13 for 1/4 max. Conclusions Quarter max (1/4 Max = 9.765mM) SMG effectively cross-linked tissue and was non-toxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects. PMID:26807905

  9. 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.

  10. 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.

  11. 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.

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

    PubMed Central

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

    2014-01-01

    A fiber-optic 3D confocal scanning microendoscope employing MEMS scanners for both lateral and axial scan was designed and constructed. The MEMS 3D scan engine achieved a lateral scan range of over ± 26° with a 2D MEMS scanning micromirror and a depth scan of over 400 μm with a 1D MEMS tunable microlens. The lateral resolution and axial resolution of this system were experimentally measured as 1.0 μm and 7.0 μm, respectively. 2D and 3D confocal reflectance images of micro-patterns, micro-particles, onion skins and acute rat brain tissue were obtained by this MEMS-based 3D confocal scanning microendoscope. PMID:25013304

  13. Scanned Laser Illuminator/Receiver

    DTIC Science & Technology

    1976-11-01

    illustrate parallel development of the PIN diode /CCD sensor hybrid and the 100W laser . Al- though a detailed cost analysis for procurement of this large...pmww^^W .m^n.m .,** ■ —ssa^ AFAL-TR-76-184 \\ SCANNED LASER ILLUMINATOR/RECEIVER ^ R. A. Honzik and F. B. Warren ^•Martin Marietta...NUMBER 4. TITLE (and Sublille) SCANNED LASER ILLUMINATOR/RECEIVER 5, TYPE OF REPORT & PERIOD COVERED Final Technical Report Dec 75

  14. Compact confocal readout system for three-dimensional memories using a laser-feedback semiconductor laser.

    PubMed

    Nakano, Masaharu; Kawata, Yoshimasa

    2003-08-01

    We present a compact confocal readout system for three-dimensional optical memories that uses the thresholding property of a semiconductor laser for feedback light. The system has higher axial resolution than a conventional confocal system with a pinhole. We demonstrate readout results for data recorded in two recording layers with the developed system.

  15. Methylene-blue aided rapid confocal laser endomicroscopy of breast cancer

    NASA Astrophysics Data System (ADS)

    Vyas, Khushi; Hughes, Michael; Leff, Daniel Richard; Yang, Guang-Zhong

    2017-02-01

    Breast conserving surgery allows complete tumor resection while maintaining acceptable cosmesis for patients. Safe and rapid intraoperative margin assessment during the procedure is important to establish the completeness of tumor excision and minimizes the need for reoperation. Confocal laser endomicroscopy has demonstrated promise for real-time intraoperative margin assessment using acriflavine staining, but it is not approved for routine in-human use. We describe a custom high-speed line-scan confocal laser endomicroscopy (LS-CLE) system at 660 nm that enables high-resolution histomorphological imaging of breast tissue stained with methylene-blue, an alternative fluorescent stain for localizing sentinel nodes during breast surgery. Preliminary imaging results on freshly excised human breast tissue specimens are presented, demonstrating the potential of methylene-blue aided rapid LS-CLE to determine the oncological status of surgical margins in-vivo.

  16. Optimum conditions for high-quality 3D reconstruction in confocal scanning microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Taehoon; Kim, Taejoong; Lee, SeungWoo; Gweon, Dae-Gab; Seo, Jungwoo

    2006-02-01

    Confocal Scanning Microscopy (CSM) is very useful to reconstruct 3D image of Bio-cells and the objects that have specification shape in higher axial and lateral resolution and widely used as measurement instrument. A 3D reconstruction is used to visualize confocal images and consists of following processes. The First process is to get 3D data by collecting a series of images at regular focus intervals (Optical Sectioning). The Second process is to fit a curve to a series of 3D data points each pixel. The Third process is to search height information that has maximum value from curve-fitting. However, because of various systematic errors (NOISE) occurred when collecting the information of images through Optical Sectioning and large peak deviation occurred from curve-fitting error, high quality 3D reconstruction is not expected. Also, it takes much time to 3d Reconstruction by using many 3D data in order to acquire high quality and much cost to improve signal-to-noise (SNR) using a higher power laser. So, we are going to define SNR, peak deviation and the order of curve-fitting as important factors and simulate the relation between the factors in order to find a optimum condition for high quality 3D reconstruction in Confoal Scanning Microscopy. If we use optimum condition obtained by this simulation, using a suitable SNR and the suitable number of data and the suitable n-th order curve-fitting, small peak deviation is expected and then, 3D reconstruction of little better quality is expected. Also, it is expected to save.

  17. Point scanning confocal microscopy facilitates 3D human hair follicle imaging in tissue sections.

    PubMed

    Kloepper, Jennifer E; Bíró, Tamás; Paus, Ralf; Cseresnyés, Zoltán

    2010-07-01

    Efficiency is a key factor in determining whether a scientific method becomes widely accepted in practical applications. In dermatology, morphological characterisation of intact hair follicles by traditional methods can be rather inefficient. Samples are embedded, sliced, imaged and digitally reconstructed, which can be time-consuming. Confocal microscopy, on the other hand, is more efficient and readily applicable to study intact hair follicles. Modern confocal microscopes deliver and collect light very efficiently and thus allow high spatial resolution imaging of relatively thick samples. In this letter, we report that we successfully imaged entire intact human hair follicles using point scanning confocal microscopy. Light delivery and light-collection were further improved by preparing the samples in 2,2'-Thiodiethanol (TDE), thus reducing refractive index gradients. The relatively short total scan times and the high quality of the acquired 3D images make confocal microscopy a desirable method for studying intact hair follicles under normal and pathological conditions.

  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. Digital laser scanning fundus camera.

    PubMed

    Plesch, A; Klingbeil, U; Bille, J

    1987-04-15

    Imaging and documentation of the human retina for clinical diagnostics are conventionally achieved by classical optical methods. We designed a digital laser scanning fundus camera. The optoelectronical instrument is based on scanning laser illumination of the retina and a modified video imaging procedure. It is coupled to a digital image buffer and a microcomputer for image storage and processing. Aside from its high sensitivity the LSF incorporates new ophthalmic imaging methods like polarization differential contrast. We give design considerations as well as a description of the instrument and its performance.

  20. 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

  1. Error analysis for a laser differential confocal radius measurement system.

    PubMed

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

    2015-02-10

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

  2. Vertically scanned laser sheet microscopy.

    PubMed

    Dong, Di; Arranz, Alicia; Zhu, Shouping; Yang, Yujie; Shi, Liangliang; Wang, Jun; Shen, Chen; Tian, Jie; Ripoll, Jorge

    2014-01-01

    Laser sheet microscopy is a widely used imaging technique for imaging the three-dimensional distribution of a fluorescence signal in fixed tissue or small organisms. In laser sheet microscopy, the stripe artifacts caused by high absorption or high scattering structures are very common, greatly affecting image quality. To solve this problem, we report here a two-step procedure which consists of continuously acquiring laser sheet images while vertically displacing the sample, and then using the variational stationary noise remover (VSNR) method to further reduce the remaining stripes. Images from a cleared murine colon acquired with a vertical scan are compared with common stitching procedures demonstrating that vertically scanned light sheet microscopy greatly improves the performance of current light sheet microscopy approaches without the need for complex changes to the imaging setup and allows imaging of elongated samples, extending the field of view in the vertical direction.

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

    NASA Astrophysics Data System (ADS)

    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 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.

  4. 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.

  5. Confocal laser endomicroscopy in inflammatory bowel diseases: Dream or reality?

    PubMed Central

    De Palma, Giovanni Domenico; Rispo, Antonio

    2013-01-01

    Confocal laser endomicroscopy (CLE) is a newly introduced procedure that provide real-time, high-resolution imaging of the gastrointestinal mucosa during endoscopy, allowing the visualization of the pathology of the mucosal epithelium with its cellular and subcellular structures. Recently, the use of CLE was reported in the study of colonic mucosa in patients with inflammatory bowel diseases and in particular in patients affected by ulcerative colitis. CLE has the potential to have an important role in management of inflammatory bowel diseases (IBD) patients as it can be used to assess the grading of colitis and in detection of microscopic colitis in endoscopically silent segments. Moreover, CLE can be used in surveillance programs especially in high-risk patients. Finally, CLE has been effectively used in diagnosing a biliary dysplasia/neoplasia in patients with primary sclerosing cholangitis, a pathological condition frequently associated with IBD, with a coexisting bile duct stricture. PMID:24039350

  6. Confocal Laser Endomicroscopy for Diagnosis of Barrett’s Esophagus

    PubMed Central

    Neumann, Helmut; Langner, Cord; Neurath, Markus F.; Vieth, Michael

    2012-01-01

    Barrett’s esophagus (BE) is established as a premalignant condition in the distal esophagus. Current surveillance guidelines recommend random biopsies every 1–2 cm at intervals of 3–5 years. Advanced endoscopic imaging of BE underwent several technical revolutions within the last decade including broad-field (red-flag) techniques (e.g., chromoendoscopy) and small-field techniques with confocal laser endomicroscopy (CLE) at the forefront. In this review we will focus on advanced endoscopic imaging using CLE for the diagnosis and characterization of BE and associated neoplasia. In addition, we will critically discuss the technique of CLE and provide some tricks and hints for the daily routine practice of CLE for diagnosis of BE. PMID:22645719

  7. 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

  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. 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.

  10. A custom CMOS imager for multi-beam laser scanning microscopy and an improvement of scanning speed

    NASA Astrophysics Data System (ADS)

    Seo, Min-Woong; Kagawa, Keiichiro; Yasutomi, Keita; Kawahito, Shoji

    2013-02-01

    Multi-beam laser scanning confocal microscopy with a 256 × 256-pixel custom CMOS imager performing focal-plane pinhole effect, in which any rotating disk is not required, is demonstrated. A specimen is illuminated by 32 × 32 diffraction limited light spots whose wavelength and pitch are 532nm and 8.4 μm, respectively. The spot array is generated by a microlens array, which is scanned by two-dimensional piezo actuator according to the scanning of the image sensor. The frame rate of the prototype is 0.17 Hz, which is limited by the actuator. The confocal effect has been confirmed by comparing the axial resolution in the confocal imaging mode with that of the normal imaging mode. The axial resolution in the confocal mode measured by the full width at half maximum (FWHM) for a planar mirror was 8.9 μm, which is showed that the confocality has been achieved with the proposed CMOS image sensor. The focal-plane pinhole effect in the confocal microscopy with the proposed CMOS imager has been demonstrated at low frame rate. An improvement of the scanning speed and a CMOS imager with photo-sensitivity modulation pixels suitable for high-speed scanning are also discussed.

  11. 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.

  12. 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.

  13. Differential Multiphoton Laser Scanning Microscopy

    SciTech Connect

    Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.

    2012-01-01

    Multifocal multiphoton laser scanning microscopy (mfMPLSM) in the biological and medical sciences has the potential to become a ubiquitous tool for obtaining high-resolution images at video rates. While current implementations of mfMPLSM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for mfMPLSM in which whole-field detection with a single detector, rather than detection with a matrix of detectors, such as a charge-coupled device (CCD) camera, is implemented. This advance makes mfMPLSM fully compatible for use in imaging through scattering media. Further, we demonstrate that this method makes it possible to simultaneously obtain multiple images and view differences in excitation parameters in a single scan of the specimen.

  14. Hand-held digital line-scanning laser ophthalmoscope (LSLO)

    NASA Astrophysics Data System (ADS)

    Hammer, Daniel X.; Ferguson, R. D.; Ustun, Teoman E.; Maislin, Gami; Webb, Robert H.

    2004-07-01

    Scanning laser ophthalmoscopy is a powerful research tool with specialized but, to date, limited use in ophthalmic clinics due in part to the size, cost, and complexity of instruments. Conversely, low-cost retinal imaging devices have limited capabilities in screening, detection, and diagnosis of diseases. To fill the niche between these two, a low-cost, hand-held, line-scanning laser ophthalmoscope (LSLO) was designed, constructed, and tested on normal human subjects. The LSLO has only one moving part, multiple imaging modes, and uses low-cost but highly sensitive complimentary metal oxide semiconductor (CMOS) linear arrays for imaging with a detector dynamic range of 12-bits. The line-scanning approach produces high contrast quasi-confocal images with nearly the same performance as a flying-spot SLO. Imaging modes include simultaneous dual wavelength illumination and live stereoscopic imaging with a split aperture. Image processing and display functions are controlled with two stacked prototype compact printed circuit boards using field-programmable gated arrays (FPGA) and other digital electronic elements. With near shot-noise limited performance, the digital LSLO camera requires low illumination power (~ 100 μW) at near-infrared wavelengths. Wide field fundus images with several imaging modes have been obtained from several human subjects. The LSLO will significantly enhance confocal scanning laser ophthalmoscopy for routine use by ophthalmologist, optometrists, general practitioners and also non-specialized emergency medical personnel and technicians in the field for retinal disease detection and other diverse applications.

  15. 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.

  16. Design of 220 GHz electronically scanned reflectarrays for confocal imaging systems

    NASA Astrophysics Data System (ADS)

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

    2012-09-01

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

  17. 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

  18. 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.

  19. Chromatic dispersive confocal technology for intra-oral scanning: first in-vitro results

    NASA Astrophysics Data System (ADS)

    Ertl, T.; Zint, M.; Konz, A.; Brauer, E.; Hörhold, H.; Hibst, R.

    2015-02-01

    Various test objects, plaster models, partially equipped with extracted teeth and pig jaws representing various clinical situations of tooth preparations were used for in-vitro scanning tests with an experimental intra-oral scanning system based on chromatic-dispersive confocal technology. Scanning results were compared against data sets of the same object captured by an industrial μCT measuring system. Compared to μCT data an average error of 18 - 30 μm was achieved for a single tooth scan area and less than 40 to 60 μm error measured over the restoration + the neighbor teeth and pontic areas up to 7 units. Mean error for a full jaw is within 100 - 140 μm. The length error for a 3 - 4 unit bridge situation form contact point to contact point is below 100 μm and excellent interproximal surface coverage and prep margin clarity was achieved.

  20. Error analysis and tolerance allocation for confocal scanning microscopy using the Monte Carlo method

    NASA Astrophysics Data System (ADS)

    Yoo, Hongki; Kang, Dong-Kyun; Lee, SeungWoo; Lee, Junhee; Gweon, Dae-Gab

    2004-07-01

    The errors can cause the serious loss of the performance of a precision machine system. In this paper, we propose the method of allocating the alignment tolerances of the components and apply this method to Confocal Scanning Microscopy (CSM) to get the optimal tolerances. CSM uses confocal aperture, which blocks the out-of-focus information. Thus, it provides images with superior resolution and has unique property of optical sectioning. Recently, due to these properties, it has been widely used for measurement in biological field, medical science, material science and semiconductor industry. In general, tight tolerances are required to maintain the performance of a system, but a high cost of manufacturing and assembling is required to preserve the tight tolerances. The purpose of allocating the optimal tolerances is minimizing the cost while keeping the performance of the system. In the optimal problem, we set the performance requirements as constraints and maximized the tolerances. The Monte Carlo Method, a statistical simulation method, is used in tolerance analysis. Alignment tolerances of optical components of the confocal scanning microscopy are optimized, to minimize the cost and to maintain the observation performance of the microscopy. We can also apply this method to the other precision machine system.

  1. 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

  2. Laparoscopic manipulation of a probe-based confocal laser endomicroscope using a steerable intravascular catheter.

    PubMed

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

    2015-04-01

    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.

  3. 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

  4. 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.

  5. Scanning laser polarimetry in glaucoma.

    PubMed

    Dada, Tanuj; Sharma, Reetika; Angmo, Dewang; Sinha, Gautam; Bhartiya, Shibal; Mishra, Sanjay K; Panda, Anita; Sihota, Ramanjit

    2014-11-01

    Glaucoma is an acquired progressive optic neuropathy which is characterized by changes in the optic nerve head and retinal nerve fiber layer (RNFL). White-on-white perimetry is the gold standard for the diagnosis of glaucoma. However, it can detect defects in the visual field only after the loss of as many as 40% of the ganglion cells. Hence, the measurement of RNFL thickness has come up. Optical coherence tomography and scanning laser polarimetry (SLP) are the techniques that utilize the evaluation of RNFL for the evaluation of glaucoma. SLP provides RNFL thickness measurements based upon the birefringence of the retinal ganglion cell axons. We have reviewed the published literature on the use of SLP in glaucoma. This review elucidates the technological principles, recent developments and the role of SLP in the diagnosis and monitoring of glaucomatous optic neuropathy, in the light of scientific evidence so far.

  6. 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.

  7. Investigations in optoelectronic image processing in scanning laser microscopy

    NASA Astrophysics Data System (ADS)

    Chaliha, Hiranya Kumar

    A considerable amount of work has been done on scann-ing laser microscopy since its applications were first pointed out by Roberts and Young[1], Minsky [2] and Davidovits et al [3]. The advent of laser has made it possible to focus an intense beam of laser light in a scanning optical microscope (SOM) [4, 5] and hence explore regions of microscopy[6] uncovered by conven-tional microscopy. In the simple SOM [7, 8, 9], the upper spatial frequency in amplitude transmittance or reflectance of an object for which transfer function is nonzero is same as that in a conventional optical microscope. However, in Type II SOM [7] or confocal SOM that employs a coherent or a point detector, the spatial frequency bandwidth is twice that obtained in a conventional microscope. Besides this confocal set-up is found to be very useful in optical sectioning and consequently in 3-D image processing[10, 11, 12] specially of biological specimens. Such systems are also suitable for studies of semiconductor materials [13], super-resolution [14] and various imaginative ways of image processing[15, 16, 17] including phase imaging[18]. A brief survey of related advances in scanning optical microscopy has been covered in the chapter 1 of the thesis. The performance of SOM may be investigated by concent-rating also on signal derived by one dimensional scan of the object specimen. This simplified mode may also be adapted to give wealth of information for biological and semiconductor specimens. Hence we have investigated the design of a scanning laser system suited specifically for studies of line scan image signals of microscopic specimens when probed through a focused laser spot. An electro-mechanical method of scanning of the object specimen has been designed with this aim in mind. Chapter 2, Part A of the thesis deals with the design consider-ations of such a system. For analysis of scan signals at a later instant of time so as to facilitate further processing, an arrangement of microprocessor

  8. 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

  9. 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.

  10. Scanning laser reflectometry of retinal and subretinal tissues

    NASA Astrophysics Data System (ADS)

    Elsner, Ann E.; Moraes, L.; Beausencourt, E.; Remky, Andreas; Weiter, J. J.; Walker, J. P.; Wing, G. L.; Burns, Stephen Allan; Raskauskas, P. A.; Kelley, L. M.

