Science.gov

Sample records for combined confocal laser

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

    PubMed Central

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

    2008-01-01

    Background Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Methods Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). Results We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes. PMID:18627634

  2. 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

  3. 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.

  4. 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.

  5. [Confocal laser scanning microscopy].

    PubMed

    Ulrich, M

    2015-07-01

    Reflectance confocal microscopy (RCM) allows the in vivo evaluation of melanocytic and nonmelanocytic skin tumours with high sensitivity and specificity. RCM represents an optical imaging technique, which enables us to examine the skin at high resolution. Today, RCM represents not only an interesting tool for dermatologic research but has also been introduced as a diagnostic tool in every day clinical practice. As such, RCM is applied for improvement of skin cancer diagnosis adjunct to clinical and dermatoscopic examination. In combination with dermatoscopy RCM has shown an increased specificity with similar sensitivity. In this regard RCM helps to decrease the rate of unnecessary biopsies of benign lesions. Despite its use in dermatooncology RCM may also be used for diagnosis and monitoring of inflammatory diseases. Future developments include technical improvements, teledermatology solutions and the application of ex vivo RCM in Moh's micrographic surgery.

  6. 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.

  7. Dual channel confocal laser scanning microscopy of lucifer yellow-microinjected human brain cells combined with Texas red immunofluorescence.

    PubMed

    Belichenko, P V; Dahlström, A

    1994-06-01

    A method for visualization of individual human brain cells and their dendritic extensions in combination with immunofluorescence is described. Microinjection of Lucifer Yellow was used to reveal the dendritic morphology of cortical brain cells. Indirect immunofluorescence with Texas Red as label was used to investigate the distribution of 3 different groups of immunogens: enzymes (monoamine oxidase A and B), receptors (beta-adrenoceptor protein), and synaptic vesicle proteins (synapsin I and synaptophysin) in each cortical slice. A dual-channel confocal laser scanning microscope with an argon/krypton laser was used for imaging these double-stained fluorescent specimens. Lucifer Yellow and Texas Red were recorded simultaneously or separately, taking advantage of the different activating lines (488 lambda and 568 lambda) of the laser and using the two filter blocks (K1 and K2) supplied with the instrument (BioRad MRC-600) for recording the emission of either fluorophore. Using this technique we have demonstrated the localization of immunoreactive material in relation to the dendritic morphology of cortical cells.

  8. New bone formation and microstructure assessed by combination of confocal laser scanning microscopy and differential interference contrast microscopy.

    PubMed

    Yang, Xiaohong; Qin, Ling; Liang, Weiguo; Wang, Wen; Tan, Jianrong; Liang, Peihong; Xu, Jiake; Li, Siming; Cui, Shuliang

    2014-03-01

    Bone is a mineralized connective tissue that is continuously and microstructurally remodeled. Altered bone formation and microstructure arise in pathological bone conditions such as osteoporosis, osteonecrosis, fracture repair, and Paget disease of bone. A proper and objective assessment of bone formation and microstructure will provide insight into the understanding of bone pathogenesis and remodeling. Here, new bone formation ex vitro and its microstructure were evaluated in in vivo multiple sequential polychrome-labeled samples using confocal laser scanning microscopy (CLSM), which generated clearer and more reliable images of thick bone sections than conventional fluorescence microscopy (CFM). Intriguingly, fine details of the bone microstructural features, including the mineralization fronts, quiescent versus active osteons, and Volkmann's channel, were elucidated using CLSM, which defines the relationship between morphological changes and function, when combined with differential interference contrast microscopy. Furthermore, CLSM provided objective evaluations of bone formation, such as the ratio of labeled areas of new bone formation in a rabbit model when compared with CFM. Altogether, new bone formation and its microstructure can be evaluated more adequately using a combination of CLSM and DIC microscopies.