    2000-06-01

    Measurements of the human ocular fundus that make use of the light returning through the pupil are called reflectometry. Early reflectometry studies were limited by poor light return from the retina and strong reflections from the anterior surface of the eye. Artifacts produced misleading results in diseases like age-related macular degeneration. Novel laser sources, scanning, confocal optics, and digital imaging provide improved sampling of the signal from the tissues of interest: photoreceptors and retinal pigment epithelial cells. A wider range of wavelengths is now compared, including the near infrared. Reflectometry now provides functional mapping, even in severe pathology.

  11. Light-sheet microscopy by confocal line scanning of dual-Bessel beams

    NASA Astrophysics Data System (ADS)

    Zhang, Pengfei; Phipps, Mary E.; Goodwin, Peter M.; Werner, James H.

    2016-10-01

    We have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the "on" state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as many photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.

  12. High spatial resolution confocal microscope with independent excitation and detection scanning capabilities.

    PubMed

    Marcet, S; Ouellet-Plamondon, C; Francoeur, S

    2009-06-01

    We present the design of a confocal microscope adapted for optical spectroscopy and imaging at cryogenic temperatures. This system is based on the existing approach of partly inserting the optical components of the microscope inside a helium-bath cryostat. It provides a spatial resolution approaching the diffraction limit with a mechanical stability allowing uninterrupted integration times exceeding 10 h and allows keeping track of a single emitter for unlimited periods of time. Furthermore, our design allows scanning the excitation spot and detection area independently of the sample position. This feature provides the means to perform probeless transport experiments on one-dimensional nanostructures. The scanning capabilities of this microscope are fully detailed and characterized using the photoluminescence of single nitrogen dyads at 4.5 K.

  13. 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

  14. Performance of full-pupil line-scanning reflectance confocal microscopy in human skin and oral mucosa in vivo

    PubMed Central

    Larson, Bjorg; Abeytunge, Sanjeewa; Rajadhyaksha, Milind

    2011-01-01

    Point-scanning reflectance confocal microscopes continue to be successfully translated for detection of skin cancer. Line-scanning, with the use of a single scanner and a linear-array detector, offers a potentially smaller, simpler and lower cost alternative approach, to accelerate widespread dissemination into the clinic. However, translation will require an understanding of imaging performance deep within scattering and aberrating human tissues. We report the results of an investigation of the performance of a full-pupil line-scanning reflectance confocal microscope in human skin and oral mucosa, in terms of resolution, optical sectioning, contrast, signal-to-noise ratio, imaging and the effect of speckle noise. PMID:21750780

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

    PubMed

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

    2013-01-10

    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. Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope.

    PubMed

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

    2011-06-01

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

  17. Large area mapping of excised breast tissue by fluorescence confocal strip scanning: a preliminary feasibility study

    NASA Astrophysics Data System (ADS)

    Larson, Bjorg A.; Abeytunge, Sanjee; Murray, Melissa; Rajadhyaksha, Milind

    2013-03-01

    Lumpectomy, in conjunction with radiation and chemotherapy drugs, together comprise breast-conserving treatment as an alternative to total mastectomy for patients with breast tumors. The tumor is removed in surgery and sent for pathology processing to assess the margins, a process that takes at minimum several hours, and generally days. If the margins are not clear of tumor, the patient must undergo a second surgery to remove residual tumor. This re-excision rate varies by institution, but can be as high as 60%. Currently, no intraoperative microscopic technique is used routinely to examine tumor margins in breast tissue. A new technique for rapidly scanning large areas of tissue has been developed, called confocal strip scanning, which provides high resolution and seamless mosaics over large areas of intact tissue, with nuclear and cellular resolution and optical sectioning of about 2 microns. Up to 3.5 x 3.5 cm2 of tissue is imaged in 13 minutes at current stage speeds. This technique is demonstrated in freshly excised breast tissue, using a mobile confocal microscope stationed in our pathology laboratory. Twenty-five lumpectomy and mastectomy cases were used as a testing ground for reflectance and fluorescence contrast modes, resolution requirements and tissue fixturing configurations. It was concluded that fluorescent imaging provides the needed contrast to distinguish ducts and lobules from surrounding stromal tissue. Therefore the system was configured with 488 nm illumination, with acridine orange fluorescent dye for nuclear contrast, with the aim of building an image library of malignant and benign breast pathologies.

  18. Integrated approach to laser delivery and confocal signal detection.

    PubMed

    Ismail, Nur; Akca, Bakiye Imran; Sun, Fei; Wörhoff, Kerstin; de Ridder, René M; Pollnau, Markus; Driessen, Alfred

    2010-08-15

    We present an on-chip arrayed waveguide grating (AWG) sensor based on the confocal arrangement of two AWGs, one acting as focusing illuminator and one as signal collector. The chip can be close to, or in direct contact with, a sample, e.g., biological tissue, without the need of external optics. The collection efficiency of our device can be more than 1 order of magnitude higher than that of a standard AWG, in which light is collected by one input channel. Experimental results on the collection efficiency and volume are presented, together with a demonstration of multiwavelength imaging.

  19. 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.

  20. 3D image reconstruction using optical sectioning in confocal scanning microscopy

    NASA Astrophysics Data System (ADS)

    Seo, Jungwoo; Kang, Dong Kyun; Park, Sunglim; Gweon, Dae gab

    2001-10-01

    Confocal scanning microscopy (CSM) has been used in biological application, materials science, semiconductor quality measurement and other non-destructive microscopic application. Small spot of light illuminates a sample, and a small detector that is ideally a point detector collects the reflected or transmitted light having the information of specimen. An image distribution can be reconstructed by a correlation analysis of spots with the high bandwidth. The mechanism for two-dimensional beam scanning and optical sectioning has an important role in CSM as the three-dimensional profiler. The parasitic motion of focus on the detector gives rise to the fatal distortion of an image profile named the extinction effect while using acousto-optical (AO) deflector. The intensity profile for the open loop scanning should be matched with its response for the standard. The non-linearity can be minimized with the optical sectioning or the optical probe of the closed loop control. This paper shows the mathematical expression of the light such as the extinction curve in the optical fields of system using AO deflector, the axial/lateral response experimentally when the error sources change, and the methods of optical sectioning. We propose the progressive methods for the high quality image as the following. At first, for having the corrected image, small spot and long scan range, this paper shows that the optimal design having the multi-objects can be used by choosing the unitary lens device in CSM. At second, in order to compensate for the intensity cancellation at the end profile that may be the cause of waviness for the optical image, this paper shows that it is efficient to schedule the frequency of scan. According to characteristics of the extinction curve and axial/lateral response having the error property, we can define the frequency and sensitivity of as their robustness. Finally, the axial response gives an important motive for the optical section, and the limit of

  1. Confocal laser spectroscopy of glasses modified by ultrashort laser pulses for waveguide fabrication

    NASA Astrophysics Data System (ADS)

    Chan, James Wai-Jeung

    2002-08-01

    The work described in this thesis involves the fabrication of waveguiding structures inside glasses using femtosecond (fs) laser pulses and the study of the different atomic scale changes associated with refractive index modification that occur in the fs laser modified glasses. This study helps elucidate the possible processes that occur during fs laser writing of waveguides in glasses. Waveguide writing inside fused silica and phosphate glass using focused fs laser pulses has been demonstrated. The modification induced inside both glasses is determined to be different. Inside fused silica, the modification involves a single high index region while inside the phosphate glass (IOG-1, Schott Glass Technologies, Inc.), the modification results in a central, low index, non-guiding region bordered by two, high index, waveguiding regions. The waveguides inside both glasses have an index change on the order of 10 -4. Color center defects have been identified in modified glasses using confocal fluorescence spectroscopy. Modified fused silica exhibits a fluorescence band at 630 nm and at 540 nm, which are attributed to the non-bridging oxygen hole center (NBOHC) and oxygen vacancy defects created by the fs pulses. A fluorescence band at 600 nm is observed in modified phosphate glass, which is assigned to the phosphorus oxygen hole center (POHC) defect. A quantitative analysis of the photobleaching of these defects with exposure to 488 nm light is conducted. Fluorescence imaging of the modified materials is performed to elucidate the location of these defects within the exposed regions in the glass. Using confocal Raman spectroscopy, atomic scale structural changes in the glass network of modified fused silica are reported and correlated to the changes in the physical properties of the material. The changes in the Raman spectrum of modified fused silica, specifically increases in the 490 cm-1 and 605 cm-1 peaks, indicate that fs pulses induce densification in fused silica

  2. Line-Scanning Reflectance Confocal Microscopy of Human Skin: Comparison of Full-pupil and Divided-pupil Configurations

    PubMed Central

    Gareau, Daniel S.; Abeytunge, Sanjee; Rajadhyaksha, Milind

    2009-01-01

    Line-scanning, with pupil engineering and the use of linear array detectors, may enable simple, small and low-cost confocal microscopes for clinical imaging of human epithelial tissues. However, a fundamental understanding of line-scanning performance within the highly scattering and aberrating conditions of human tissue is necessary, to translate from benchtop instrumentation to clinical implementation. The results of a preliminary investigation for reflectance imaging in skin are reported. PMID:19838284

  3. 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.

  4. High-speed adaptive optics line scan confocal retinal imaging for human eye

    PubMed Central

    Wang, Xiaolin; Zhang, Yuhua

    2017-01-01

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

  5. 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.

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

    PubMed Central

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

    2015-01-01

    Abstract. 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

  7. 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.

  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. Defect Detection Using a Scanning Laser Source

    NASA Astrophysics Data System (ADS)

    Burrows, S. E.; Dixon, S.

    2011-06-01

    Surface breaking defects are identified using a scanning laser source. A Q-switched Nd-YAG laser is used as a non-contact source of ultrasound and an electromagnetic acoustic transducer (EMAT) employed as detector. For a thin plate, an increase in frequency content of the received wave is observed when the laser spot is situated directly over the defect. Time-frequency analysis using a Wigner transform has enabled individual Lamb wave modes to be identified, while propagation of Lamb waves through aluminium sheet is studied by finite element analysis.

  10. 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.

  11. Lateral resolution enhancement of laser scanning microscopy by a higher-order radially polarized mode beam

    NASA Astrophysics Data System (ADS)

    Kozawa, Yuichi; Hibi, Terumasa; Sato, Aya; Horanai, Hibiki; Kurihara, Makoto; Hashimoto, Nobuyuki; Yokoyama, Hiroyuki; Nemoto, Tomomi; Sato, Shunichi

    2011-08-01

    We demonstrate that the lateral resolution of confocal laser scanning microscopy is dramatically improved by a higher-order radially polarized (HRP) beam with six concentric rings. This beam was generated simply by inserting liquid crystal devices in front of an objective lens. An HRP beam visualized aggregated 0.17 μm beads individually and is also applicable to biological imaging. This method can extend the capability of conventional laser scanning microscopes without modification of the system, with the exception of the addition of the liquid crystal devices in the optical path.

  12. 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.

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

    PubMed

    Gheonea, Dan Ionuţ; Cârţână, Tatiana; Ciurea, Tudorel; Popescu, Carmen; Bădărău, Anca; Săftoiu, Adrian

    2011-01-07

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

  14. 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.

  15. Fiber optic confocal laser Doppler velocimeter using an all-fiber laser source for high resolution measurements.

    PubMed

    Sharma, Utkarsh; Chen, Gang; Kang, Jin; Ilev, Ilko; Waynant, Ronald

    2005-08-08

    We demonstrate and analyze a novel fiber optic confocal laser Doppler velocimeter using an ultra-narrow linewidth all-fiber laser source centered at around 1550 nm (eye-safe region). The narrow spectral linewidth of the fiber laser (<10 kHz) is used to achieve an extremely high velocity resolution (~0.0075 m/s), which is an order of magnitude better as compared to the commonly used semiconductor diode lasers or He-Ne lasers based systems. The directional optical circulator based design used in our system is much simpler to implement and is power conserving compared to the conventional Michelson interferometer based designs. We perform Gaussian beam propagation analysis by using the ABCD law to study the performance of the confocal design. The analysis is in good accord with our experimental results. The confocal design is capable of providing ultrahigh spatial resolution (~5microm, in both lateral and longitudinal directions) for high-precision velocity distribution measurement applications.

  16. Motion-compensated blind deconvolution of scanning laser opthalmoscope imagery

    NASA Astrophysics Data System (ADS)

    O'Connor, Nathan J.; Bartsch, Dirk-Uwe G.; Freeman, William R.; Holmes, Timothy J.

    1998-06-01

    A deconvolution algorithm for use with scanning laser ophthalmoscope (SLO) data is being developed. The SLO is fundamentally a confocal microscope in which the objective lens is the human ocular lens. 3D data is collected by raster scanning to form images at different depths in retinal and choroidal layers. In this way, 3D anatomy may be imaged and stored as a series of optical sections.Given the poor optical quality of the human lens and random eye motion during data acquisition, any deconvolution method applied to SLO data must be able to account for distortions present in the observed data. The algorithm presented compensates for image warping and frame-to-frame displacement due to random eye motion, smearing along the optic axis, sensor saturation, and other problems. A preprocessing step is first used to compensate for frame-to-frame image displacement. The image warping, caused by random eye motion during raster scanning, is corrected. Finally, a maximum likelihood based blind deconvolution algorithm is used to correct severe blurring along the optic axis. The blind deconvolution algorithm contains an iterative search for subpixel displacements remaining after image warping and frame-to-frame displacements are corrected. This iterative search is formulated to ensure that the likelihood functional is non-decreasing.

  17. 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.

  18. 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.

  19. Lens based adaptive optics scanning laser ophthalmoscope.

    PubMed

    Felberer, Franz; Kroisamer, Julia-Sophie; Hitzenberger, Christoph K; Pircher, Michael

    2012-07-30

    We present an alternative approach for an adaptive optics scanning laser ophthalmoscope (AO-SLO). In contrast to other commonly used AO-SLO instruments, the imaging optics consist of lenses. Images of the fovea region of 5 healthy volunteers are recorded. The system is capable to resolve human foveal cones in 3 out of 5 healthy volunteers. Additionally, we investigated the capability of the system to support larger scanning angles (up to 5°) on the retina. Finally, in order to demonstrate the performance of the instrument images of rod photoreceptors are presented.

  20. 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.

  1. 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.

  2. 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

  3. A preliminary study of spatial resolution enhancement of confocal and triangulation displacement meters using contact mode scanning probes.

    PubMed

    Gaitas, Angelo

    2008-02-01

    This paper presents a method for the spatial resolution enhancement of confocal and triangulation meters using cantilever probes. Integration of a cantilever with existing commercially available meters is substantially eased by the absence of feedback control of the cantilever position. Confocal and triangulation meters are used for a number of applications in research and industrial settings including thickness measurements, topography measurements, step height measurements, flatness measurements, and profile measurements. These instruments provide a vertical (out-of-plane) resolution of a few nanometers. However, they are limited in their spatial resolution to the laser beam diameter, which is typically larger than 2 microm and often about 20 microm. Using a cantilever probe to make contact with the sample, the lateral resolution of standard commercial instruments can be improved to less than 1 microm.

  4. Design, operation and applications of a visible-light confocal scanning Fourier transform Raman microscope for volumetric Raman spectrochemical imaging

    NASA Astrophysics Data System (ADS)

    Brenan, Colin John Herbert

    A new type of confocal Raman microscope called a Fourier transform confocal Raman microscope (FT-CRM) was designed, built and characterized with respect to its spatio-spectral imaging properties. Several different applications of the FT-CRM are presented that take advantage of its unique spectral and spatial imaging characteristics. The instrument combines focused illumination with spatially-filtered detection in a confocal optical configuration to collect photons scattered from a diffraction-limited volume in the sample (typically [<]5×10-18/ m3) and reject photons from outside that region. The molecular vibrational information encoded in the inelastic, or Raman, spectral component of light scattered from the confocal volume is measured with a visible light Fourier transform Raman spectrometer. By scanning the sample relative to the confocal volume, a volumetric Raman spectrochemical image of the sample can be constructed. Raman scattering is an inherently inefficient process; hence an optimal radius pinhole must be found that balances the FT-CRM optical throughput against the microscope spatial resolution and image contrast. Detailed experimental measurements mapped out the FT-CRM spatial response (axial and lateral), optical throughput and image signal-to-background and signal-to-noise ratios as a function of pinhole radius. Excellent agreement was found between these measurements and the predictions of a theoretical microscope model also developed as part of this thesis. Several applications of the FT-CRM included volumetric compositional imaging of three-dimensional chemically inhomogeneous materials such as cellulose and polyester fibers in water or two immiscible optically- similar liquids, water and trichloroehthylene, in a porous quartz sandstone matrix. The potential of the FT- CRM for non-invasive spectrochemical detection and imaging through a turbid tissue-like medium was demonstrated and a new spectral estimator, Fast Orthogonal Search, was evaluated

  5. Nonlinear femtosecond laser induced scanning tunneling microscopy.

    PubMed

    Dey, Shirshendu; Mirell, Daniel; Perez, Alejandro Rodriguez; Lee, Joonhee; Apkarian, V Ara

    2013-04-21

    We demonstrate ultrafast laser driven nonlinear scanning tunneling microscopy (STM), under ambient conditions. The design is an adaptation of the recently introduced cross-polarized double beat method, whereby z-polarized phase modulated fields are tightly focused at a tunneling junction consisting of a sharp tungsten tip and an optically transparent gold film as substrate. We demonstrate the prerequisites for ultrafast time-resolved STM through an operative mechanism of nonlinear laser field-driven tunneling. The spatial resolution of the nonlinear laser driven STM is determined by the local field intensity. Resolution of 0.3 nm-10 nm is demonstrated for the intensity dependent, exponential tunneling range. The demonstration is carried out on a junction consisting of tungsten tip and gold substrate. Nano-structured gold is used for imaging purposes, to highlight junction plasmon controlled tunneling in the conductivity limit.

  6. 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.

  7. Efficiency of the confocal method of laser endomicroscopy in complex diagnoses of diseases of common bile duct

    NASA Astrophysics Data System (ADS)

    Anaskin, S. G.; Panchenkov, D. N.; Chertyuk, V. B.; Sazonov, D. V.; Zabozlayev, F. G.; Danilevskaya, O. V.; Mokshina, N. V.; Korniletsky, I. D.

    2017-01-01

    One of the more frequent manifestations of diseases of the bile ducts are its’ strictures or stenoses that could be of either malignant or benign nature. Current methods of diagnosing this pathology include computer tomography (CT) scan, magnetic resonance cholangiopancreatography (MRCP), endoscopic ultrasound (EUS) and endoscopic retrograde cholangiopancreatography (ERCP). However, these methods are not always informative, which makes this a current and topical problem. A fundamentally new method that broadens the capabilities of ERCP when diagnosing diseases of the bile duct accompanied by the development of strictures or stenoses is probe-based confocal laser endomicroscopy (pCLE). The method is based on the principle of confocal fluorescence microscopy. The most elaborate complications arise with the presence of the pre-existing pancreatobiliary pathology: pseudotumoral chronic pancreatitis, acute cholangitis, etc. Early stage cholangiocarcinoma diagnosis can be difficult (and not always possible) even with the help of modern research methods. For the timely diagnostic it is advantageous to conduct pCLE and targeted biopsy of the zone with most manifested changes. In all instances, the first use of the pCLE method for diagnostic purposes allowed us to clarify and correctly verify the diagnosis. When concerning the diseases of the bile duct, the modern stage of pCLE development can be of critical importance when other methods are not effective.

  8. 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.

  9. 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. PMID:27446641

  10. 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...

  11. Low-power laser effects at the single-cell level: a confocal microscopy study

    NASA Astrophysics Data System (ADS)

    Alexandratou, Eleni; Yova, Dido M.; Atlamazoglou, Vassilis; Handris, Panagiotis; Kletsas, Dimitris; Loukas, Spyros

    2000-11-01

    Confocal microscopy was used for irradiation and observation of the same area of interest, allowing the imaging of low power laser effects in subcellular components and functions, at the single cell level. Coverslips cultures of human fetal foreskin fibroblasts (HFFF2) were placed in a small incubation chamber for in vivo microscopic observation. Cells were stimulated by the 647 nm line of the Argon- Krypton laser of the confocal microscope (0.1 mW/cm2). Membrane permeability, mitochondrial membrane potential ((delta) Psim), intracellular pHi, calcium alterations and nuclear chromatin accessibility were monitored, at different times after irradiation, using specific fluorescent vital probes. Images were stored to the computer and quantitative evaluation was performed using image- processing software. After irradiation, influx and efflux of the appropriate dyes monitored changes in cell membrane permeability. Laser irradiation caused alkalizatoin of the cytosolic pHi and increase of the mitochondrial membrane potential ((delta) Psim). Temporary global Ca2+ responses were also observed. No such effects were noted in microscopic fields other than the irradiated ones. No toxic effects were observed, during time course of the experiment.

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  13. Patterned retinal coagulation with a scanning laser

    NASA Astrophysics Data System (ADS)

    Palanker, Daniel; Jain, ATul; Paulus, Yannis; Andersen, Dan; Blumenkranz, Mark S.

    2007-02-01

    Pan-retinal photocoagulation in patients with diabetic retinopathy typically involves application of more than 1000 laser spots; often resulting in physician fatigue and patient discomfort. We present a semi-automated patterned scanning laser photocoagulator that rapidly applies predetermined patterns of lesions; thus, greatly improving the comfort, efficiency and precision of the treatment. Patterns selected from a graphical user interface are displayed on the retina with an aiming beam, and treatment can be initiated and interrupted by depressing a foot pedal. To deliver a significant number of burns during the eye's fixation time, each pulse should be considerably shorter than conventional 100ms pulse duration. We measured coagulation thresholds and studied clinical and histological outcomes of the application of laser pulses in the range of 1-200ms in pigmented rabbits. Laser power required for producing ophthalmoscopically visible lesions with a laser spot of 132μm decreased from 360 to 37mW with pulse durations increasing from 1 to 100ms. In the range of 10-100ms clinically and histologically equivalent light burns could be produced. The safe therapeutic range of coagulation (ratio of the laser power required to produce a rupture to that for a light burn) decreased with decreasing pulse duration: from 3.8 at 100ms, to 3.0 at 20ms, to 2.5 at 10ms, and to 1.1 at 1ms. Histology demonstrated increased confinement of the thermal damage with shorter pulses, with coagulation zone limited to the photoreceptor layer at pulses shorter than 10ms. Durations of 10-20ms appear to be a good compromise between the speed and safety of retinal coagulation. Rapid application of multiple lesions greatly improves the speed, precision, and reduces pain in retinal photocoagulation.

  14. Slate characterization using 3D laser scanning

    NASA Astrophysics Data System (ADS)

    López, M.; Taboada, J.; Martínez, J.; Matías, J. M.; Vilán, J. A.

    2012-12-01

    Quality control is a necessary component of the slate slab manufacturing process so as to evaluate defects as defined by the current standard for slate. Quality control has traditionally been performed manually by an expert in the field, with the consequent human subjectivity. We studied the feasibility of using a 3D laser scanner to measure slate slabs and analyze possible defects that would lead to the rejection of slabs for particular industrial processes. The application requires slate characterization to be performed in real time and thereby requires a short computation time. We describe an optimized calibration method based on Tsai's approach that reduces calculation complexity and cost in this key 3D laser scanning stage. Configured and implemented for slate slab characterization, the system produces the required information in real time during the production process.

  15. Elastic extracellular matrix of the embryonic chick heart: an immunohistological study using laser confocal microscopy.

    PubMed

    Hurle, J M; Kitten, G T; Sakai, L Y; Volpin, D; Solursh, M

    1994-08-01

    The "elastic matrix" constitutes a specialized component of the extracellular matrix which confers resiliency to tissues and organs subjected to repeated deformations. The role of the elastic matrix in living organisms appears to be of key importance since diseases characterized by expression of defective inherited genes which encode components of the elastic matrix lead to premature death. While the elastic matrix of adult organs has received a great deal of attention, little is known about when it first appears in embryonic tissues or its possible role in developing organs. In the present study we have performed an immunohistochemical study of the distribution of elastin and three additional components often associated with elastic matrices in adult tissues (i.e., fibrillin, emilin, and type VI collagen) during the development of the chicken embryonic heart. The three-dimensional arrangement of these components was established through the observation of whole-amount specimens with scanning laser confocal microscopy. Our results revealed three different periods of heart development regarding the composition of the elastic matrix. Prior to stage 21 the embryonic heart lacks elastin but exhibits a matrix scaffold of fibrillin and emilin associated with the endocardium and the developing cardiac jelly. Between stages 22 and 29 the heart shows a transient elastic scaffold in the outflow tract which contains elastin, fibrillin, and emilin. Elastin-positive fibrillar material is also observed during these stages in the base of the atrioventricular cushion adjacent to the myocardial wall. In addition, emilin-positive material appears to be associated with the zones of formation of ventricular trabeculae. Collagen type VI was not detected during these early stages. From stage 30 to stage 40 a progressive modification of the pattern of distribution of elastin, fibrillin, emilin, and collagen type VI is observed in association with the formation of the definitive four

  16. 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.

  17. Damage detection using scanning laser vibrometer

    NASA Astrophysics Data System (ADS)

    Chen, Shen-En; Venkatappa, Suhas; Petro, Samer H.; Gangarao, Hota V.

    1998-06-01

    A damage detection algorithm based on the principle of curvature changes has been developed at CFC-WVU. However, the algorithm requires accurate mode shapes with adequate spatial density. Existing contact sensors can not provide adequate spatial density without adding excessive mass. Hence, non-contact scanning techniques, such as scanning laser vibrometer (SLV) has adequate sensitivity and accuracy is yet to be proven. The applicability of SLV on large structures is also questionable. To assess the suitability of using SLV for damage detection, a beam specimen has been tested using an existing system. The results confirm that damage detection using vibration measurements from SLV is successful. Due to more spatial density, the SLV data is shown to be more sensitive than the contact sensor test.

  18. 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.

  19. 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.

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

    PubMed

    Bhutani, Manoop S; Koduru, Pramoda; Joshi, Virendra; Karstensen, John G; Saftoiu, Adrian; Vilmann, Peter; Giovannini, Marc

    2015-04-01

    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.

  1. 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.

  2. Cross-Sectional Shape of Rat Mesenteric Arterioles at Branching Studied by Confocal Laser Microscopy

    NASA Astrophysics Data System (ADS)

    Nakano, Atushi; Minamiyama, Motomu; Niimi, Hideyuki

    This study was aimed to investigate the cross-sectional shape of mesenteric arterioles at branching, using confocal laser microscopy. Wistar rats (8 weeks, male) were anesthetized with thiobutabarbital sodium. Blood flow and microvascular network in the mesentery were observed using video microscopy. The rat intestine with mesentery was extracted and the intestinal vasculature was perfused with Krebs-Ringer and then fixed with paraformaldehyde under a static pressure of 100mmHg. A section of mesentery was isolated from the intestine, and spread up to the in vivo geometry based on the intravital microscopic observation. The mesentery section was stained with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin. The samples were observed under a confocal laser microscope. The cross-sectional image was re-sliced to measure the cross-sectional area and major/minor axes of the best fitting ellipse. The aspect ratio was defined in terms of the minor/major diameter ratio. The extended focus image of mesenteric arterioles showed that the cross-sectional shape was not circular but elliptic-like. The cross-sectional area of the parent vessel decreased from proximal to distal positions. The mean aspect ratio of the parent vessel was approximately 0.5, while that of the branching vessel was approximately 0.8. The flattened shape and variation of the cross-sectional area of arterioles requires some correction of in vivo data of the two-dimensional mesenteric microvasculature obtained using intravital microscopy.

  3. 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.

  4. Scanning Optics Enabled Possibilities and Challenges in Laser Cladding

    NASA Astrophysics Data System (ADS)

    Pekkarinen, Joonas

    Laser cladding using a scanned beam is quite a similar process than laser cladding using static optics (e.g. lens or mirror optics). The main difference comes from the manipulation of the laser beam. In laser cladding with scanning optics the laser beam is manipulated with a scanner so that the laser's area of influence can be shaped numerically. This increases cladding process flexibility. Scanning optics enable laser beam modification considerably versatile way than normal static optics can. This is due to possibility of numerical adjustment of scanning amplitude, laser power and scanning frequency. By modifying these parameters clad beads geometry can by modified quite freely. However scanned laser beam in surface modification process creates some restricting factors to the process. Mainly limitations for the process parameter values come from the dual characteristics of the energy input. This paper treats usability of scanning optics in laser cladding process in general level. In this paper is discussed how scanned beam can be used to increase the flexibility but also maters that limit the usage of scanned beam in cladding process. Process possibilities and limitations are presented in trough experimental data and examples.

  5. Fundus imaging in patients with cataract: role for a variable wavelength scanning laser ophthalmoscope.

    PubMed Central

    Kirkpatrick, J N; Manivannan, A; Gupta, A K; Hipwell, J; Forrester, J V; Sharp, P F

    1995-01-01

    AIMS--An investigation was carried out to compare the image quality of the ocular fundus obtained clinically, photographically, and with the scanning laser ophthalmoscope (SLO) at visible and infrared wavelengths in patients with significant cataract. METHODS--Nineteen patients admitted for routine cataract extraction were examined clinically by two independent observers to ascertain cataract type and clarity of fundus view with an indirect ophthalmoscope. Fundus photography and both confocal and direct (non-confocal) SLO imaging at 590 nm, 670 nm, and 830 nm were carried out after pupillary dilatation. Images obtained were graded independently using a recognised grading system. RESULTS--Quality of SLO images appeared to be superior to indirect ophthalmoscopy (p < 0.01) and fundus photography (p < 0.001) when graded subjectively. Quantitative analysis of contrast of retinal vessels demonstrated significantly higher contrast for the SLO compared with digitised fundus photographs at all wavelengths tested (p < 0.001), with highest contrast at 590 nm. Use of a confocal aperture significantly improved vessel contrast but may reduce overall image intensity. CONCLUSIONS--Scanning laser ophthalmoscopy may offer a method to observe and record fine fundus detail in patients who have marked cataract. Images PMID:7488576

  6. Terrestrial Laser Scanning for Vegetation Sampling

    PubMed Central

    Richardson, Jeffrey J; Moskal, L. Monika; Bakker, Jonathan D.

    2014-01-01

    We developed new vegetation indices utilizing terrestrial laser scanning (TLS) to quantify the three-dimensional spatial configuration of plant communities. These indices leverage the novelty of TLS data and rely on the spatially biased arrangement of a TLS point cloud. We calculated these indices from TLS data acquired within an existing long term manipulation of forest structure in Central Oregon, USA, and used these data to test for differences in vegetation structure. Results provided quantitative evidence of a significant difference in vegetation density due to thinning and burning, and a marginally significant difference in vegetation patchiness due to grazing. A comparison to traditional field sampling highlighted the novelty of the TLS based method. By creating a linkage between traditional field sampling and landscape ecology, these indices enable field investigations of fine-scale spatial patterns. Applications include experimental assessment, long-term monitoring, and habitat characterization. PMID:25353981

  7. Terrestrial laser scanning for vegetation sampling.

    PubMed

    Richardson, Jeffrey J; Moskal, L Monika; Bakker, Jonathan D

    2014-10-28

    We developed new vegetation indices utilizing terrestrial laser scanning (TLS) to quantify the three-dimensional spatial configuration of plant communities. These indices leverage the novelty of TLS data and rely on the spatially biased arrangement of a TLS point cloud. We calculated these indices from TLS data acquired within an existing long term manipulation of forest structure in Central Oregon, USA, and used these data to test for differences in vegetation structure. Results provided quantitative evidence of a significant difference in vegetation density due to thinning and burning, and a marginally significant difference in vegetation patchiness due to grazing. A comparison to traditional field sampling highlighted the novelty of the TLS based method. By creating a linkage between traditional field sampling and landscape ecology, these indices enable field investigations of fine-scale spatial patterns. Applications include experimental assessment, long-term monitoring, and habitat characterization.

  8. A Pilot Study of In Vivo Confocal Laser Endomicroscopy of Upper Tract Urothelial Carcinoma

    PubMed Central

    Bui, Daniel; Mach, Kathleen E.; Zlatev, Dimitar V.; Rouse, Robert V.; Leppert, John T.

    2015-01-01

    Abstract Purpose: Tissue diagnosis of upper tract urothelial carcinoma (UTUC) is limited by variance in tumor sampling by standard ureteroscopic biopsy. Optical imaging technologies can potentially improve UTUC diagnosis, surveillance, and endoscopic treatment. We previously demonstrated in vivo optical biopsy of urothelial carcinoma of the bladder using confocal laser endomicroscopy (CLE). In this study, we evaluated a new 0.85-mm imaging probe in the upper urinary tract and demonstrated feasibility and compatibility with standard ureteroscopes to achieve in vivo optical biopsy of UTUC. Materials and Methods: Fourteen patients scheduled for ureteroscopy of suspected upper tract lesions or surveillance of UTUC were recruited. After intravenous (IV) administration of fluorescein, CLE was performed using a 0.85-mm-diameter imaging probe inserted through the working channel of standard ureteroscopes. Acquired confocal video sequences were reviewed and analyzed. A mosaicing algorithm was used to compile a series of images into a single larger composite image. Processed CLE images were compared with standard histopathologic analysis. Results: Optical biopsy of the UTUC using CLE was effectively achieved during standard ureteroscopy. There were no adverse events related to IV fluorescein administration or image acquisition. Confocal imaging of UTUC showed characteristic features similar to urothelial carcinoma of the bladder, including papillary structure, fibrovascular stalks, and pleomorphism. Lamina propria in normal areas of the renal pelvis and ureter was also identified. Conclusions: We report an initial feasibility of CLE of UTUC. Pending further clinical investigation, CLE may become a useful adjunct to ureteroscopic biopsy, endoscopic ablation, and surveillance of UTUC. PMID:26413927

  9. Scanning laser retinoscopy: a new technique for evaluating optical properties of the cornea after refractive surgery

    NASA Astrophysics Data System (ADS)

    Van de Velde, Frans J.; Tassignon, Marie J.; Trau, Rene

    1997-12-01

    We present a new technique, scanning laser retinoscopy, to spatially resolve in two dimensions the optical aberrations and refractive power of the ocular media. For this purpose, the Maxwellian view of a confocal scanning laser ophthalmoscope (SLO) is configured to scan simultaneously the posterior and the anterior segment of the eye at different levels of prefocussing. This set-up allows retinal imaging and psychophysics through different optical zones of the cornea and lens. In addition, the size of the anatomical pupil can be dynamically controlled by adjusting the colinear infrared and visible light intensities of the illuminating system. In retinoscopic images we can see a part of the retina superimposed by distinctive patterns of shadows in the pupillary area. The variable patterns of shadows in the retinoscopic images change with the level of prefocussing of the SLO. The patterns are the result of local variations in refraction or wavefront aberrations within the lens and cornea. In cases of excimer laser refractive surgery, for example, scanning laser retinoscopy is able to distinguish between a treated central area, transition zone and peripheral cornea. As a corollary, we can document differences between excimer laser delivery systems and also correlate the retinoscopic images with the subjective complaints of refractive surgery patients. These include monocular diplopia, glare, loss of contrast sensitivity besides reduced visual acuity.

  10. Confocal and scanning electron microscopic study of teeth restored with fiber posts, metal posts, and composite resins.

    PubMed

    Mannocci, F; Innocenti, M; Ferrari, M; Watson, T F

    1999-12-01

    Forty-two single-rooted lower premolars, extracted for periodontal reasons, were endodontically treated and divided into 7 groups of 6 teeth each. In five of the groups, three different types of carbon fiber posts, quartz fiber posts, and titanium posts were used in combination with All Bond 2 dental adhesive. In two groups, two types of carbon fiber posts were also cemented with Panavia 21 dental adhesive. After 3 wk storage in saline, the teeth were longitudinally sectioned; one half was observed using confocal microscopy and the other by scanning electron microscopy. The specimens were evaluated for the presence of a resin dentin interdiffusion zone for the presence of voids at post-resin-dentin interfaces and for the determination of the fiber posts' structure. Upon examination with the confocal microscope, the interfaces of teeth restored with All Bond 2 showed a higher percentage (p < 0.05) of resin dentin interdiffusion zone than those treated with panavia. The fiber size and the post structure were similar in all the fiber posts observed. Some voids were present inside the fiber post structure.

  11. 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.

  12. Visualizing epithelial expression of EGFR in vivo with distal scanning side-viewing confocal endomicroscope

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  13. Visualizing epithelial expression of EGFR in vivo with distal scanning side-viewing confocal endomicroscope

    PubMed Central

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

    2016-01-01

    Confocal endomicroscopy is an emerging imaging technology that has recently been introduced into the clinic to instantaneously collect “optical biopsies” in vivo with histology-like quality. Here, we demonstrate a fast scanner located in the distal end of a side-viewing instrument using a compact lens assembly with numerical aperture of 0.5 to achieve a working distance of 100 μm and field-of-view of 300 × 400 μm2. The microelectromechanical systems (MEMS) mirror was designed based on the principle of parametric resonance and images at 5 frames per second. The instrument has a 4.2 mm outer diameter and 3 cm rigid length, and can pass through the biopsy channel of a medical endoscope. We achieved real time optical sections of NIR fluorescence with 0.87 μm lateral resolution, and were able to visualize in vivo binding of a Cy5.5-labeled peptide specific for EGFR to the cell surface of pre-cancerous colonocytes within the epithelium of dysplastic crypts in mouse colon. By performing targeted imaging with endomicroscopy, we can visualize molecular expression patterns in vivo that provide a biological basis for disease detection. PMID:27874037

  14. Noninvasive Ultrasound Imaging for Bone Quality Assessment Using Scanning Confocal Acoustic Diagnosis, μCT, DXA Measurements, and Mechanical Testing

    NASA Astrophysics Data System (ADS)

    Qin, Yi-Xian; Xia, Yi; Lin, Wei; Mittra, Erik; Rubin, Clint; Gruber, Barry

    Osteoporosis is a disease characterized by decreased bone mass and progressive deterioration of the microstructure, affecting both mineral density and bone's fragility. Current diagnoses are only measuring apparent bone mineral density (AppBMD). Using our newly developed scanning confocal acoustic diagnostic (SCAD) system, we evaluated the ability of quantitative ultrasound in noninvasively predicting bone's quantity and quality on 19 human cadaver calcanei. Results show that ultrasound attenuation image on intact calcaneus represents bone mass distribution. High correlation (R=0.82) exists between SCAD determined broadband ultrasound attenuation (BUA) and DXA determined AppBMD at the calcaneus, as well as in the AppBMD result at femoral neck (R=0.81). SCAD determined BUA and ultrasound velocity (UV) are highly correlated with the micro-CT and mechanical testing determined bone quantity and quality parameters. These results suggest that image-based quantitative ultrasound is able to identify ROI and predict both bone mass and strength.