  9. Effects of the Combined PDL/Nd:YAG Laser on Surgical Scars: Vascularity and Collagen Changes Evaluated by In Vivo Confocal Microscopy

    PubMed Central

    Vas, Krisztina; Gaál, Magdolna; Varga, Erika; Kovács, Réka; Bende, Balázs; Kocsis, Ádám; Kemény, Lajos

    2014-01-01

    The aim of this study was to investigate the efficacy of the sequential combined 585 nm PDL and the 1064 nm neodymium:yttrium-aluminium-garnet laser (PDL/Nd:YAG) in the treatment of surgical scars and to evaluate the short-term effects by in vivo confocal microscopy (RCM) and the long-term effects by clinical assessment of the scars. Twenty-five patients were enrolled with 39 postoperative linear scars; each scar was divided into two fields. One half was treated with the combined PDL/Nd:YAG laser, whereas the other half remained untreated. Each scar was treated three times at monthly intervals. Scars were evaluated by an independent examiner, using the Vancouver Scar Scale. The combined PDL/Nd:YAG laser significantly improved the appearance of the scars. In order to study the short-term effects of combined laser treatment, six additional patients were enrolled with 7 postoperative linear scars. One half of scars was treated once with the combined PDL/Nd:YAG laser. One week after this laser treatment, both the treated and the nontreated parts of the scars were examined by dermoscopy and RCM. The dermoscopic pictures revealed improvements even in treated areas. In conclusion, the combined PDL/Nd:YAG laser was found to be effective in improving the quality and appearance of the surgical scars. PMID:25276770

  10. 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

  11. 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 ...

  12. 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 ...

  13. Antimicrobial effectiveness of oxidant and chelating agents combination in infected dentine: an ex vivo confocal laser scanning microscopy study.

    PubMed

    Giardino, L; Del Fabbro, M; Cesario, F; Fernandes, F S; Andrade, F B

    2017-09-27

    To evaluate the intratubular antimicrobial activity of several oxidant and chelating agents associated or not with surfactants in experimentally infected root canals, using confocal laser scanning microscopy. Twenty-four dentine blocks from bovine incisors were contaminated for five days with Enterococcus faecalis (ATCC- 29212). Ten contaminated dentine specimens were irrigated for 5 min with 5.25% NaOCl followed by 17% EDTA for 2 min, and the other 10 with Hypoclean for 5 min followed by Tetraclean NA for 2 min. The remaining four specimens were used as positive and negative controls (2 samples each).Then, dentine blocks were stained with Live/Dead BacLight for analysis of the remaining live or dead bacteria using confocal laser scanning microscope. Comparison between and within groups was performed using the Mann Whitney test for independent samples and the Wilcoxon signed-rank test, respectively. After exposure to irrigants, the positive control group had a median of 67.41% of viable bacteria (95% CI: 48.15, 78.9) of viable bacteria, while NaOCl+EDTA group and Hypoclean+Tetraclean NA group had 3.77% (1.28, 15.92) and 0.87% (-0.42, 4.30) of viable bacteria, respectively. These results were significantly different each other, both overall and distinct by region (cervical and medium third), or depth (superficial and deep layer) (p<0.01 in all cases). The use of adjunctive agents reducing the surface tension associated with oxidant and chelating agents improved the antimicrobial activity of irrigating solutions and intra-tubular decontamination against Enterococcus faecalis, possibly due to a better removal of the smear layer and deeper penetration into dentinal tubules. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. Confocal unstable-resonator semiconductor laser

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  15. 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.

  16. 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

  17. Combining confocal microscopy with precise force-scope optical tweezers

    NASA Astrophysics Data System (ADS)

    Richardson, Andrew C.; Reihani, Nader; Oddershede, Lene B.

    2006-08-01

    We demonstrate an example of 'confocal-tweezers' wherein confocal images and precise optical force measurements, using photodiodes, are obtained simultaneously in the x-y plane without moving the objective lens. The optical trap is produced using a 1.064μm cw laser and is combined with Leica's TCS SP5 broadband confocal microscope to trap and image living cells. The unique method by which the confocal images are created facilitates the acquisition of images in areas far from the trapping location. In addition, because the scanning process involves moving galvanic mirrors independently of the objective, the trap is held stable in position and is not subject to any error in position for the x-y scan. We have successfully trapped and confocally imaged 80nm gold colloids, 150nm gold colloids and 1μm polystyrene beads whilst making quantitative measurements of the force applied by the trap on each bead. To the best of our knowledge this is the first time that anyone has combined precise force measuring optical tweezers with confocal microscopy. We also discuss some of the technical challenges involved in advancing the experimental set up to make quantitative force measurements in combination with 3D stacking. Having proven the potential of this system in 2D, we hope to develop it further to investigate the nano-mechanics of cell division through the attachment of gold beads to fluorescently labelled organelles in S. pombe yeast cells.

  18. 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.

  19. Combined Confocal and Magnetic Resonance Microscopy

    SciTech Connect

    Wind, Robert A.; Majors, Paul D.; Minard, Kevin R.; Ackerman, Eric J.; Daly, Don S.; Holtom, Gary R.; Thrall, Brian D.; Weber, Thomas J.

    2002-05-12

    Confocal and magnetic resonance microscopy are both used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each of the microscopes individually. In this paper two configurations of a combined confocal and magnetic resonance microscope described. In both cases the sample compartment is part of a temperature regulated perfusion system. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas with the second configuration monolayers of mammalian cells can be investigated . Combined images are shown of Xenopus laevis frog oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.

  20. Analysis of dehydration kinetics, status of water and oil distribution of microwave-assisted vacuum frying potato chips combined with NMR and confocal laser scanning microscopy.

    PubMed

    Su, Ya; Zhang, Min; Fang, Zhongxiang; Zhang, Weiming

    2017-11-01

    In this study, the dehydration kinetics, status of water and oil distribution of microwave-assisted vacuum frying (MVF) potato chips were analyzed combining with NMR and confocal laser scanning microscopy (CLSM). Results showed that the MVF markedly increased the moisture evaporation kinetics and effective moisture diffusivity compared to vacuum frying (VF). The higher microwave power level (1000W) used achieved higher moisture evaporation rate and higher effective moisture diffusivity, The Logarithmic model exhibited the best fit for the obtained data of MR versus frying time in the MVF. The NMR analysis showed the free water in the samples firstly evaporate and the linkage between water and structure of materials becomes tighter with the frying time. The total oil content, surface and structural oil content were all significantly lower in MVF samples than that in VF samples. The CLSM analysis confirmed that less oil adhere to the surface and less oil trapped in the structure in MVF slices. The surface morphology showed that there were less ruptured and damaged cells in MVF samples and helped to explain the reduction of oil content. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. 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.

  2. Attempt of correlative observation of morphological synaptic connectivity by combining confocal laser-scanning microscope and FIB-SEM for immunohistochemical staining technique.

    PubMed

    Sonomura, Takahiro; Furuta, Takahiro; Nakatani, Ikuko; Yamamoto, Yo; Honma, Satoru; Kaneko, Takeshi

    2014-11-01

    Ten years have passed since a serial block-face scanning electron microscopy (SBF-SEM) method was developed [1]. In this innovative method, samples were automatically sectioned with an ultramicrotome placed inside a scanning electron microscope column, and the block surfaces were imaged one after another by SEM to capture back-scattered electrons. The contrast-inverted images obtained by the SBF-SEM were very similar to those acquired using conventional TEM. SFB-SEM has made easy to acquire image stacks of the transmission electron microscopy (TEM) in the mesoscale, which is taken with the confocal laser-scanning microcopy(CF-LSM).Furthermore, serial-section SEM has been combined with the focused ion beam (FIB) milling method [2]. FIB-incorporated SEM (FIB-SEM) has enabled the acquisition of three-dimensional images with a higher z-axis resolution com- pared to ultramicrotome-equipped SEM.We tried immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in CF-LSM. Dendrites of neurons in the rat neostriatum were visualized using a recombinant viral vector. 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.We showed that conventional immuno-cytochemical staining for TEM was applicable to FIB-SEM. Furthermore, several synaptic contacts, which were thought to exist on the basis of CF-LSM findings, were confirmed with FIB-SEM, revealing the usefulness of the combined method of CF-LSM and FIB-SEM. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  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 RAT FOLLICLE DEVELOPMENT

    EPA Science Inventory

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

  5. 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

  6. Confocal backscatter laser velocimeter with on-axis sensitivity.

    NASA Technical Reports Server (NTRS)

    Orloff, K. L.; Logan, S. E.