  15. In vivo analysis of solar lentigines by reflectance confocal microscopy before and after Q-switched ruby laser treatment.

    PubMed

    Richtig, Erika; Hofmann-Wellenhof, Rainer; Kopera, Daisy; El-Shabrawi-Caelen, Laila; Ahlgrimm-Siess, Verena

    2011-03-01

    Solar lentigines are benign lesions usually found on sun-damaged skin. We investigated twelve cases of solar lentigines through dermoscopy and reflectance confocal microscopy, performed before, and 30 min and 10 days after, a single treatment with a Q-switched ruby laser. At baseline, all lesions showed characteristic features of solar lentigines in reflectance confocal microscopy analysis: regular honeycomb patterns, edged dermal papillae and cord-like rete ridges at the dermoepidermal junction. Thirty minutes post-laser treatment, blurred epidermal intercellular connections, dark structureless areas of different sizes and shapes in the lower epidermal layers, and hyporeflective dermal papillae, reflecting epidermal and dermal oedema, were observed. Ten days post-treatment highly reflective round-to polygonal areas and aggregated granules, representing extracellular melanin, were detected in all epidermal layers featuring regular honeycomb patterns. Reflectance confocal microscopy can be used to visualise dynamic skin processes, allowing non-invasive in vivo follow-up of skin lesions after treatment.

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

    SciTech Connect

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

    2009-11-20

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

  17. A Review of Probe-Based Confocal Laser Endomicroscopy for Pancreaticobiliary Disease.

    PubMed

    Karia, Kunal; Kahaleh, Michel

    2016-09-01

    Confocal laser endomicroscopy (CLE) is a novel in vivo imaging technique that can provide real-time optical biopsies in the evaluation of pancreaticobiliary strictures and pancreatic cystic lesions (PCLs), both of which are plagued by low sensitivities of routine evaluation techniques. Compared to pathology alone, CLE is associated with a higher sensitivity and accuracy for the evaluation of indeterminate pancreaticobiliary strictures. CLE has the ability to determine the malignant potential of PCLs. As such, CLE can increase the diagnostic yield of endoscopic retrograde cholangiopancreatography and endoscopic ultrasound, reducing the need for repeat procedures. It has been shown to be safe, with an adverse event rate of ≤1%. Published literature regarding its cost-effectiveness is needed.

  18. A Review of Probe-Based Confocal Laser Endomicroscopy for Pancreaticobiliary Disease

    PubMed Central

    Karia, Kunal; Kahaleh, Michel

    2016-01-01

    Confocal laser endomicroscopy (CLE) is a novel in vivo imaging technique that can provide real-time optical biopsies in the evaluation of pancreaticobiliary strictures and pancreatic cystic lesions (PCLs), both of which are plagued by low sensitivities of routine evaluation techniques. Compared to pathology alone, CLE is associated with a higher sensitivity and accuracy for the evaluation of indeterminate pancreaticobiliary strictures. CLE has the ability to determine the malignant potential of PCLs. As such, CLE can increase the diagnostic yield of endoscopic retrograde cholangiopancreatography and endoscopic ultrasound, reducing the need for repeat procedures. It has been shown to be safe, with an adverse event rate of ≤1%. Published literature regarding its cost-effectiveness is needed. PMID:27642847

  19. Mathematical model for light scanning system based on circular laser

    NASA Astrophysics Data System (ADS)

    Xu, Peiquan; Yao, Shun; Lu, Fenggui; Tang, Xinhua; Zhang, Wei

    2005-11-01

    A novel light scanning system based on circular laser trajectory for welding robot is developed. With the help of image processing technique, intelligent laser welding could be realized. According to laser triangulation algorithm and Scheimpflug condition, mathematical model for circular laser vision is built. This scanning system projects circular laser onto welded seams and recovers the depth of the welded seams, escapes from shortcomings of less information, explains ambiguity and single tracking direction inherent in "spot" or "line" type laser trajectory. Three-dimensional (3D) model for welded seams could be recognized after depth recovery. The imaging error is investigated also.

  20. 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.

  1. Effect of EndoActivator and Er,Cr:YSGG laser activation of Qmix, as final endodontic irrigant, on sealer penetration: A Confocal microscopic study

    PubMed Central

    Yadav, Sudha; Talwar, Sangeeta; Verma, Mahesh

    2017-01-01

    Background Through chemomechanical debridement of the root canal is a primary requisite for successful endodontic therapy. Thus the aim of this study was to evaluate the effects of using QmiX alone, QmiX with EndoActivator and QmiX with Er,Cr:YSGG laser for final irrigation on sealer penetration into the dentinal tubules. Material and Methods 75 extracted human mandibular premolar teeth were treated with sodium hypochlorite (NaOCl) irrigation. The samples were divided into 5 groups according to the final irrigation solution used: (1) 17% EDTA and 2.5% NaOCl, (2) QmiX (3) QmiX with Er,Cr:YSGG laser and (4) QmiX with EndoActivator (5) 2.5%NaOCl. All teeth were obturated using cold lateral condensation technique with gutta percha and AH 26 sealer (Dentsply; DeTrey,Konstanz, Germany) labeled with Rhodamine B dye. The teeth were sectioned at distances of 2 and 5 from root apex. Total percentage and maximum depth of sealer penetration were measured using confocal laser scanning microscopy. Results Results of one way Anova analysis showed that there was a significant difference in the percentage and depth of sealer penetration among all groups at 3 and 5 mm level sections (P < .05). Within the groups maximum sealer penetration was recorded for Er,Cr:YSGG laser activated group. Greater depth of sealer penetration was recorded at 5mm as compared to 3mm in all the groups. Conclusions Activation of QMix using EndoActivator and Er,Cr:YSGG laser enhanced the sealer penetration at apical and middle third. Thus Er,Cr:YSGG laser and EndoActivator may act as an appropriate adjunct during chemomechanical preparation of the root canal. Key words:EndoActivator, Er,Cr:YSGG laser, Qmix, confocal microscopy, sealer penetration. PMID:28210439

  2. [Application of confocal micro-beam X-ray fluorescence in nondestructive scanning analysis of the distribution of elements in a single hair].

    PubMed

    Liu, He-He; Liu, Zhi-Guo; Sun, Tian-Xi; Peng, Song; Zhao, Wei-Gang; Sun, Wei-Yuan; Li, Yu-De; Lin, Xiao-Yan; Zhao, Guang-Cui; Luo, Ping; Ding, Xun-Liang

    2013-11-01

    The confocal micro X-ray fluorescence (XRF) based on polycapillary X-ray lens and conventional X-ray source was used to carry out the scanning analysis of the distribution of the elements in a single hair. The elemental distribution in the single hair was obtained. In the confocal micro XRF technology, the output focal spot of the polycapillary focusing X-ray lens and the input focal spot of the polycapillary parallel X-ray lens were adjusted confocally. The detector could only detect the X-rays from the overlapping foci. This confocal structure decreased the effects of the background on the X-ray spectra, and was accordingly helpful for improving the accuracy of this XRF technology. A polycapillary focusing X-ray lens with a high gain in power density was used to decrease the requirement of power of the X-ray source used in this confocal technology, and made it possible to perform such confocal micro XRF analysis by using the conventional X-ray source with low cost. Experimental results indicated that the confocal micro X-ray fluorescence based on polycapillary X-ray lens had potential applications in analyzing the elemental distribution of individual hairs.

  3. 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.

  4. 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.

  5. 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.

  6. Multi-point scanning two-photon excitation microscopy by utilizing a high-peak-power 1042-nm laser.

    PubMed

    Otomo, Kohei; Hibi, Terumasa; Murata, Takashi; Watanabe, Hirotaka; Kawakami, Ryosuke; Nakayama, Hiroshi; Hasebe, Mitsuyasu; Nemoto, Tomomi

    2015-01-01

    The temporal resolution of a two-photon excitation laser scanning microscopy (TPLSM) system is limited by the excitation laser beam's scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti:sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV. This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells.

  7. 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

  8. SLATE: scanning laser automatic threat extraction

    NASA Astrophysics Data System (ADS)

    Clark, David J.; Prickett, Shaun L.; Napier, Ashley A.; Mellor, Matthew P.

    2016-10-01

    SLATE is an Autonomous Sensor Module (ASM) designed to work with the SAPIENT system providing accurate location tracking and classifications of targets that pass through its field of view. The concept behind the SLATE ASM is to produce a sensor module that provides a complementary view of the world to the camera-based systems that are usually used for wide area surveillance. Cameras provide a hi-fidelity, human understandable view of the world with which tracking and identification algorithms can be used. Unfortunately, positioning and tracking in a 3D environment is difficult to implement robustly, making location-based threat assessment challenging. SLATE uses a Scanning Laser Rangefinder (SLR) that provides precise (<1cm) positions, sizes, shapes and velocities of targets within its field-of-view (FoV). In this paper we will discuss the development of the SLATE ASM including the techniques used to track and classify detections that move through the field of view of the sensor providing the accurate tracking information to the SAPIENT system. SLATE's ability to locate targets precisely allows subtle boundary-crossing judgements, e.g. on which side of a chain-link fence a target is. SLATE's ability to track targets in 3D throughout its FoV enables behavior classification such as running and walking which can provide an indication of intent and help reduce false alarm rates.

  9. 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.

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

    PubMed Central

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

    2015-01-01

    Abstract. 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. PMID:26509413

  11. Rapid scanning autocorrelator for measurements of picosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Harde, H.; Burggraf, H.

    1981-08-01

    A rapid scanning autocorrelation interferometer for measurements of picosecond laser pulses is described which uses a rotating prism as scanning device in one arm of the interferometer to permit continuous display of autocorrelation traces at audio frequencies on an oscilloscope. Scan widths of more than 500 ps with high linearity can be achieved. Autocorrelation measurements of picosecond pulses from a synchronously pumped mode-locked dye laser are presented.

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

    NASA Astrophysics Data System (ADS)

    Kotlarchyk, M. A.; Botvinick, E. L.; Putnam, A. J.

    2010-05-01

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

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

    PubMed

    Kotlarchyk, M A; Botvinick, E L; Putnam, A J

    2010-05-19

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

  14. 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.

  15. Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces

    PubMed Central

    Anguish, L. J.; Ghiorse, W. C.

    1997-01-01

    A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10(sup2) oocysts(middot)g [dry weight](sup-1)) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical sections revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence. PMID:16535523

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. A semi-automatic 3D laser scan system design

    NASA Astrophysics Data System (ADS)

    Xiong, Hanwei; Pan, Ming; Zhang, Xiangwei

    2009-11-01

    Digital 3D models are now used everywhere, from traditional fields of industrial design, artistic design, to heritage conservation. Although laser scan is very useful to get densely samples of the objects, nowadays, such an instrument is expensive and always need to be connected to a computer with stable power supply, which prevent it from usage for fieldworks. In this paper, a new semi-automatic 3D laser scan method is proposed using two line laser sources. The planes projected from the laser sources are orthogonal, one of which is fixed relative to the camera, and the other can be rotated along a settled axis. Before scanning, the system must be calibrated, from which the parameters of the camera, the position of the fixed laser plane and the settled axis are introduced. In scanning process, the fixed laser plane and the camera form a conventional structured light system, and the 3d positions of the intersection curves of the fixed laser plane with the object can be computed. The other laser plane is rotated manually or mechanically, and its position can be determined from the cross point intersecting with the fixed laser plane on the object, so the coordinates of sweeping points can be obtained. The new system can be used without a computer (The data can be processed later), which make it suitable for fieldworks. A scanning case is given in the end.

  3. 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 ...

  4. Damage visualization using synchronized noncontact laser ultrasonic scanning

    NASA Astrophysics Data System (ADS)

    Liu, Peipei; Sunarsa, Timotius Yonathan; Sohn, Hoon

    2016-04-01

    This paper presents a damage visualization technique using a fully noncontact laser ultrasonic measurement system and a synchronized scanning strategy. The noncontact laser ultrasonic measurement system is composed of a Q-switched Nd:YAG laser for ultrasonic wave generation and a laser Doppler vibrometer (LDV) for ultrasonic wave detection. The laser beams for ultrasonic wave generation and detection are shot on the target structure with a constant and tiny distance, and these two laser beams are synchronously moved over the scanning area. Compared with conventional laser scanning strategies, the ultrasonic responses detected through the synchronized scanning strategy owns a much higher and more stable signal to noise ratio and the scanning time can be significantly reduced with less time averaging. By spatial comparison in the scanning area, damage can be detected and visualized without relying on baseline data obtained from the pristine condition of the target structure. In this paper, the developed technique is validated by visualization hidden corrosion in a steel straight pipe and a steel elbow pipe.

  5. 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).

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Excitation beyond the monochromatic laser limit: simultaneous 3-D confocal and multiphoton microscopy with a tapered fiber as white-light laser source.

    PubMed

    Betz, Timo; Teipel, Jörn; Koch, Daniel; Härtig, Wolfgang; Guck, Jochen; Käs, Josef; Giessen, Harald

    2005-01-01

    Confocal and multiphoton microscopy are essential tools in modern life sciences. They allow fast and highly resolved imaging of a steadily growing number of fluorescent markers, ranging from fluorescent proteins to quantum dots and other fluorophores, used for the localization of molecules and the quantitative detection of molecular properties within living cells and organisms. Up to now, only one physical limitation seemed to be unavoidable. Both confocal and multiphoton microscopy rely on lasers as excitation sources, and their monochromatic radiation allows only a limited number of simultaneously usable dyes, which depends on the specific number of laser lines available in the used microscope. We have overcome this limitation by successfully replacing all excitation lasers in a standard confocal microscope with pulsed white light ranging from 430 to 1300 nm generated in a tapered silica fiber. With this easily reproducible method, simultaneous confocal and multiphoton microscopy was demonstrated. By developing a coherent and intense laser source with spectral width comparable to a mercury lamp, we provide the flexibility to excite any desired fluorophore combination.

  13. 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.

  14. 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

  15. Three-dimensional imaging of interphase cell nuclei with laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Boecker, Wilfried; Radtke, Thomas; Streffer, Christian

    1998-10-01

    During the past decade 3D image processing has become an important key component in biological research mainly due to two different developments. The first is based on an optical instrument, the so-called confocal laser scanning microscope, allowing optical sectioning of the biological specimen. The second is a biological preparatory method, the so-called FISH-technique (Fluorescence-In-Situ- Hybridization), allowing labeling of certain cellular and sub-cellular compartments with highly specific fluorescent dyes. Both methods make it possible to investigate the 3D biological framework within cells and nuclei. Image acquisition with confocal laser scanning microscopy must deal with different limits of resolution along and across the optical axis. Although lateral resolution is about 0.7 times better than in non-confocal arrangements, axial resolution is more than 3 - 4 times poorer than that of the lateral (depending on the pinhole size). For 3D reconstruction it is desirable to improve axial resolution in order to provide nearly identical image information across the 3D specimen space. This presentation will give an overview of some of the most popular restoration and deblurring algorithms used in 3D image microscopy. After 3D image restoration, segmentation of certain details of the cell structure is usually the next step in image processing. We compared two different kinds of algorithms for segmentation of chromosome territories in interphase cell nuclei. One is based on Mathematical Morphology, the other on Split & Merge methods. The segmented image regions provided the basis for chromosome domain reconstruction as well as for regional localization for subsequent quantitative measurements. As a result the chromatin density within certain chromosome domains as well as some terminal DNA sequences (telomere signals) could be measured.

  16. Comparison of three-dimensional retinal imaging methods: the method of scanning laser triangulation.

    PubMed

    Milbocker, M T; Reznichenko, Y M

    1991-10-01

    Three methods of three-dimensional imaging of the vitreous and the fundus of the human eye are compared. Equations are derived for the theoretical depth resolution of stereophotogrammetry, scanning laser tomography, and scanning laser triangulation. Scanning laser triangulation provides superior depth resolution without requiring axial scanning. A description of a prototype scanning laser triangulator is given.

  17. 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

  18. Stereo vision based hand-held laser scanning system design

    NASA Astrophysics Data System (ADS)

    Xiong, Hanwei; Xu, Jun; Wang, Jinming

    2011-11-01

    Although 3D scanning system is used more and more broadly in many fields, such computer animate, computer aided design, digital museums, and so on, a convenient scanning device is expansive for most people to afford. In another hand, imaging devices are becoming cheaper, a stereo vision system with two video cameras cost little. In this paper, a hand held laser scanning system is design based on stereo vision principle. The two video cameras are fixed tighter, and are all calibrated in advance. The scanned object attached with some coded markers is in front of the stereo system, and can be changed its position and direction freely upon the need of scanning. When scanning, the operator swept a line laser source, and projected it on the object. At the same time, the stereo vision system captured the projected lines, and reconstructed their 3D shapes. The code markers are used to translate the coordinate system between scanned points under different view. Two methods are used to get more accurate results. One is to use NURBS curves to interpolate the sections of the laser lines to obtain accurate central points, and a thin plate spline is used to approximate the central points, and so, an exact laser central line is got, which guards an accurate correspondence between tow cameras. Another way is to incorporate the constraint of laser swept plane on the reconstructed 3D curves by a PCA (Principle Component Analysis) algorithm, and more accurate results are obtained. Some examples are given to verify the system.

  19. Automated pressure scanning of tunable dye lasers

    NASA Astrophysics Data System (ADS)

    Gottscho, R. A.