    1973-01-01

    A confocal backscatter laser Doppler velocimeter (LDV) that measures two velocity components has been developed. This device requires only two incident beams polarized normally to one another. Moreover, the velocity components sensed are nearly orthogonal. The velocimeter employs a combined dual-scatter, local oscillator arrangement to obtain the bidirectional sensitivity. Two photodetectors are used, each sensing only one Doppler frequency proportional to one of the very nearly orthogonal velocity components. In addition, a single Bragg cell serves to frequency bias both velocity components in order to eliminate directional ambiguity. A differencing technique has also been incorporated to enhance the dual-scatter Doppler signal corresponding to the transverse velocity.

  7. Combined In Vivo Confocal Raman Spectroscopy and Confocal Microscopy of Human Skin

    PubMed Central

    Caspers, P. J.; Lucassen, G. W.; Puppels, G. J.

    2003-01-01

    In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose. PMID:12829511

  8. Optimization of confocal scanning laser ophthalmoscope design.

    PubMed

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

    2013-07-01

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

  9. 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...

  10. 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...

  11. 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

  12. Probe based confocal laser endomicroscopy of the pancreatobiliary system.

    PubMed

    Almadi, Majid A; Neumann, Helmut

    2015-11-28

    To review applications of confocal laser endomicroscopy (CLE) in pancreatobiliary lesions and studies that assessed training and interpretation of images. 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. 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. The role of pCLE in the evaluation of pancreatobiliary disorders might be better

  13. 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

  14. 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. Copyright © 2013 Wiley Periodicals, Inc.

  15. 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

  16. 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.

  17. Ocular mucin visualization by confocal laser scanning microscopy.

    PubMed

    Peral, Assumpta; Pintor, Jesús

    2008-05-01

    To describe a new method of visualizing human conjunctiva goblet cell mucin secretion by using a combination of impression cytology and laser scanning microscopy. By assembling a Z-stack of confocal microscopy images taken from human impression cytology samples, we obtained 3-dimensional information about the release and spread of goblet cell secretions above the conjunctival surface. After reconstruction and rendering of these images, analysis of the shape and spreading characteristics of the mucins permitted definition of the following parameters related to goblet cell secretion: mucin cloud height as the height of the top of the cloudlike mucin structure visible above the goblet cell opening and spread mucin thickness, which is the thickness of the mucin layer distributed over the surface of the conjunctiva. Several impression cytology samples of control and muco-deficient patients have been analyzed through the confocal laser scanning technique, and significant differences between these groups were found. Mucin cloud height and spread mucin thickness values for controls were 8.81 +/- 4.00 and 2.77 +/- 1.00 microm, respectively (n = 25). These values decreased by approximately 70% and 40%, respectively, for moderately mucodeficient subjects and by 84% and 48% for those with severe mucodeficiency. Classifying those individuals having mucin-related pathology may thus be possible on the basis of application of these techniques. In summary, we present a method of objectively identifying those individuals with problems associated with either a lack of mucins or a reduction in the distribution of these proteins over the ocular surface.

  18. [Combined endoscopic diagnostics with catheter confocal endomicroscopy for gastric neoplasia detection].

    PubMed

    Shuleshova, A G; Zav'ialov, M O; Ul'ianov, D N; Kanareĭtseva, T D

    2014-01-01

    The analysis of combined endoscopic diagnostics with catheter confocal laser endomicroscopy (CCLE) for detection of gastric neoplasia in 103 patients is presented in the article. It was described the main principles of catheter confocal laser endomicroscopy by using of Cellvizio-system ("Mauna Kea Technologies", France). All patients underwent esophagogastroduodenoscopy before catheter confocal laser endomicroscopy. Such modes as HRE-endoscopy, NBI-endoscopy and Zoom-endoscopy were used. It was revealed different neoplastic changes of stomach mucous coat and early cancer forms of stomach in 185 cases. It was noted expediency and high informational content of CCLE which leads to detect the foci of intestinal metaplasia by colonic type, foci of dysplasia and early cancer of stomach mucous coat. The role of conventional morphological study for verification of changes detected with CCLE was shown.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. 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...

  1. 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...

  2. Calibration of diode laser spectra using a confocal etalon

    NASA Technical Reports Server (NTRS)

    Jennings, D. E.

    1984-01-01

    The dual-beam diode laser spectrometer described by Jennings (1980) is adapted to use a 50-cm confocal etalon for frequency calibration. The collimated radiation from the laser is split at a wedged ZnSe window, and the reference beam is then focused at the midpoint of the etalon length. After the etalon, the reference beam is recollimated and continues its regular path to the monochromator and detectors. An aperture is placed before the etalon in order to limit the entrance beam diameter to approximately 5 mm. Both ends of the etalon are furnished with two-axis adjustments. Initial alignment is achieved using an He-Ne laser, and final optimization involves adjustment of the cavity length as well as the etalon pitch and yaw. The 50-cm confocal etalon produces fringes separated by 150 MHz (0.005/cm). With the aid of a CO2 laser, it is found to have fringe widths (FWHM) of 2 MHz. The confocal etalon makes it possible to improve the accuracy of relative frequency measurements in diode laser spectra and to check the spectral purity and stability of the laser during the recording of spectra.

  3. Three-dimensional scanning confocal laser microscope

    DOEpatents

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

    1999-01-01

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

  4. 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.

  5. Confocal volume in laser Raman microscopy depth profiling

    SciTech Connect

    Maruyama, Yutaka; Kanematsu, Wataru

    2011-11-15

    To clarify the degradation of confocality in laser Raman microscopy depth profiling (optical sectioning) and the influence of pinhole filtering on it, we investigate the confocal volume in detail based on Gaussian beam optics and scalar wave optics. Theoretical depth profiles of a homogeneous transparent sample for four different pinhole sizes, which are computed using the measured incident beam waist radius w{sub 0} and only a few optical system specific parameters such as a numerical aperture (NA) and a focal length, show a good agreement with the corresponding measured depth profiles. The computed confocal volume demonstrates that the pinhole size affects the actual probe depth as well as the axial resolution and the total intensity loss.

  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. 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.

  8. Electrostatically driven micromirrors for a miniaturized confocal laser scanning microscope

    NASA Astrophysics Data System (ADS)

    Hofmann, Ulrich; Muehlmann, Sascha; Witt, Martin; Doerschel, Klaus; Schuetz, Rijk; Wagner, Bernd

    1999-09-01

    A compact two-mirror microscanner has been fabricated to build the central part of a miniaturized confocal laser scanning microscope. This microscope shall be mounted at the tip of an endoscope to provide high resolution imaging for medical diagnostics. In order to achieve a resolution of 500 X 500 image elements large scan angles and also large mirror dimensions have to be realized within a spatially strong limited housing. While bulk silicon technology on the one hand enables fabrication of micromirrors with nearly ideal elastical behavior, those actuators on the other hand often are too fragile for a lot of applications. This paper describes the design, fabrication and assembling of electrostatically driven torsional micromirrors that meet the requirements of fast two-dimensional scanning with high angular precision over large scan angles, compact design and also high shock resistance. This is achieved with the combination of bulk silicon technology with metal surface micromachining. Besides medical diagnostics these microscanners can be used in a wider range of applications such as displays, two-dimensional barcode scanning, multiplexing of fiber optics, etc.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Confocal laser endomicroscopy in breast surgery: a pilot study.

    PubMed

    De Palma, Giovanni D; Esposito, Dario; Luglio, Gaetano; Limite, Gennaro; Accurso, Antonello; Sollazzo, Viviana; Maione, Francesco; Cassese, Gianluca; Siciliano, Saverio; Gennarelli, Nicola; Ilardi, Gennaro; Paternoster, Mariano; Giglio, Mariano C; Forestieri, Pietro