    1985-04-01

    A method for the remote control of tunable laser frequency tuning is proposed in the framework of real-time monitoring of the chemistry and physics of plasma, combustion, and chemical vapor deposition reactions. The technique presented involves indirect frequency tuning and stabilization by direct control of the laser cavity pressure. The long-term drift in power, resulting from the grating and etalon misalignment is suggested to be correctable by using a second feedback circuit which would optimize laser power by finely tuning the etalon or grating. Experimental results obtained with a dye laser of Hansch type are included; a maximum variation in LIF signal of + or - 7 percent, which corresponds to a frequency drift of + or - 0.005/cm, over a 30-min interval was achieved. A block diagram of the feedback loop and the LIF apparatus are included.

  20. 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

  1. Geodetic Laser Scanning: Refractive Optics Offer Wide Variety of Scan Patterns

    NASA Astrophysics Data System (ADS)

    Carter, W. E.; Shrestha, R. L.; Slatton, C. K.; Shrestha, K. Y.; Cossio, T.

    2005-12-01

    Most commercial geodetic laser mapping instruments use reflective element scanners, often a single nutating or oscillating mirror, and sometimes dual axis units, to create a specific pattern of laser spots on the surface being mapped. The user may be able to set the scanning speed (scan lines per second) and field of coverage (range of scan angles), but the basic pattern of points sampled is fixed. Engineers developing scanners for a surprisingly diverse set of applications, ranging from bar code scanning, to compensating for image motion in astronomical telescopes, to scanning spectrometers, have increasingly turned to refractive scanners-most particularly to scanners that utilize "Risley prisms." Samuel Doty Risley (1845-1920), an ophthalmologist, invented an optometer that contained a pair of thin prisms that rotated in opposite directions about their optical axes to change the convergence of light rays from a single source. He used his optometer measure the visual acuity of patients eyes, as a function of distance. In this original application, both prisms were driven by a common gear assembly, which resulted in a nearly linear scan line. But if the prisms are driven independently in both direction and angular speed, a wide variety of scan patterns can be generated. The University of Florida is developing, a photon counting geodetic laser scanning instrument that will use a Risley prism scanner. The scanner, being built by Sigma Space Inc., will be capable of producing nearly linear scan lines (saw tooth pattern from moving platform), circular scans lines (helical pattern from a moving platform) and any number of rosette scan patterns that are particularly interesting for fixed ground based work. The flexibility provided by the scanner offers the possibility of using the same sensor for airborne and ground based geodetic laser scanning. Examples of the scanner patterns and the initial results from laboratory and early field tests will be presented.

  2. Laser-scanning Doppler photoacoustic microscopy based on temporal correlation

    NASA Astrophysics Data System (ADS)

    Song, Wei; Liu, Wenzhong; Zhang, Hao F.

    2013-05-01

    We present a methodology to measure absolute flow velocity using laser-scanning photoacoustic microscopy. To obtain the Doppler angle, the angle between ultrasonic detection axis and flow direction, we extracted the distances between the transducer and three adjacent scanning points along the flow and repeatedly applied the law of cosines. To measure flow velocity along the ultrasonic detection axis, we calculated the time shift between two consecutive photoacoustic waves at the same scanning point, then converted the time shift to velocity according to the sound velocity and time interval between two laser illuminations. We verified our method by imaging flow phantoms.

  3. 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.

  4. Phase relation recovery for scanning laser Doppler vibrometry

    NASA Astrophysics Data System (ADS)

    Alveringh, D.; Sanders, R. G. P.; Wiegerink, R. J.; Lötters, J. C.

    2017-02-01

    Laser Doppler vibrometers are able to measure the velocity of a single point compared to a reference point by analyzing the Doppler shift of the laser beams. In many commercially available laser Doppler vibrometers, the laser point can be scanned to obtain an out-of-plane velocity profile of a surface. It is essential that the phase information of the velocities between points is measured as well to be able to fully reproduce the velocity profile of the surface. If this cannot be done by triggering on the actuation signal, the proposed two stage method can be used. This method measures the surface in two stages: one scan with the reference beam at a fixed point and one scan with the reference beam on a moving point. The algorithm in this article calculates the phase and reconstructs the velocity of each point. This is experimentally verified on three different micro structures. The postprocessing algorithm is not intensive in computing power.

  5. 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).

  6. 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

  7. 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...

  8. Confocal Microscopy Of The Eye.

    NASA Astrophysics Data System (ADS)

    Masters, Barry R.

    1989-12-01

    A laser scanning confocal microscope was used to study the structure of human donor eyes and enucleated rabbit eyes. Reflected light confocal images were obtained with a Leitz water immersion objective (50X, NA 1.0). A drop of bicarbonate Ringer's was placed between the objective and the tissue to optically couple the tissue. The confocal microscope was used to image the following objects within the eye: superficial epithelial cells, super basal and basal epithelial cells, basement membrane, stromal nerve plexus, nerve fibers, nuclei and cell bodies of stromal keratocytes, cell processes of stromal keratocytes, Descemet's membrane, and the endothelial cells. In addition, the ocular lens and excised retina were imaged. The confocal microscope produces images of the eye with the following enhanced features: increased lateral resolution, decreased depth of field, and increased contrast of transparent ocular structures. It is concluded that confocal imaging systems are an improvement over traditional optical instruments, and they may develop into a new tool for basic visual science and clinical ophthalmology.

  9. Concurrent Reflectance Confocal Microscopy and Laser Doppler Flowmetry to Improve Skin Cancer Imaging: A Monte Carlo Model and Experimental Validation

    PubMed Central

    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

  10. 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)

  11. A Laser Sheet Self-Calibration Method for Scanning PIV

    NASA Astrophysics Data System (ADS)

    Knutsen, Anna N.; Dawson, James R.; Lawson, John M.; Worth, Nicholas A.

    2016-11-01

    A laser sheet self-calibration method for scanning PIV has been developed to replace the current laser sheet calibration, which is complex, time consuming and very sensitive to misalignment of the optics or cameras during experiments. The new calibration method is simpler, faster and crucially more robust. The concept behind the method is to traverse a laser sheet through the measurement volume, take a series of images from two different views, and calculate the global 3D particle locations. This information is used to find the real space coordinates of the measurement volume and the orientation and width of the laser sheets. The spatial location of the particles is found by object matching and triangulation. The light intensity in the laser sheet has an approximately Gaussian shape, and the illumination of one particle which will be illuminated multiple times during the scan will thus vary as the sheet is scanned across the measurement volume. The thickness of the laser sheet is calculated by identifying the variation of illumination of the particles during a scan and fitting this to a Gaussian shaped curve, while the orientation is found using a least square fit. The accuracy of the new method will be presented with respect to both synthetic and experimental data.

  12. Applying RANSAC Algorithm for Fitting Scanning Strips from Airborne Laser Scanning

    NASA Astrophysics Data System (ADS)

    Błaszczak-Bąk, Wioleta; Janicka, Joanna; Sobieraj-Żłobińska, Anna

    2016-12-01

    During the development of the data acquired by airborne laser scanning the important issue is the fitting and georeferencing of ALS point clouds by means of the tie surfaces and the reference planes. The process of scanning strips adjustment is based on mutual integration of point clouds (scanning strips) and their adaptation to the reference planes. In simultaneous adjustment all strips are combined into one geometrically coherent block, to which the coordinates are given. In the process of determining discrepancies between scanning strips it is important to determine the correct size of the shifts (offsets). Authors propose to do this by using RANSAC algorithm.

  13. Diffractive elements performance in chromatic confocal microscopy

    NASA Astrophysics Data System (ADS)

    Garzón, J.; Duque, D.; Alean, A.; Toledo, M.; Meneses, J.; Gharbi, T.

    2011-01-01

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

  14. 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.

  15. 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

  16. 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}.

  17. 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}.

  18. 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

  19. Validation of diagnostic characteristics of needle based confocal laser endomicroscopy in differentiation of pancreatic cystic lesions

    PubMed Central

    Krishna, Somashekar G.; Swanson, Benjamin; Hart, Phil A.; El-Dika, Samer; Walker, Jon P.; McCarthy, Sean T.; Malli, Ahmad; Shah, Zarine K.; Conwell, Darwin L.

    2016-01-01

    Background and aims: Endoscopic ultrasound (EUS)-guided needle-based Confocal Laser Endomicroscopy (nCLE) characteristics of pancreatic cystic lesions (PCLs) have been identified in studies where the gold standard surgical histopathology was available in a minority of patients. There are diverging reports of interobserver agreement (IOA) and paucity of intraobserver reliability (IOR). Thus, we sought to validate current EUS-nCLE criteria of PCLs in a larger consecutive series of surgical patients. Methods: A retrospective analysis of patients who underwent EUS-nCLE at a single center was performed. For calculation of IOA (Fleiss’ kappa) and IOR (Cohen’s kappa), blinded nCLE-naïve observers (n = 6) reviewed nCLE videos of PCLs in two phases separated by a 2-week washout period. Results: EUS-nCLE was performed in 49 subjects, and a definitive diagnosis was available in 26 patients. The overall sensitivity, specificity, and accuracy for diagnosing a mucinous PCL were 94 %, 82 %, and 89 %, respectively. The IOA for differentiating mucinous vs. non-mucinous PCL was “substantial” (κ = 0.67, 95 %CI 0.57, 0.77). The mean (± standard deviation) IOR was “substantial” (κ = 0.78 ± 0.13) for diagnosing mucinous PCLs. Both the IOAs and mean IORs were “substantial” for detection of known nCLE image patterns of papillae/epithelial bands of mucinous PCLs (IOA κ = 0.63; IOR κ = 0.76 ± 0.11), bright particles on a dark background of pseudocysts (IOA κ = 0.71; IOR κ = 0.78 ± 0.12), and fern-pattern or superficial vascular network of serous cystadenomas (IOA κ = 0.62; IOR κ = 0.68 ± 0.20). Three (6.1 % of 49) patients developed post-fine needle aspiration (FNA) pancreatitis. Conclusion: Characteristic EUS-nCLE patterns can be consistently identified and improve the diagnostic accuracy of PCLs. These results support further investigations to optimize EUS-nCLE while minimizing adverse events

  20. 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

  1. 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.

  2. 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

  3. Two-Photon Laser Scanning Stereomicroscopy for Fast Volumetric Imaging

    PubMed Central

    Yang, Yanlong; Yao, Baoli; Lei, Ming; Dan, Dan; Li, Runze; Horn, Mark Van; Chen, Xun; Li, Yang; Ye, Tong

    2016-01-01

    Bessel beams have been successfully used in two-photon laser scanning fluorescence microscopy to extend the depth of field (EDF), which makes it possible to observe fast events volumetrically. However, the depth information is lost due to integration of fluorescence signals along the propagation direction. We describe the design and implementation of two-photon lasers scanning stereomicroscopy, which allows viewing dynamic processes in three-dimensional (3D) space stereoscopically in real-time with shutter glasses at the speed of 1.4 volumes per second. The depth information can be appreciated by human visual system or be recovered with correspondence algorithms for some cases. PMID:27997624

  4. 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.

  5. 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

  6. 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%.

  7. Nonmechanical axial scanning laser Doppler velocimeter with directional discrimination.

    PubMed

    Maru, Koichi; Hata, Takahiro

    2012-07-10

    An axial scanning laser Doppler velocimeter (LDV) with directional discrimination not requiring any moving mechanism in its probe is proposed. The proposed LDV utilizes frequency shift induced by acousto-optic modulators (AOMs) for discriminating the direction of velocity. The measurement position is axially scanned by changing the wavelength of the light input to the probe. The experimental result reveals that both the axial scan and the directional discrimination can be realized by using the proposed method without any moving element in the probe.

  8. 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.

  9. Handheld confocal laser endomicroscopic imaging utilizing tumor-specific fluorescent labeling to identify experimental glioma cells in vivo

    PubMed Central

    Martirosyan, Nikolay L.; Georges, Joseph; Kalani, M. Yashar S.; Nakaji, Peter; Spetzler, Robert F.; Feuerstein, Burt G.; Preul, Mark C.

    2016-01-01

    Background: We have reported that handheld confocal laser endomicroscopy (CLE) can be used with various nonspecific fluorescent dyes to improve the microscopic identification of brain tumor and its boundaries. Here, we show that CLE can be used experimentally with tumor-specific fluorescent labeling to define glioma margins in vivo. Methods: Thirteen rats underwent craniectomy and in vivo imaging 21 days after implantation with green fluorescent protein (GFP)-labeled U251 (n = 7) cells or epidermal growth factor receptor (EGFR) overexpressing F98 cells (n = 6). Fluorescein isothiocyanate (FITC) conjugated EGFR fluorescent antibody (FITC-EGFR) was applied for contrast in F98 tumors. Confocal images of normal brain, obvious tumor, and peritumoral zones were collected using the CLE system. Bench-top confocal microscopy and hematoxylin and eosin-stained sections were correlated with CLE images. Results: GFP and FITC-EGFR fluorescence of glioma cells were detected by in vivo visible-wavelength fluorescence CLE. CLE of GFP-labeled tumors revealed bright individual satellite tumor cells within peritumoral tissue, a definitive tumor border, and subcellular structures. Imaging with FITC-EGFR labeling provided weaker contrast in F98-EGFR tumors but was able to delineate tumor cells. Imaging with both methods in various tumor regions correlated with standard confocal imaging and clinical histology. Conclusions: These data suggest that in vivo CLE of selectively tagged neoplasms could allow specific interactive identification of tumoral areas. Imaging of GFP and FITC-EGFR provides real-time histologic information precisely related to the site of microscopic imaging of tumor. PMID:28144472

  10. 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.

  11. Automating laser scanning of 3D surfaces for reverse engineering

    NASA Astrophysics Data System (ADS)

    Chan, Vincent H.; Bradley, Colin H.; Vickers, Geoffrey W.

    1997-12-01

    Application of current 3-D laser scanning systems to reverse engineering is limited by two obstacles. The meticulous guidance of the laser scanner over the surface of the object being scanned and the segmentation of the cloud data which is collected by the laser scanner. Presently, both obstacles are being manually solved. The guidance of the laser scanning sensor at the correct surface to sensor distance is dependent on operator judgement and the segmentation of the collected data is reliant on the user to manually define surface boundaries on a computer screen. By applying a 2-D CCD camera, both of these problems can be resolved. Depth information on the location of the object surface can be derived from a pair of stereo images from the CCD camera. Using this depth information, the scanner path can be automatically calculated. Segmentation of the object surface can be accomplished by employing a Kohonen neural network into the CCD image. Successful segmentation of the image is conditional on the locations selected to start neural nodes as well as the prevention of the neuron connectors from bleeding onto neighboring patches. Thus the CCD camera allows for the automatic path planning of the laser scanner as well as the segmentation of the surface into patches defined along its natural boundaries.

  12. Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells.

    PubMed

    Amor, Rumelo; Mahajan, Sumeet; Amos, William Bradshaw; McConnell, Gail

    2014-12-08

    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.

  13. 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.

  14. Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

    PubMed Central

    Heinonen, J T; Sidhu, J S; Reilly, M T; Farin, F M; Omiecinski, C J; Eaton, D L; Kavanagh, T J

    1996-01-01

    Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur. Images Figure 1. Figure 2. Figure 3. Figure 3. Figure 4. Figure 4. Figure 5. Figure 6. PMID:8743442

  15. 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

  16. Confocal microscopy to guide erbium:yttrium aluminum garnet laser ablation of basal cell carcinoma: an ex vivo feasibility study.

    PubMed

    Sierra, Heidy; Larson, Bjorg A; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

    2013-09-01

    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.

  17. 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.

  18. 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)

  19. 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

  20. Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection.

    PubMed

    Zhi, Yanan; Wang, Benquan; Yao, Xincheng

    2015-01-01

    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.

  1. 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

  2. Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes.

    PubMed

    Kress, Matthias; Meier, Thomas; Steiner, Rudolf; Dolp, Frank; Erdmann, Rainer; Ortmann, Uwe; Rück, Angelika

    2003-01-01

    This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging

  3. 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

  4. Confocal microscopy in microgravity research.

    PubMed

    Goede, A P; Brakenhoff, G J; Woldringh, C L; Aalders, J W; Imhof, J P; van Kralingen, P; Mels, W A; Schreinemakers, P; Zegers, A

    1992-01-01

    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.

  5. [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.

  6. High-definition laser display system using multibeam scanning

    NASA Astrophysics Data System (ADS)

    Zhao, Zhenming; Li, Yongda; Lang, Baihe

    2000-10-01

    The design and principles of a high definition laser display system with multi-beam scanning are described. The system employs 4 laser beams each being composed of red, green and blue components. The four beams from one scanner are scanned simultaneously by a rotating polygonal mirror for horizontal deflection and by a galvanometer for vertical deflection. Compared with conventional single-beam scanning, the new design has the following advantages: 1) The rotational speed of the polygonal mirrors can be reduced by a factor of 4, which would improve the system performance and decrease the difficulties of the manufacture of the system. The size of the polygonal facet and, therefore, the laser beam diameter can be increased which would decrease the pixel diffusion. 2) The simultaneous operation of the 4 modulators would improve the horizontal resolution by a factor of 4. 3) For the same screen brightness, the single pixel power density can be reduced by a factor of 4 which would decrease the hazardous laser radiation.

  7. 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.

  8. Optics designs and system MTF for laser scanning displays

    NASA Astrophysics Data System (ADS)

    Urey, Hakan; Nestorovic, Ned; Ng, Baldwin S.; Gross, Abraham A.

    1999-07-01

    The Virtual Retinal DisplayTM (VRDTM) technology is a new display technology being developed at Microvision Inc. The displayed image is scanned onto the viewer's retina using low- power red, green, and blue light sources. Microvision's proprietary miniaturized scanner designs make VRD system very well suited for head-mounted displays. In this paper we discuss some of the advantages of the VRD technology, various ocular designs for HMD and other applications, and details of constructing a system MTF budget for laser scanning systems that includes electronics, modulators, scanners, and optics.

  9. Scanning laser system to determine the corneal shape

    NASA Astrophysics Data System (ADS)

    Ascanio, Gabriel; Caballero-Ruiz, Alberto; Ruiz-Huerta, Leopoldo; Gonzalez-Cardel, Mario; Diaz-Uribe, Rufino

    2005-07-01

    The development and tests of a scanning system to be used to determine the corneal topography with the laser deflectometry method are presented. In this equipment, a He-Ne laser beam scans the cornea by describing a spiral trajectory generated by two components: radial and angular. The first component is produced by the displacement of a plane mirror moved by a linear pneumatic actuator. The second component is produced by passing the beam through a Dove prism which is rotating by means of a belt drive coupled to a high-speed electric motor. Tests were first performed by analyzing both components independently and then they were characterized by combining the two components. Results are discussed and compared to those of an earlier cited work.

  10. 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

  11. Compact scanning-force microscope using a laser diode

    NASA Astrophysics Data System (ADS)

    Sarid, Dror; Iams, Doug; Weissenberger, Volker; Bell, L. Stephen

    1988-12-01

    The paper describes the operation of a compact scanning-force microscope in which the gradient of force acting on a vibrating tip is monitored by a diode laser and its integrated photodiode. The system does not require reflecting or focusing elements or complicated electronics. Experimental results using this system with magnetic domains on a magnetooptic storage medium attest to the feasibility of this concept.