    2015-04-10

    Breast neoplasms include different histopathological entities, varying from benign tumors to highly aggressive cancers. Despite the key role of imaging, traditional histology is still required for a definitive diagnosis. Confocal Laser Endomicroscopy (CLE) is a new technique, which enables to obtain histopathological images in vivo, currently used in the diagnosis of gastrointestinal diseases. This is a single-center pilot feasibility study; the main aim is to describe the basic morphological patterns of Confocal Laser Endomicroscopy in normal breast tissue besides benign and malignant lesions. Thirteen female patients (mean age 52.7, range from 22 to 86) who underwent surgical resection for a palpable breast nodule were enrolled. CLE was performed soon after resection with the Cellvizio® Endomicroscopy System (Mauna Kea Technologies, Paris, France), by using a Coloflex UHD-type probe; intravenous fluorescein was used as contrast-enhancing agent. The surgical specimen was cut along the main axis; dynamic images were obtained and recorded using a hand-held probe directly applied both to the internal part of the lesion and to several areas of surrounding normal tissue. Each specimen was then sent for definitive histologic examination. Histopathology revealed a benign lesion in six patients (46%), while a breast cancer was diagnosed in seven women (54%). Confocal laser endomicroscopy showed some peculiar morphological patterns. Normal breast tissue was characterized by a honeycomb appearance with regular, dark, round or hexagonal glandular lobules on a bright stroma background; tubular structures, representing ducts or blood vessels, were also visible in some frames. Benign lesions were characterized by a well-demarcated "slit-like" structure or by lobular structures in abundant bright stroma. Finally, breast cancer was characterized by a complete architectural subversion: ductal carcinoma was characterized by ill-defined structures, with dark borders and irregular

  17. [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.

  18. Liquid level sensing based on laser differential confocal detectors

    NASA Astrophysics Data System (ADS)

    Gao, Haibo; Fan, Chunshi; Zhang, Li; Hu, Yao

    2015-01-01

    Liquid level measurement plays an important part in industry and daily life. Applications include oil tanks, gasoline stations and public water supplies. Traditional electronic sensors cannot satisfy the demands in harsh environments. Recently, optical sensors have been particularly attractive in these applications. We propose a sensing method based on laser differential confocal detectors for discrete or continuous liquid level sensing. No target or supplementary device need to be immersed into the liquid. The sensitivity of the liquid level is about 0.01 mm with current systematic parameters. Measurement experiment of simulated liquid surface with a reflective mirror is carried out to verify the method.

  19. Fluorescence (Multiwave) Confocal Microscopy.

    PubMed

    Welzel, J; Kästle, Raphaela; Sattler, Elke C

    2016-10-01

    In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. 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.

  1. 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.

  2. Passport examination by a confocal-type laser profile microscope.

    PubMed

    Sugawara, Shigeru

    2008-06-10

    The author proposes a nondestructive and highly precise method of measuring the thickness of a film pasted on a passport using a confocal-type laser profile microscope. The effectiveness of this method in passport examination is demonstrated. A confocal-type laser profile microscope is used to create profiles of the film surface and film-paper interface; these profiles are used to calculate the film thickness by employing an algorithm developed by the author. The film thicknesses of the passport samples--35 genuine and 80 counterfeit Japanese passports--are measured nondestructively. The intra-sample standard deviation of the film thicknesses of the genuine and counterfeit Japanese passports was of the order of 1 microm The intersample standard deviations of the film thicknesses of passports forged using the same tools and techniques are expected to be of the order of 1 microm. The thickness values of the films on the machine-readable genuine passports ranged between 31.95 microm and 36.95 microm. The likelihood ratio of this method in the authentication of machine-readable Japanese genuine passports is 11.7. Therefore, this method is effective for the authentification of genuine passports. Since the distribution of the film thickness of all forged passports was considerably larger than the accuracy of this method, this method is considered effective also for revealing the relation among the forged passports and acquiring proof of the crime.

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

    NASA Astrophysics Data System (ADS)

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

    1996-02-01

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

  4. 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.

  5. 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.

  6. Diagnosing Helicobacter pylori in vivo by confocal laser endoscopy.

    PubMed

    Kiesslich, Ralf; Goetz, Martin; Burg, Juergen; Stolte, Manfred; Siegel, Ekkehard; Maeurer, Markus J; Thomas, Steven; Strand, Dennis; Galle, Peter R; Neurath, Markus F