  12. Evaluation of microvision SD2500 scanning laser display

    NASA Astrophysics Data System (ADS)

    Harding, Thomas H.; Rash, Clarence E.; Dennis, Scott J.

    2006-05-01

    Microvision's Spectrum TM SD2500 is a candidate technology for the Modular Integrated Helmet Display System (MIHDS)program. This HMD design is intended to provide a full-color, see-through, daylight and night-readable, moderate-resolution (800X600 pixels) display. The employed technology is that of scanning lasers. This paper presents the testing results for the latest version of this prototype system.

  13. Superconducting Magnet System for a Low Temperature Laser Scanning Microscope

    DTIC Science & Technology

    2006-09-22

    Our initial studies with the LTLSM bought with this equipment grant show that the intragrain critical current density crosses over with the...SUBTITLE 5a. CONTRACT NUMBER Superconducting Magnet System for a Low Temperature Laser Scanning Microscope 5b. GRANT NUMBER FA9550-05-1-0425 5c...ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER Applied Superconductivity Center 1500 Engineering Drive University of Wisconsin -Madison Room 909

  14. Precision Targeting With a Tracking Adaptive Optics Scanning Laser Ophthalmoscope

    DTIC Science & Technology

    2006-01-01

    galvanometers placed at appropriate conjugates within the path of the adaptive optics scanning laser ophthalmoscope. The input to the “master” control loop is...loop is the scaled position signals from the master galvanometers . The slave tracking mirrors are placed at conjugates to the center of rotation of the...slave systems), and analog-to-digital and digital-to- analog converters (ADC and DACs) to receive reflectometer signals and drive galvanometers . The

  15. 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.

  16. 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.

  17. Velocity gradients in spatially-resolved laser Doppler flowmetry and dynamic light scattering with confocal and coherence gating

    PubMed Central

    Uribe-Patarroyo, Néstor; Bouma, Brett E.

    2016-01-01

    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, non-turbulent 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

  18. Wedge-Filtering of Geomorphologic Terrestrial Laser Scan Data

    PubMed Central

    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

  19. Primary detection of hardwood log defects using laser surface scanning

    NASA Astrophysics Data System (ADS)

    Thomas, Edward; Thomas, Liya; Mili, Lamine; Ehrich, Roger W.; Abbott, A. Lynn; Shaffer, Clifford

    2003-05-01

    The use of laser technology to scan hardwood log surfaces for defects holds great promise for improving processing efficiency and the value and volume of lumber produced. External and internal defect detection to optimize hardwood log and lumber processing is one of the top four technological needs in the nation"s hardwood industry. The location, type, and severity of defects on hardwood logs are the key indicators of log quality and value. These visual cues provide information about internal log characteristics and products for which the log is suitable. We scanned 162 logs with a high-resolution industrial four-head laser surface scanner. The resulting data sets contain hundreds of thousands of three-dimensional coordinate points. The size of the data and noise presented special problems during processing. Robust regression models were used to fit geometric shapes to the data. The estimated orthogonal distances between the fitted model and the log surface are converted to a two-dimensional image to facilitate defect detection. Using robust regression methods and standard image processing tools we have demonstrated that severe surface defects on hardwood logs can be detected using height and contour analyses of three-dimensional laser scan data.

  20. 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.

  1. Categorisation of full waveform data provided by laser scanning devices

    NASA Astrophysics Data System (ADS)

    Ullrich, Andreas; Pfennigbauer, Martin

    2011-11-01

    In 2004, a laser scanner device for commercial airborne laser scanning applications, the RIEGL LMS-Q560, was introduced to the market, making use of a radical alternative approach to the traditional analogue signal detection and processing schemes found in LIDAR instruments so far: digitizing the echo signals received by the instrument for every laser pulse and analysing these echo signals off-line in a so-called full waveform analysis in order to retrieve almost all information contained in the echo signal using transparent algorithms adaptable to specific applications. In the field of laser scanning the somewhat unspecific term "full waveform data" has since been established. We attempt a categorisation of the different types of the full waveform data found in the market. We discuss the challenges in echo digitization and waveform analysis from an instrument designer's point of view and we will address the benefits to be gained by using this technique, especially with respect to the so-called multi-target capability of pulsed time-of-flight LIDAR instruments.

  2. Laser Brazing with Beam Scanning: Experimental and Simulative Analysis

    NASA Astrophysics Data System (ADS)

    Heitmanek, M.; Dobler, M.; Graudenz, M.; Perret, W.; Göbel, G.; Schmidt, M.; Beyer, E.

    Laser beam brazing with copper based filler wire is a widely established technology for joining zinc-coated steel plates in the body-shop. Successful applications are the divided tailgate or the zero-gap joint, which represents the joint between the side panel and the roof-top of the body-in-white. These joints are in direct view to the customer, and therefore have to fulfil highest optical quality requirements. For this reason a stable and efficient laser brazing process is essential. In this paper the current results on quality improvement due to one dimensional laser beam deflections in feed direction are presented. Additionally to the experimental results a transient three-dimensional simulation model for the laser beam brazing process is taken into account. With this model the influence of scanning parameters on filler wire temperature and melt pool characteristics is analyzed. The theoretical predictions are in good accordance with the experimental results. They show that the beam scanning approach is a very promising method to increase process stability and seam quality.

  3. Novel approach towards colour imaging using a scanning laser ophthalmoscope

    PubMed Central

    Manivannan, A; Kirkpatrick, J; Sharp, P; Forrester, J

    1998-01-01

    AIMS—Conventional fundus imaging using a fundus camera produces colour fundus pictures. The scanning laser ophthalmoscope (SLO) has the advantages of lower levels of light exposure, improved contrast, and direct digital imaging but until now has produced monochromatic images as a laser of single wavelength is used. True representation of the fundus is possible by combining images taken using blue, green, and red lasers.
METHODS—A custom built SLO was used to capture blue, green, and red fundus images from suitable volunteers and patients with fundus disease. Images were corrected for eye movement and combined to form a colour image. Colour fundus photographs were taken using a fundus camera for comparison with the SLO image.
RESULTS—The background fundus and retinal vasculature had similar appearances with the two imaging modalities. Internal limiting membrane reflections were prominent with the SLO. Identification of new vessels in the diabetic fundus was easier with the SLO than the colour fundus photographs.
CONCLUSION—A colour SLO offers all the advantages of the present monochromatic imaging system with the added advantage of true colour representation of the fundus.

 Keywords: scanning laser ophthalmoscope; fundus imaging; digital colour fundus images PMID:9640178

  4. Detection and characterisation of surface cracking using scanning laser techniques

    NASA Astrophysics Data System (ADS)

    Edwards, R. S.; Clough, A. R.; Rosli, M. H.; Hernandez-Valle, J. F.; Dutton, B.

    2012-05-01

    The use of lasers for generating and detecting ultrasound is becoming more established in non-destructive testing. However, there is still scope in developing the techniques to fully realise the benefits of non-contact measurements. One application is the detection of surface defects in metals; for example, rolling contact fatigue in rails, and surface cracking on billets or plates. We present measurements using a pulsed Nd:YAG laser to generate surface ultrasonic waves and an interferometer to detect the surface displacement on the sample, and investigate the interaction of Rayleigh or Lamb waves with surface defects. Signal enhancement in the near-field is observed for Rayleigh waves when either the generator or detector is close to a defect. For a scanned detector measurement, enhancement is observed due to constructive interference of the incident and reflected waves. For a scanned generator measurement, the change in generation conditions when the laser is over the defect also lead to an enhancement. In measurements of plate samples we observe similar enhancement effects whereby higher order modes are observed when the laser is above a defect. We discuss the implications of signal enhancements for detecting and characterising surface cracking.

  5. SEMI-QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY APPLIED TO MARINE INVERTEBRATE ECOTOXICOLOGY. (R827397)

    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...

  6. 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...

  7. 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...

  8. The application of in vivo laser confocal microscopy to the diagnosis and evaluation of meibomian gland dysfunction

    PubMed Central

    Matsumoto, Yukihiro; Sato, Enrique Adan; Ibrahim, Osama M.A.; Tsubota, Kazuo

    2008-01-01

    Purpose To evaluate the morphological changes of the meibomian glands (MG) in patients with meibomian gland dysfunction (MGD) compared to normal subjects by in vivo confocal microscopy and to investigate the relation of these changes to the clinical ocular surface findings and tear functions. Methods Twenty MGD patients and 15 normal subjects were recruited into this prospective study. Patients and controls underwent slit lamp examinations, tear film break-up time (BUT) measurements, fluorescein and Rose-Bengal stainings, Schirmer test I without anesthesia, tear evaporation rate assessment (TEROS), tear film lipid layer interferometry (DR-1), transillumination of the lids (meibography), MG expressibility test, and in vivo laser confocal microscopy of the lids (HRTII-RCM). Results The BUT, DR-1 tear film lipid layer interferometry grades, fluorescein and Rose-Bengal staining scores, MG drop out grade in meibography, and MG expressibility grades were significantly worse in MGD patients compared to normal controls (p<0.05). The severity of both MG dropout and MG expressibility related significantly with the BUT, DR-1 grades, and TEROS (p<0.05). The mean density of acinar units of MGs as measured by HRTII-RCM was significantly lower in MGD patients (47.6±26.6/mm2) than in control subjects (101.3±33.8/mm2; p<0.05). The mean acinar unit diameter as determined by HRTII-RCM was significantly larger in MGD patients (98.2±53.3 μm) than in controls (41.6±11.9 μm; p<0.05). Both the density and diameter of MG acinar units related significantly with the severity of MG dropout and MG expression grades (p<0.05). Conclusions In vivo confocal microscopy can effectively demonstrate the morphological changes of the MG in patients with MGD. Glandular acinar density and acinar unit diameter seemed to be promising new parameters of in vivo confocal microscopy, which is significantly related to the clinical ocular surface and tear function findings of MGD. PMID:18618006

  9. 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.

  10. Super-Resolution Laser Scanning Microscopy through Spatiotemporal Modulation

    PubMed Central

    Lu, Ju; Min, Wei; Conchello, José-Angel; Xie, Xiaoliang Sunney; Lichtman, Jeff W.

    2009-01-01

    Super-resolution optical microscopy has attracted great interest among researchers in many fields, especially in biology where the scale of physical structures and molecular processes fall below the diffraction limit of resolution for light. As one of the emerging techniques, structured illumination microscopy can double the resolution by shifting unresolvable spatial frequencies into the pass-band of the microscope through spatial frequency mixing with a wide-field structured illumination pattern. However, such a wide-field scheme typically can only image optically thin samples and is incompatible with multiphoton processes such as two-photon fluorescence, which require point scanning with a focused laser beam. Here, we propose two new super-resolution schemes for laser scanning microscopy by generalizing the concept of a spatially nonuniform imaging system. One scheme, scanning patterned illumination (SPIN) microscopy, employs modulation of the excitation combined with temporally cumulative imaging by a nondescanned array detector. The other scheme, scanning patterned detection (SPADE) microscopy, utilizes detection modulation together with spatially cumulative imaging, in this case by a nondescanned single-element detector. When combined with multiphoton excitation, both schemes can image thick samples with three-dimensional optical sectioning and much improved resolution. PMID:19743870

  11. 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.

  12. An Integrated Laser-Induced Piezoelectric/Differential Confocal Surface Acoustic Wave System for Measurement of Thin Film Young's Modulus

    PubMed Central

    Yang, Fei; Dorantes-Gonzalez, Dante J.; Chen, Kun; Lu, Zimo; Jin, Baoyin; Li, Yanning; Chen, Zhi; Hu, Xiaotang

    2012-01-01

    The present paper presents the design and development results of a system setup for measuring Young's modulus of thin films by laser-induced surface acoustic waves based on the integration of two detection methods, namely, piezoelectric transducer detection and differential confocal detection, which may be used for conducting consecutive or simultaneous measurements. After demonstrating the capabilities of each detection approach, it is shown how, depending on a wider range of applications, sample materials and measurement environments, the developed integrated system inherits and harnesses the main characteristics of its detection channels, resulting in an more practical and flexible equipment for determining Young's modulus than traditional nanoindentation equipment, and also suitable for cross-validation purposes.

  13. 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).

  14. 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.

  15. 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...

  16. 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...

  17. 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

  18. Application of reflectance confocal microscopy to evaluate skin damage after irradiation with an yttrium-scandium-gallium-garnet (YSGG) laser.

    PubMed

    Yue, Xueping; Wang, Hongwei; Li, Qing; Li, Linfeng

    2017-02-01

    The objective of this study was to observe the characteristics of the skin after irradiation with a 2790-nm yttrium-scandium-gallium-garnet (YSGG) laser using reflectance confocal microscopy (RCM). A 2790-nm YSGG laser was used to irradiate fresh foreskin (four doses, at spot density 3) in vitro. The characteristics of microscopic ablative columns (MAC), thermal coagulation zone (TCZ), and microscopic treatment zones (MTZ) were observed immediately after irradiation using digital microscope and RCM. The characteristics of MAC, TCZ, and MTZ with variations in pulse energy were comparatively analyzed. After irradiation, MAC, TCZ, and MTZ characteristics and undamaged skin between MTZs can be observed by RCM. The depth and width of MTZ obviously increased with the increase in pulse energy. At 80, 120, and 160 mJ/microbeam (MB), the MTZ actual area and proportion were about two times that of the theoretical value and three times at 200 mJ/MB. With increases in depth, the single MAC gradually decreased in a fingertip-shaped model, with TCZ slowly increasing, and MTZ slightly decreasing in a columnar shape. RCM was able to determine the characteristics of thermal injury on the skin after the 2790-nm YSGG laser irradiation with different pulse energies. Pulse energy higher than 200 mJ/MB may have much larger thermal injury and side effect. RCM could be used in the clinic in future.

  19. 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

  20. Adsorption of alexa-labeled Bt toxin on mica, glass, and hydrophobized glass: study by normal scanning confocal fluorescence.

    PubMed

    Janot, Jean-Marc; Boissière, Michel; Thami, Thierry; Tronel-Peyroz, Emmanuel; Helassa, Nordine; Noinville, Sylvie; Quiquampoix, Hervé; Staunton, Siobhán; Déjardin, Philippe

    2010-06-14

    We studied the kinetics of adsorption of alexa-labeled Bt toxin Cry1Aa, in monomer and oligomer states, on muscovite mica, acid-treated hydrophilic glass, and hydrophobized glass, in the configuration of laminar flow of solution in a slit. Normal confocal fluorescence through the liquid volume allows the visualization of the concentration in solution over the time of adsorption, in addition to the signal due to the adsorbed molecules at the interface. The solution signal is used as calibration for estimation of interfacial concentration. We found low adsorption of the monomer compared to oligomers on the three types of surface. The kinetic adsorption barrier for oligomers increases in the order hydrophobized glass, muscovite mica, acid-treated hydrophilic glass. This suggests enhanced immobilization in soil if toxin is under oligomer state.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Land-Based Mobile Laser Scanning Systems: a Review

    NASA Astrophysics Data System (ADS)

    Puente, I.; González-Jorge, H.; Arias, P.; Armesto, J.

    2011-09-01

    Mobile mapping has been using various photogrammetric techniques for many years. In recent years, there has been an increase in the number of mobile mapping systems using laser scanners available in the market, partially because of the improvement in GNSS/INS performance for direct georeferencing. In this article, some of the most important land-based mobile laser scanning (MLS) systems are reviewed. Firstly, the main characteristics of MLS systems vs. airborne (ALS) and terrestrial laser scanning (TLS) systems are compared. Secondly, a short overview of the mobile mapping technology is also provided so that the reader can fully grasp the complexity and operation of these devices. As we put forward in this paper, a comparison of different systems is briefly carried out regarding specifications provided by the manufacturers. Focuses on the current research are also addressed with emphasis on the practical applications of these systems. Most of them have been utilized for data collection on road infrastructures or building façades. This article shows that MLS technology is nowadays well established and proven, since the demand has grown to the point that there are several systems suppliers offering their products to satisfy this particular market.

  6. Three-dimensional dental cast analyzing system using laser scanning.

    PubMed

    Kuroda, T; Motohashi, N; Tominaga, R; Iwata, K

    1996-10-01

    The purpose of this article is to introduce the outline of our newly developed three-dimensional dental cast analyzing system with laser scanning, and its preliminary clinical applications. The system is composed of a measuring device with a slit-ray laser projector and two sets of coupled charged devised video cameras, an image processing unit, a 16-bit personal computer as a controller, and an engineering workstation as a post processor. The dental cast is projected and scanned with a slit-ray laser beam. The raster coordinates of the target are determined with an image processor. Triangulation is applied to determine the location of each point. Generation of three-dimensional graphics of the dental cast takes approximately 40 minutes. About 90,000 sets of X, Y, Z coordinates are stored in the main memory of the microcomputer. The measurement error is less than 0.05 mm. Besides the conventional linear and angular measurements of the dental cast, we are also able to demonstrate the size of the palatal surface area and the volume of the oral cavity. The advantage of this system is that it facilitates the otherwise complicated and time-consuming mock surgery necessary for treatment planning in orthognathic surgery.

  7. 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.

  8. 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...

  9. Noninvasive spatially resolved kinetic study of hydrolyzable camptothecin antitumor drugs in vitro and in vivo by confocal scanning microspectrofluorometry

    NASA Astrophysics Data System (ADS)

    Chourpa, Igor; Charonov, Serguei; Kokota, Alexandre; Riou, Jean-Francois; Manfait, Michel

    1998-04-01

    Hydrolysis of the lactone ring of camptothecins (CPTs) leads to a loss of their antitumor activity. The non-hydrolyzable derivatives are also inactive. Thus, the state of the lactone ring during the drug interaction with biological partners is of a great interest. High performance liquid chromatography currently employed to study the lactone hydrolysis in free CPTs can not be applied to the drug- target complexes and in vivo measurements. We followed kinetics of the lactone hydrolysis in CPTs using hydrolysis- induced time-dependent evolution of their fluorescence spectra. Spectra were obtained from micro-volumes of the samples under the microscope of a computer-controlled confocal microspectrofluorometer (M51, DILOR, France). Spectral recording and treatment (filtering, decomposition into model spectra of the intact and hydrolyzed forms, etc.) were performed using a software package developed in our laboratory. Data obtained for a series of CPTs at very low concentrations, ca. 10-7 M, demonstrated a good reproducibility, even at basic pH, where the hydrolysis is fast. Then the kinetics studies were extended to CPTs in complexes with their potential biological targets, DNA and topoisomerase I, in vitro. The in vivo studies of the lactone status at the level of single living cancer cells treated with CPTs are actually in progress.

  10. Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging.

    PubMed

    Mancuso, James J; Larson, Adam M; Wensel, Theodore G; Saggau, Peter

    2009-01-01

    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.

  11. 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.

  12. Angular line scanning deflectometry using a laser pico projector

    NASA Astrophysics Data System (ADS)

    Zhan, Hao-Xun; Liang, Chao-Wen; Chien, Shih-Che

    2016-08-01

    In our previous publications, we had successfully made a deflectometry measurement by using a portable laser projector. In this research, we propose the beam weighting centroid method rather than previous the phase shifting method for quantification of the angular direction of the testing beam in the tested optics entrance pupil. By projecting the angular sequential lines on tested optics entrance pupil, the wavefront aberration is reconstructed from two orthogonal directions measurements, in a similar way to the line scanning deflectometry. The limited gray scale problem of laser projector during the phase shifting measurement is therefore eliminated. The reconstructed wavefront is proven to yield a more accurate result than the phase shifting methods at the cost of more image frames and acquisition time.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. Scanning laser beam displays based on a 2D MEMS

    NASA Astrophysics Data System (ADS)

    Niesten, Maarten; Masood, Taha; Miller, Josh; Tauscher, Jason

    2010-05-01

    The combination of laser light sources and MEMS technology enables a range of display systems such as ultra small projectors for mobile devices, head-up displays for vehicles, wearable near-eye displays and projection systems for 3D imaging. Images are created by scanning red, green and blue lasers horizontally and vertically with a single two-dimensional MEMS. Due to the excellent beam quality of laser beams, the optical designs are efficient and compact. In addition, the laser illumination enables saturated display colors that are desirable for augmented reality applications where a virtual image is used. With this technology, the smallest projector engine for high volume manufacturing to date has been developed. This projector module has a height of 7 mm and a volume of 5 cc. The resolution of this projector is WVGA. No additional projection optics is required, resulting in an infinite focus depth. Unlike with micro-display projection displays, an increase in resolution will not lead to an increase in size or a decrease in efficiency. Therefore future projectors can be developed that combine a higher resolution in an even smaller and thinner form factor with increased efficiencies that will lead to lower power consumption.

  18. Theory of the laser diode interaction in scanning force microscopy

    SciTech Connect

    Sarid, D.; Iams, D.A.; Ingle, J.T.; Weissenberger, V.

    1989-08-01

    The theory of interaction of a vibrating cantilever and a laser diode used in a scanning force microscope is given in terms of a feedback-dependent parameter C, which determines the gain associated with this interaction. It is shown that both C and the amplitude of vibrations can be determined experimentally from the measurement of the first and second harmonics. Experimental results, which are in good agreement with the theory, yield a value for C which is 0.045. Under these weak feedback conditions, it is found that the interaction can be modeled approximately as a simple homodyne process.

  19. Precision Targeting with a Tracking adaptive Optics Scanning Laser Ophthalmoscope

    DTIC Science & Technology

    2006-02-01

    in Figure 2) but drives two galvanometers placed at appropriate conjugates within the path of the adaptive optics scanning laser ophthalmoscope...reflectometer. The input to the "slave" control loop is the scaled position signals from the master galvanometers . The slave tracking mirrors are placed at...signals and drive galvanometers . The DSP has a loop rate of 62.5 kHz (compared to 16 kHz in the previously-used real-time processing board) for a

  20. Automatic road edge detection from Mobile Laser Scanning (MLS)

    NASA Astrophysics Data System (ADS)

    Cabo, Carlos; García-Cortés, Silverio; Menéndez-Díaz, Agustín.; Ordoñez, Celestino

    2016-11-01

    In this article we present an algorithm for automatic road edge detection from MLS (Mobile Laser Scanning) data. The method takes advantage of linear structures derived from MLS point clouds. These lines are extracted from the point cloud and grouped following geometric restrictions. Then, the outlines of the groups are extracted as road edges. Finally, a moving window filter is applied to those points in order to remove outliers and delineate the road edge. The method was tested on an 800m stretch of road, and the results were checked through visual inspection. Correctness and completeness were 99.1% and 97.5%, respectively.

  1. Coastal Benthic Optical Properties Fluorescence Imaging Laser Line Scan Sensor

    DTIC Science & Technology

    2002-09-30

    Coral reefs are a prime example of an environment where current acoustic methods can be expected to have great difficulty. Our prototype Fluorescence Imaging Laser Line Scan (FILLS) sensor[1,2,3,4] has demonstrated that fluorescence imagery provides strong signatures which may be used to separate the coral clutter from mines. The image above demonstrates the ease with which a human observer can differentiate the mine like objects (MLOs) from the natural clutter in an environment that is difficult for sonars. Accordingly, this technology is a leading

  2. Marking of organic materials by CO2 laser beam scanning

    NASA Astrophysics Data System (ADS)

    Dumitras, Dan C.; Chitu, Livia; Blanaru, Constantin; Cernat, Ramona C.; Bucatica, Irina Alexandra L.; Puiu, Adriana P.

    2003-11-01

    CO2 laser beam scanning method was used for marking of organic materials (leather, paper, wood) both in continuous wave and in pulsed regime. The computer controlled X-Y galvometric scanner and the software developed for this application control every parameter of irradiation and allow programmable marking of simple marks, logos, alphanumeric characters, filled text, codes, graphics, or highly complex drawings and images. The factors influencing the quality of the marking were analyzed and the irradiation conditions were optimized to produce marks on organic materials with a quality imposed by industry standards.

  3. 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.

  4. Laser scanning microscopy of broad freezing interfaces with applications to biological cells

    NASA Astrophysics Data System (ADS)

    Neils, Christopher Martin

    2000-09-01

    A new, vertical cryostage was used for microscopic observation of broad-front freezing in aqueous solutions. This cryostage complements traditional studies of cell behavior and interface morphology in cryobiology. Traditional systems directionally solidify thin samples perpendicular to the optical axis. Thin samples confer thermal and optical advantages for video brightfield microscopy. However, sample thickness can affect the interface morphology. In the new cryostage, ice propagates parallel to the microscope optical axis. The sample cup is 1 cm tall and 1.5 cm in diameter, with insulated sides and a nitrogen-cooled base to freeze the solution upward. The top of the solution is warmed passively through a cover glass or immersion objective. The freezing solutions contain dilute fluorescein dye, which is visible where it is concentrated by exclusion from the ice. The stage is mounted on a confocal laser-scanning microscope, and thermal control and image capture routines are centralized in a LabView user interface. Filtered water, physiological saline, 9.5% glycerol, and 10% glycerol with PBS were frozen at rates between -2°C/min and -10°C/min and sequential images at one plane were captured. Images distinctly revealed a lamellar interface but could not resolve 3-D morphology. The average lamellar spacing was quantified using image analysis. Physiological saline was frozen in flat glass capillary tubes with 0.05 to 0.4 mm path length, mounted vertically to observe internal ice in cross-section. Lamellae were randomly oriented with respect to the glass, suggesting caution when measuring dendrite spacing in a horizontal cryostage. No correlation between capillary size and lamellar spacing was noted. Cell monolayers and synthetic membranes were mounted horizontally to let a well-developed ice front approach the layer broadly. In transparent membranes, ice-membrane interaction was visible until ice grew over and obscured the membrane. The vertical cryostage improved

  5. 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

  6. 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.

  7. 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.

  8. Antecedents of two-photon excitation laser scanning microscopy.

    PubMed

    Masters, Barry R; So, Peter T C

    2004-01-01

    In 1931, Maria Göppert-Mayer published her doctoral dissertation on the theory of two-photon quantum transitions (two-photon absorption and emission) in atoms. This report describes and analyzes the theoretical and experimental work on nonlinear optics, in particular two-photon excitation processes, that occurred between 1931 and the experimental implementation of two-photon excitation microscopy by the group of Webb in 1990. In addition to Maria Göppert-Mayer's theoretical work, the invention of the laser has a key role in the development of two-photon microscopy. Nonlinear effects were previously observed in different frequency domains (low-frequency electric and magnetic fields and magnetization), but the high electric field strength afforded by lasers was necessary to demonstrate many nonlinear effects in the optical frequency range. In 1978, the first high-resolution nonlinear microscope with depth resolution was described by the Oxford group. Sheppard and Kompfner published a study in Applied Optics describing microscopic imaging based on second-harmonic generation. In their report, they further proposed that other nonlinear optical effects, such as two-photon fluorescence, could also be applied. However, the developments in the field of nonlinear optical stalled due to a lack of a suitable laser source. This obstacle was removed with the advent of femtosecond lasers in the 1980s. In 1990, the seminal study of Denk, Strickler, and Webb on two-photon laser scanning fluorescence microscopy was published in Science. Their paper clearly demonstrated the capability of two-photon excitation microscopy for biology, and it served to convince a wide audience of scientists of the potential capability of the technique.

  9. Determination of foveal location using scanning laser polarimetry

    PubMed Central

    VanNasdale, Dean A.; Elsner, Ann E.; Weber, Anke; Miura, Masahiro; Haggerty, Bryan P.

    2009-01-01

    The fovea is the retinal location responsible for our most acute vision. There are several methods used to localize the fovea, but the fovea is not always easily identifiable. Landmarks used to determine the foveal location are variable in normal subjects and localization becomes even more difficult in instances of retinal disease. In normal subjects, the photoreceptor axons that make up the Henle fiber layer are cylindrical and the radial orientation of these fibers is centered on the fovea. The Henle fiber layer exhibits form birefringence, which predictably changes polarized light in scanning laser polarimetry imaging. In this study 3 graders were able to repeatably identify the fovea in 35 normal subjects using near infrared image types with differing polarization content. There was little intra-grader, inter-grader, and inter-image variability in the graded foveal position for 5 of the 6 image types examined, with accuracy sufficient for clinical purposes. This study demonstrates that scanning laser polarimetry imaging can localize the fovea by using structural properties inherent in the central macula. PMID:19757960

  10. Ligand-Receptor Binding Measured by Laser-Scanning Imaging

    NASA Astrophysics Data System (ADS)

    Zuck, Paul; Lao, Zhege; Skwish, Stephen; Fraser Glickman, J.; Yang, Ke; Burbaum, Jonathan; Inglese, James

    1999-09-01

    This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.

  11. Adaptive optics scanning laser ophthalmoscope imaging: technology update

    PubMed Central

    Merino, David; Loza-Alvarez, Pablo

    2016-01-01

    Adaptive optics (AO) retinal imaging has become very popular in the past few years, especially within the ophthalmic research community. Several different retinal techniques, such as fundus imaging cameras or optical coherence tomography systems, have been coupled with AO in order to produce impressive images showing individual cell mosaics over different layers of the in vivo human retina. The combination of AO with scanning laser ophthalmoscopy has been extensively used to generate impressive images of the human retina with unprecedented resolution, showing individual photoreceptor cells, retinal pigment epithelium cells, as well as microscopic capillary vessels, or the nerve fiber layer. Over the past few years, the technique has evolved to develop several different applications not only in the clinic but also in different animal models, thanks to technological developments in the field. These developments have specific applications to different fields of investigation, which are not limited to the study of retinal diseases but also to the understanding of the retinal function and vision science. This review is an attempt to summarize these developments in an understandable and brief manner in order to guide the reader into the possibilities that AO scanning laser ophthalmoscopy offers, as well as its limitations, which should be taken into account when planning on using it. PMID:27175057

  12. An omnidirectional 3D sensor with line laser scanning

    NASA Astrophysics Data System (ADS)

    Xu, Jing; Gao, Bingtuan; Liu, Chuande; Wang, Peng; Gao, Shuanglei

    2016-09-01

    An active omnidirectional vision owns the advantages of the wide field of view (FOV) imaging, resulting in an entire 3D environment scene, which is promising in the field of robot navigation. However, the existing omnidirectional vision sensors based on line laser can measure points only located on the optical plane of the line laser beam, resulting in the low-resolution reconstruction. Whereas, to improve resolution, some other omnidirectional vision sensors with the capability of projecting 2D encode pattern from projector and curved mirror. However, the astigmatism property of curve mirror causes the low-accuracy reconstruction. To solve the above problems, a rotating polygon scanning mirror is used to scan the object in the vertical direction so that an entire profile of the observed scene can be obtained at high accuracy, without of astigmatism phenomenon. Then, the proposed method is calibrated by a conventional 2D checkerboard plate. The experimental results show that the measurement error of the 3D omnidirectional sensor is approximately 1 mm. Moreover, the reconstruction of objects with different shapes based on the developed sensor is also verified.

  13. Extraction of power lines from mobile laser scanning data

    NASA Astrophysics Data System (ADS)

    Xiang, Qing; Li, Jonathan; Wen, Chenglu; Huang, Pengdi

    2016-03-01

    Modern urban life is becoming increasingly more dependent on reliable electric power supply. Since power outages cause substantial financial losses to producers, distributors and consumers of electric power, it is in the common interest to minimize failures of power lines. In order to detect defects as early as possible and to plan efficiently the maintenance activities, distribution networks are regularly inspected. Carrying out foot patrols or climbing the structures to visually inspect transmission lines and aerial surveys (e.g., digital imaging or most recent airborne laser scanning (ALS) are the two most commonly used methods of power line inspection. Although much faster in comparison to the foot patrol inspection, aerial inspection is more expensive and usually less accurate, in complex urban areas particularly. This paper presents a scientific work that is done in the use of mobile laser scanning (MLS) point clouds for automated extraction of power lines. In the proposed method, 2D power lines are extracted using Hough transform in the projected XOY plane and the 3D power line points are visualized after the point searching. Filtering based on an elevation threshold is applied, which is combined with the vehicle's trajectory in the horizontal section.

  14. Determination of Percent Body Fat Using 3D Whole Body Laser Scanning: A Preliminary Investigation

    DTIC Science & Technology

    2006-11-01

    circumferences, 3D whole body laser scans and DEXA scans were performed on fifty-one men and women age 18-62. Mean percent body fat was not statistically...3D whole body laser scan , and DEXA scan to measure individuals during a one hour measurement session. 1 Report Documentation Page Form...underwent a 6 minute whole body DEXA scan using a GE Lunar Prodigy DEXA scanner running software version 7.53. Percent body fat was calculated from the

  15. Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope.

    PubMed

    Mytskaniuk, Vasyl; Bardin, Fabrice; Boukhaddaoui, Hassan; Rigneault, Herve; Tricaud, Nicolas

    2016-07-17

    Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique

  16. Test field for airborne laser scanning in Finland

    NASA Astrophysics Data System (ADS)

    Ahokas, E.; Kaartinen, H.; Kukko, A.; Litkey, P.

    2014-11-01

    Airborne laser scanning (ALS) is a widely spread operational measurement tool for obtaining 3D coordinates of the ground surface. There is a need for calibrating the ALS system and a test field for ALS was established at the end of 2013. The test field is situated in the city of Lahti, about 100 km to the north of Helsinki. The size of the area is approximately 3.5 km × 3.2 km. Reference data was collected with a mobile laser scanning (MLS) system assembled on a car roof. Some streets were measured both ways and most of them in one driving direction only. The MLS system of the Finnish Geodetic Institute (FGI) consists of a navigation system (NovAtel SPAN GNSS-IMU) and a laser scanner (FARO Focus3D 120). In addition to the MLS measurements more than 800 reference points were measured using a Trimble R8 VRS-GNSS system. Reference points are along the streets, on parking lots, and white pedestrian crossing line corners which can be used as reference targets. The National Land Survey of Finland has already used this test field this spring for calibrating their Leica ALS-70 scanner. Especially it was easier to determine the encoder scale factor parameter using this test field. Accuracy analysis of the MLS points showed that the point height RMSE is 2.8 cm and standard deviation is 2.6 cm. Our purpose is to measure both more MLS data and more reference points in the test field area to get a better spatial coverage. Calibration flight heights are planned to be 1000 m and 2500 m above ground level. A cross pattern, southwest-northeast and northwest-southeast, will be flown both in opposite directions.

  17. Quantification of telomere length by FISH and laser scanning cytometry

    NASA Astrophysics Data System (ADS)

    Mahoney, John E.; Sahin, Ergun; Jaskelioff, Mariela; Chin, Lynda; DePinho, Ronald A.; Protopopov, Alexei I.

    2008-02-01

    Telomeres play a critical role in the maintenance of chromosomal stability. Telomere erosion, coupled with loss of DNA damage checkpoint function, results in genomic instability that promotes the development of cancer. The critical role of telomere dynamics in cancer has motivated the development of technologies designed to monitor telomere reserves in a highly quantitative and high-throughput manner in humans and model organisms. To this end, we have adapted and modified two established technologies, telomere-FISH and laser scanning cytometry. Specifically, we have produced a number of enhancements to the iCys LSC (CompuCyte) package including software updates, use of 60X dry objectives, and increased spatial resolution by 0.2 um size of stage steps. In addition, the 633 nm HeNe laser was replaced with a 532 nm green diode laser to better match the viewing options. Utilization of telomere-deficient mouse cells with short dysfunctional telomeres and matched telomerase reconstituted cultures demonstrated significantly higher mean integral specific fluorescence values for mTR transfectants relative to empty vector controls: 4.485M vs. 1.362M (p<0.0001). Histograms of average telomere intensities for individual cells were obtained and demonstrated intercellular heterogeneity in telomere lengths. The validation of the approach derives from a strong correlation between iCys LSC values and Southern blotting. This validated method greatly increases our experimental throughput and objectivity.

  18. Automated laser registration in image-guided surgery: evaluation of the correlation between laser scan resolution and navigation accuracy.

    PubMed

    Marmulla, R; Lüth, T; Mühling, J; Hassfeld, S

    2004-10-01

    Markerless patient registration based on the facial skin surface makes logistics prior to image-guided surgery much easier, as it is not necessary to place and measure registration markers. A laser scan registration of the surgical site takes the place of conventional marker-based registration. In a clinical study, the stability and accuracy of markerless patient registration was evaluated in 12 patients. Intraoral titanium markers served as targets for the infrared-pointer of the navigation system in order to check the accuracy of the markerless registration process. The correlation between laser scan resolution and navigation accuracy was checked using seven different laser scan resolutions (a cloud of 300,000 laser scan points down to 3750 laser scan points of the surgical site). The markerless patient registration was successful as long as high laser scan resolution was used (30,000 laser scan points and more): the titanium markers were detected with a mean deviation of 1.1 +/- 0.2 mm. Low resolution laser scans (6000 laser scan points of the surgical site and less) revealed inaccuracies up to 6 mm.

  19. Pavement cracking measurements using 3D laser-scan images

    NASA Astrophysics Data System (ADS)

    Ouyang, W.; Xu, B.