    2005-06-01

    Confocal laser endomicroscopy enables subsurface microscopic imaging of living tissue during ongoing endoscopy. This case report describes the in vivo detection of Helicobacter pylori by endomicroscopy. Endomicroscopy (Pentax, Tokyo, EC-3870CIFK) was performed by using two different contrast stains: Topical Acriflavine in addition to intravenously applied fluorescein netted the surface and allowed identification of focal accumulation of Helicobacter pylori at the surface and in deeper layer of the gastric epithelium. Biopsies were performed at the antrum and corpus for urease testing and histology. In addition, biopsies were cultured for Helicobacter pylori. Cultured bacteria were re-assessed ex vivo using confocal microscopy with and without acriflavine staining. Helicobacter pylori infection could be detected in a 70-year-old male by endomicroscopy. Accumulated, as well as single bacteria, could be observed and the distinct shape and flagella of Helicobacter pylori could be identified. Helicobacter pylori infection was proved by histology. Furthermore, ex vivo examination of cultures proved the presence of Helicobacter pylori and the active uptake of acriflavine into the bacteria. Endomicroscopy is a new diagnostic approach, which enables the immediate diagnosis of Helicobacter pylori in vivo during standard video endoscopy.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Diffusion of photoacid generators by laser scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

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

    1998-06-01

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

  13. The free-electron laser in a symmetrical confocal resonator

    NASA Technical Reports Server (NTRS)

    Ozcan, Meric; Pantell, Richard H.

    1993-01-01

    A tapered wiggler is used in a FEL oscillator to improve the saturation efficiency. During signal buildup the tapered wiggler does not provide optimum phase synchronism between the electron beam and the electromagnetic wave, resulting in an appreciable loss in small-signal gain. This problem can be ameliorated by using a multicomponent wiggler, which is a combination of a uniform wiggler and a tapered section. During buildup, gain is primarily contributed by the linear element, and at high power levels the gain and efficiency are enhanced by the taper. Ideally, one would like to have the optical waist location near the linear section at small-signal levels and at near the tapered section at high power levels. Placing the FEL in a symmetrical confocal resonator approaches this desired effect automatically since it has the unique characteristic that a stable mode exists for all locations of the waist of a Gaussian beam along the axis of the interferometer.

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

    PubMed

    Hovis, D B; Heuer, A H

    2010-12-01

    Laser scanning confocal microscopes are essential and ubiquitous tools in the biological, biochemical and biomedical sciences, and play a similar role to scanning electron microscopes in materials science. However, modern laser scanning confocal microscopes have a number of advantages for the study of materials, in addition to their obvious uses for high resolution reflected and transmitted light optical microscopy. In this paper, we provide several examples that exploit the laser scanning confocal microscope's capabilities of pseudo-infinite depth of field imaging, topographic imaging, photo-stimulated luminescence imaging and Raman spectroscopic imaging. © 2010 The Authors Journal of Microscopy © 2010 The Royal Microscopical Society.

  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. 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.

  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. Applications of confocal laser scanning microscopy to dental bonding.

    PubMed

    Pioch, T; Stotz, S; Staehle, H J; Duschner, H

    1997-11-01

    The introduction of confocal laser scanning microscopy (CLSM) has provided a valuable new technique for the visualization of bonding structures such as a hybrid layer in dentin (Watson, 1989, 1991). In the case of seven commercially-available dentin bonding systems, it could be demonstrated that the CLSM renders considerably more detailed information than the SEM because of its non-destructive nature and because of the possibility of a distinction between components of bonding agents. With most of the bonding systems, measurements of the thickness of the hybrid layer could be carried out when the primer component was labeled with rhodamine B. It was found that this thickness is significantly increased by increases in etching time and only slightly decreased by increases in the drying time of the dentin and of the primer. When rhodamine B was used for dye penetration tests on four different dentin bonding systems, a leakage within the demineralized zone in the dentin was found in each of the specimens. This structure appears similar to that which Sano et al. (1995) called "nanoleakage". The amount of nanoleakage could not be measured by this method. In the case of enamel or ceramic bonding, a penetration zone was found which corresponded to the etching patterns found in enamel and ceramics, respectively. We conclude that CLSM can offer a wealth of new information about bonding morphology and, therefore, should be used in addition to conventional methods so that the maximum information can be obtained.

  19. Confocal laser microscopic analysis of biofilm on newer feldspar ceramic.

    PubMed

    Brentel, A S; Kantorski, K Z; Valandro, L F; Fúcio, S B; Puppin-Rontani, R M; Bottino, M A

    2011-01-01

    This study evaluated the surface roughness, hydrophobicity and in situ dental biofilm associated with microfilled feldspar ceramics submitted to the different finishing and polishing procedures. Samples were made according to the manufacturer's instructions and allocated to groups as follows: glaze (G1); glaze and diamond bur (G2); glaze, diamond bur and rubber tips (G3) and glaze, diamond bur, rubber tips and felt disks impregnated with a fine-aluminum oxide particle based paste (G4). Roughness was evaluated with a roughness analyzer (Ra). Hydrophobicity was determined by the contact angle of deionized water on samples. Biofilm was evaluated eight hours after formation in the oral environment using confocal laser-scanning microscopy (CLSM) and scanning-electron microscopy (SEM). Significant differences were found related to roughness (G1

  20. 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).