    2013-10-01

    Pavement condition surveying is vital for pavement maintenance programs that ensure ride quality and traffic safety. This paper first introduces an automated pavement inspection system which uses a three-dimensional (3D) camera and a structured laser light to acquire dense transverse profiles of a pavement lane surface when it carries a moving vehicle. After the calibration, the 3D system can yield a depth resolution of 0.5 mm and a transverse resolution of 1.56 mm pixel-1 at 1.4 m camera height from the ground. The scanning rate of the camera can be set to its maximum at 5000 lines s-1, allowing the density of scanned profiles to vary with the vehicle's speed. The paper then illustrates the algorithms that utilize 3D information to detect pavement distress, such as transverse, longitudinal and alligator cracking, and presents the field tests on the system's repeatability when scanning a sample pavement in multiple runs at the same vehicle speed, at different vehicle speeds and under different weather conditions. The results show that this dedicated 3D system can capture accurate pavement images that detail surface distress, and obtain consistent crack measurements in repeated tests and under different driving and lighting conditions.

  20. Street environment change detection from mobile laser scanning point clouds

    NASA Astrophysics Data System (ADS)

    Xiao, Wen; Vallet, Bruno; Brédif, Mathieu; Paparoditis, Nicolas

    2015-09-01

    Mobile laser scanning (MLS) has become a popular technique for road inventory, building modelling, infrastructure management, mobility assessment, etc. Meanwhile, due to the high mobility of MLS systems, it is easy to revisit interested areas. However, change detection using MLS data of street environment has seldom been studied. In this paper, an approach that combines occupancy grids and a distance-based method for change detection from MLS point clouds is proposed. Unlike conventional occupancy grids, our occupancy-based method models space based on scanning rays and local point distributions in 3D without voxelization. A local cylindrical reference frame is presented for the interpolation of occupancy between rays according to the scanning geometry. The Dempster-Shafer theory (DST) is utilized for both intra-data evidence fusion and inter-data consistency assessment. Occupancy of reference point cloud is fused at the location of target points and then the consistency is evaluated directly on the points. A point-to-triangle (PTT) distance-based method is combined to improve the occupancy-based method. Because it is robust to penetrable objects, e.g. vegetation, which cause self-conflicts when modelling occupancy. The combined method tackles irregular point density and occlusion problems, also eliminates false detections on penetrable objects.

  1. Divided-aperture differential confocal fast-imaging microscopy

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  2. Assessment of trabecular bone quality in human cadaver calcaneus using scanning confocal ultrasound and dual x-ray absorptiometry (DEXA) measurements

    NASA Astrophysics Data System (ADS)

    Qin, Yixian; Xia, Yi; Lin, Wei; Rubin, Clinton; Gruber, Barry

    2004-10-01

    Microgravity and aging induced bone loss is a critical skeleton complication, occurring particularly in the weight-supporting skeleton, which leads to osteoporosis and fracture. Advents in quantitative ultrasound (QUS) provide a unique method for evaluating bone strength and density. Using a newly developed scanning confocal acoustic diagnostic (SCAD) system, QUS assessment for bone quality in the real body region was evaluated. A total of 19 human cadaver calcanei, age 66 to 97 years old, were tested by both SCAD and nonscan mode. The scanning region covered an approximate 40×40 mm2 with 0.5 mm resolution. Broadband ultrasound attenuation (BUA, dB/MHz), energy attenuation (ATT, dB), and ultrasound velocity (UV, m/s) were measured. The QUS properties were then correlated to the bone mineral density (BMD) measured by DEXA. Correlations between BMD and QUS parameters were significantly improved by using SCAD as compared to nonscan mode, yielding correlations between BMD and SCAD QUS parameters as R=0.82 (BUA), and R=0.86 (est. BMD). It is suggested that SCAD is feasible for in vivo bone quality mapping. It can be potentially used for monitoring instant changes of bone strength and density. [Work supported by the National Space Biomedical Research Institute (TD00207), and New York Center for Biotechnology.

  3. Confocal sample-scanning microscope for single-molecule spectroscopy and microscopy with fast sample exchange at cryogenic temperatures.

    PubMed

    Hussels, Martin; Konrad, Alexander; Brecht, Marc

    2012-12-01

    The construction of a microscope with fast sample transfer system for single-molecule spectroscopy and microscopy at low temperatures using 2D/3D sample-scanning is reported. The presented construction enables the insertion of a sample from the outside (room temperature) into the cooled (4.2 K) cryostat within seconds. We describe the mechanical and optical design and present data from individual Photosystem I complexes. With the described setup numerous samples can be investigated within one cooling cycle. It opens the possibility to investigate biological samples (i) without artifacts introduced by prolonged cooling procedures and (ii) samples that require preparation steps like plunge-freezing or specific illumination procedures prior to the insertion into the cryostat.

  4. Analysis of reactive oxygen species in the guard cell of wheat stoma with confocal microscope.

    PubMed

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

    2011-09-01

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

  5. An adaptive-optics scanning laser ophthalmoscope for imaging murine retinal microstructure

    NASA Astrophysics Data System (ADS)

    Alt, Clemens; Biss, David P.; Tajouri, Nadja; Jakobs, Tatjana C.; Lin, Charles P.

    2010-02-01

    In vivo retinal imaging is an outstanding tool to observe biological processes unfold in real-time. The ability to image microstructure in vivo can greatly enhance our understanding of function in retinal microanatomy under normal conditions and in disease. Transgenic mice are frequently used for mouse models of retinal diseases. However, commercially available retinal imaging instruments lack the optical resolution and spectral flexibility necessary to visualize detail comprehensively. We developed an adaptive optics scanning laser ophthalmoscope (AO-SLO) specifically for mouse eyes. Our SLO is a sensor-less adaptive optics system (no Shack Hartmann sensor) that employs a stochastic parallel gradient descent algorithm to modulate a deformable mirror, ultimately aiming to correct wavefront aberrations by optimizing confocal image sharpness. The resulting resolution allows detailed observation of retinal microstructure. The AO-SLO can resolve retinal microglia and their moving processes, demonstrating that microglia processes are highly motile, constantly probing their immediate environment. Similarly, retinal ganglion cells are imaged along with their axons and sprouting dendrites. Retinal blood vessels are imaged both using evans blue fluorescence and backscattering contrast.

  6. Determination of the thickness and structure of the skin barrier by in vivo laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Lademann, J.; Richter, H.; Astner, S.; Patzelt, A.; Knorr, F.; Sterry, W.; Antoniou, Ch

    2008-04-01

    Normal skin barrier function is an essential aspect of skin homeostasis and regeneration. Dynamic inflammatory, proliferative and neoplastic skin processes such as wound healing, psoriasis and contact dermatitis are associated with a significant disruption of the skin barrier. In recent years, there has been increasing interest in evaluating cosmetic and pharmacologic products for their ability to restore these protective properties. The gold standard for characterization of barrier function has been the measurement of the transepidermal water loss, however the disadvantage of this method is its interference with several endogenous and exogenous factors such as hydration, perspiration and topically applied substances. This study was aimed to test the clinical applicability of a fluorescence confocal laser scanning microscope (LSM) for a systematic morphologic analysis of the structure, integrity and thickness of the stratum corneum in 10 otherwise healthy volunteers. The influence of skin treatment with commercial moisturizing cream on skin barrier function was evaluated in serial non-invasive examinations. Our findings showed that in vivo LSM may represent a simple and efficient method for the characterization of skin barrier properties, such as the thickness and hydration of the stratum corneum.

  7. 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.

  8. Acquisition of a Modular, Multi-laser, Raman-AFM Instrument for Multdisciplinary Research

    DTIC Science & Technology

    2015-04-28

    vapor deposition on copper foils. The four lasers range from the blue to 785 nm and provides a unique handle to determine excitation dependence of...Acquisition of a Modular, Multi- laser , Raman- AFM Instrument for Multdisciplinary Research A four- laser , confocal Raman/Atomic Force Scanning... laser , Raman-AFM Instrument for Multdisciplinary Research Report Title A four- laser , confocal Raman/Atomic Force Scanning microscope (Raman-AFM

  9. Laser-scanning photoacoustic microscopy with ultrasonic phased array transducer.

    PubMed

    Zheng, Fan; Zhang, Xiangyang; Chiu, Chi Tat; Zhou, Bill L; Shung, K Kirk; Zhang, Hao F; Jiao, Shuliang

    2012-11-01

    In this paper, we report our latest progress on proving the concept that ultrasonic phased array can improve the detection sensitivity and field of view (FOV) in laser-scanning photoacoustic microscopy (LS-PAM). A LS-PAM system with a one-dimensional (1D) ultrasonic phased array was built for the experiments. The 1D phased array transducer consists of 64 active elements with an overall active dimension of 3.2 mm × 2 mm. The system was tested on imaging phantom and mouse ear in vivo. Experiments showed a 15 dB increase of the signal-to-noise ratio (SNR) when beamforming was employed compared to the images acquired with each single element. The experimental results demonstrated that ultrasonic phased array can be a better candidate for LS-PAM in high sensitivity applications like ophthalmic imaging.

  10. Quantitative flaw characterization with scanning laser acoustic microscopy

    NASA Technical Reports Server (NTRS)

    Generazio, E. R.; Roth, D. J.

    1986-01-01

    Surface roughness and diffraction are two factors that have been observed to affect the accuracy of flaw characterization with scanning laser acoustic microscopy. In accuracies can arise when the surface of the test sample is acoustically rough. It is shown that, in this case, Snell's law is no longer valid for determining the direction of sound propagation within the sample. The relationship between the direction of sound propagation within the sample, the apparent flaw depth, and the sample's surface roughness is investigated. Diffraction effects can mask the acoustic images of minute flaws and make it difficult to establish their size, depth, and other characteristics. It is shown that for Fraunhofer diffraction conditions the acoustic image of a subsurface defect corresponds to a two-dimensional Fourier transform. Transforms based on simulated flaws are used to infer the size and shape of the actual flaw.

  11. Nondestructive monitoring damage in composites using scanning laser acoustic microscopy

    NASA Technical Reports Server (NTRS)

    Wey, A. C.; Kessler, L. W.; Dos Reis, H. L. M.

    1992-01-01

    Several Nicalon fiber reinforced LAS (lithium alumino-silicate) glass matrix composites were tested to study the relation between the residual strength and the different amounts of damage. The samples were fatigued by four-point cyclic loading at a 5 Hz rate at 500 C for a different number of cycles. 10 MHz scanning laser acoustic microscope (SLAM) images were taken to monitor damage on the samples. Our SLAM results indicate that there were defects already existing throughout the sample before fatigue, and the resultant damage pattern from fatigue could be related to the initial defect distribution in the sample. Finally, the fatigued samples were fractured and the residual strength data could not be explained by the cyclic fatigue alone. Rather, the damage patterns evident in the SLAM images were needed to explain the scatter in the data. The results show that SLAM is useful in nondestructively monitoring damage and estimating residual strength of fatigued ceramic composites.

  12. Laser-scanning photoacoustic microscopy with ultrasonic phased array transducer

    PubMed Central

    Zheng, Fan; Zhang, Xiangyang; Chiu, Chi Tat; Zhou, Bill L.; Shung, K. Kirk; Zhang, Hao F.; Jiao, Shuliang

    2012-01-01

    In this paper, we report our latest progress on proving the concept that ultrasonic phased array can improve the detection sensitivity and field of view (FOV) in laser-scanning photoacoustic microscopy (LS-PAM). A LS-PAM system with a one-dimensional (1D) ultrasonic phased array was built for the experiments. The 1D phased array transducer consists of 64 active elements with an overall active dimension of 3.2 mm × 2 mm. The system was tested on imaging phantom and mouse ear in vivo. Experiments showed a 15 dB increase of the signal-to-noise ratio (SNR) when beamforming was employed compared to the images acquired with each single element. The experimental results demonstrated that ultrasonic phased array can be a better candidate for LS-PAM in high sensitivity applications like ophthalmic imaging. PMID:23162708

  13. Multicomponent wavefield characterization with a novel scanning laser interferometer.

    PubMed

    Blum, Thomas E; van Wijk, Kasper; Pouet, Bruno; Wartelle, Alexis

    2010-07-01

    The in-plane component of the wavefield provides valuable information about media properties from seismology to nondestructive testing. A new compact scanning laser ultrasonic interferometer collects light scattered away from the angle of incidence to provide the absolute ultrasonic displacement for both the out-of-plane and an in-plane components. This new system is tested by measuring the radial and vertical polarization of a Rayleigh wave in an aluminum half-space. The estimated amplitude ratio of the horizontal and vertical displacement agrees well with the theoretical value. The phase difference exhibits a small bias between the two components due to a slightly different frequency response between the two processing channels of the prototype electronic circuitry.

  14. Multicomponent wavefield characterization with a novel scanning laser interferometer

    SciTech Connect

    Blum, Thomas E.; Wijk, Kasper van; Pouet, Bruno; Wartelle, Alexis

    2010-07-15

    The in-plane component of the wavefield provides valuable information about media properties from seismology to nondestructive testing. A new compact scanning laser ultrasonic interferometer collects light scattered away from the angle of incidence to provide the absolute ultrasonic displacement for both the out-of-plane and an in-plane components. This new system is tested by measuring the radial and vertical polarization of a Rayleigh wave in an aluminum half-space. The estimated amplitude ratio of the horizontal and vertical displacement agrees well with the theoretical value. The phase difference exhibits a small bias between the two components due to a slightly different frequency response between the two processing channels of the prototype electronic circuitry.

  15. Quantitative flaw characterization with scanning laser acoustic microscopy

    SciTech Connect

    Generazio, E.R.; Roth, D.J.

    1986-06-01

    Surface roughness and diffraction are two factors that have been observed to affect the accuracy of flaw characterization with scanning laser acoustic microscopy. Inaccuracies can arise when the surface of the test sample is acoustically rough. It is shown that, in this case, Snell's law is no longer valid for determining the direction of sound propagation within the sample. The relationship between the direction of sound propagation within the sample, the apparent flaw depth, and the sample's surface roughness is investigated. Diffraction effects can mask the acoustic images of minute flaws and make it difficult to establish their size, depth, and other characteristics. It is shown that for Fraunhofer diffraction conditions the acoustic image of a subsurface defect corresponds to a two-dimensional Fourier transform. Transforms based on simulated flaws are used to infer the size and shape of the actual flaw. 15 references.

  16. Quantitative flaw characterization with scanning laser acoustic microscopy

    NASA Technical Reports Server (NTRS)

    Generazio, E. R.; Roth, D. J.

    1986-01-01

    Surface roughness and diffraction are two factors that have been observed to affect the accuracy of flaw characterization with scanning laser acoustic microscopy. Inaccuracies can arise when the surface of the test sample is acoustically rough. It is shown that, in this case, Snell's law is no longer valid for determining the direction of sound propagation within the sample. The relationship between the direction of sound propagation within the sample, the apparent flaw depth, and the sample's surface roughness is investigated. Diffraction effects can mask the acoustic images of minute flaws and make it difficult to establish their size, depth, and other characteristics. It is shown that for Fraunhofer diffraction conditions the acoustic image of a subsurface defect corresponds to a two-dimensional Fourier transform. Transforms based on simulated flaws are used to infer the size and shape of the actual flaw.

  17. System Design Considerations In Bar-Code Laser Scanning

    NASA Astrophysics Data System (ADS)

    Barkan, Eric; Swartz, Jerome

    1984-08-01

    The unified transfer function approach to the design of laser barcode scanner signal acquisition hardware is considered. The treatment of seemingly disparate system areas such as the optical train, the scanning spot, the electrical filter circuits, the effects of noise, and printing errors is presented using linear systems theory. Such important issues as determination of depth of modulation, filter specification, tolerancing of optical components, and optimi-zation of system performance in the presence of noise are discussed. The concept of effective spot size to allow for impact of optical system and analog processing circuitry upon depth of modulation is introduced. Considerations are limited primarily to Gaussian spot profiles, but also apply to more general cases. Attention is paid to realistic bar-code symbol models and to implications with respect to printing tolerances.

  18. Urban Tree Classification Using Full-Waveform Airborne Laser Scanning

    NASA Astrophysics Data System (ADS)

    Koma, Zs.; Koenig, K.; Höfle, B.

    2016-06-01

    Vegetation mapping in urban environments plays an important role in biological research and urban management. Airborne laser scanning provides detailed 3D geodata, which allows to classify single trees into different taxa. Until now, research dealing with tree classification focused on forest environments. This study investigates the object-based classification of urban trees at taxonomic family level, using full-waveform airborne laser scanning data captured in the city centre of Vienna (Austria). The data set is characterised by a variety of taxa, including deciduous trees (beeches, mallows, plane trees and soapberries) and the coniferous pine species. A workflow for tree object classification is presented using geometric and radiometric features. The derived features are related to point density, crown shape and radiometric characteristics. For the derivation of crown features, a prior detection of the crown base is performed. The effects of interfering objects (e.g. fences and cars which are typical in urban areas) on the feature characteristics and the subsequent classification accuracy are investigated. The applicability of the features is evaluated by Random Forest classification and exploratory analysis. The most reliable classification is achieved by using the combination of geometric and radiometric features, resulting in 87.5% overall accuracy. By using radiometric features only, a reliable classification with accuracy of 86.3% can be achieved. The influence of interfering objects on feature characteristics is identified, in particular for the radiometric features. The results indicate the potential of using radiometric features in urban tree classification and show its limitations due to anthropogenic influences at the same time.

  19. Control electronics for a multi-laser/multi-detector scanning system

    NASA Technical Reports Server (NTRS)

    Kennedy, W.

    1980-01-01

    The Mars Rover Laser Scanning system uses a precision laser pointing mechanism, a photodetector array, and the concept of triangulation to perform three dimensional scene analysis. The system is used for real time terrain sensing and vision. The Multi-Laser/Multi-Detector laser scanning system is controlled by a digital device called the ML/MD controller. A next generation laser scanning system, based on the Level 2 controller, is microprocessor based. The new controller capabilities far exceed those of the ML/MD device. The first draft circuit details and general software structure are presented.

  20. High-sensitive scanning laser magneto-optical imaging system.

    PubMed

    Murakami, Hironaru; Tonouchi, Masayoshi

    2010-01-01

    A high-sensitive scanning laser magneto-optical (MO) imaging system has been developed. The system is mainly composed of a laser source, galvano meters, and a high-sensitive differential optical-detector. Preliminary evaluation of system performance by using a Faraday indicator with a Faraday rotation coefficient of 3.47 x 10(-5) rad/microm Oe shows a magnetic sensitivity of about 5 microT, without any need for accumulation or averaging processing. Using the developed MO system we have succeeded in the fast and quantitative imaging of a rotationally symmetric magnetic field distribution around an YBa(2)Cu(3)O(7-delta) (YBCO) strip line applied with dc-biased current, and also succeeded in the detection of quantized fine signals corresponding to magnetic flux quantum generation in a superconducting loop of an YBCO Josephson vortex flow transistor. Thus, the developed system enables us not only to do fast imaging and local signal detection but also to directly evaluate both the strength and direction of a magnetic signal.