  1. 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.

  2. CCDiode: an optimal detector for laser confocal microscopes

    NASA Astrophysics Data System (ADS)

    Pawley, James B.; Blouke, Morley M.; Janesick, James R.

    1996-04-01

    The laser confocal microscope (LCM) is now an established research tool in biology and materials science. In biological applications, it is usually employed to detect the location of fluorescent market molecules and, under these conditions, signal levels from bright areas are often < 20 photons/pixel (from the specimen, assuming a standard 512 X 768, 1 sec. scan). Although this data rate limits the speed at which information can be derived from the specimen, saturation of the fluorophor, photobleaching of the dye, and phototoxicity prevent it being increased. Currently, most LCMs use photomultiplier tubes (PMT, QE equals 1 - 30% 400 - 900 nm). By contrast, rear-illuminated, scientific charge-coupled devices (CCD) now routinely readout the signal from square sensors approximately 30 micrometers on a side with a QE of 80 - 90%, a noise of only +/- 3 e-/pix and with no multiplicative noise. For this reason, in 1989, one of us (JJ) developed a rear-illuminated, single-channel Si sensor, called the Turbodiode, employing some of the sophisticated readout techniques used to measure charge in a scientific CCD. We are now extending this work to a device in which a single 36 X 36 micrometers sensor is read out through a low-noise FET charge amplifier with a reset circuit and then passed to a correlated, double-sampling digitizer. To maintain the desired +/- 3 e noise level at the relatively high data rate of 1 MHz, our new device utilizes 64 separate readout amplifier/digitizer systems, operating in sequence. The resulting detector is more compact, efficient and reliable than the PMT it replaces but as its sensitive area is smaller than that of a PMT, it will require auxiliary optics when used with any LCM having a large (mm) pinhole. As the signal light is parallel, a simple lens mounted axially and with the CCDiode at its focus would suffice. Future versions may use 3 X 3 or 5 X 5 arrays of sensors to `track' the confocal spot as it is deflected by inhomogeneities of the

  3. 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 ...

  4. 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 ...

  5. 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.

  6. 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

  7. 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.

  8. In vivo laser and white-light confocal microscopic findings of human conjunctiva.

    PubMed

    Kobayashi, Akira; Yoshita, Tsuyoshi; Sugiyama, Kazuhisa

    2004-01-01

    To report in vivo laser and white-light confocal microscopic findings of human conjunctiva to investigate the potential application of these confocal microscopes as diagnostic devices for normal and pathologic conjunctiva. Two healthy volunteers, 28- and 35-year-old men, participated in this study. The temporal bulbar conjunctiva, which was approximately 5 mm away from the limbus, was examined by in vivo laser and white-light confocal microscopic analysis. Using laser confocal microscopy, at least two different cell types could be distinguished in both subjects: conjunctival superficial epithelial cells and conjunctival basal epithelial cells. In contrast, conjunctival epithelial cells could not be visualized using white-light confocal microscopy. These results indicate that laser confocal microscopy, but not white-light confocal microscopy, can be used to visualize in vivo human conjunctiva. Further investigations in a large number of normal subjects and in patients with conjunctival pathologies are required to fully evaluate the usefulness of this device in daily clinical ophthalmology.

  9. Probe-based confocal laser endomicroscopy in double balloon enteroscopy.

    PubMed

    Miehlke, Stephan; Morgner, A; Aust, D; Baretton, G; Madisch, A

    2011-07-01

    Probe-based confocal laser endomicroscopy (pCLE) allows in-vivo assessment of the gastrointestinal mucosal architecture during ongoing endoscopy. We investigated the feasibility and safety of pCLE during double balloon enteroscopy (DBE). DBE was performed using the Fujinon EN-450P5. pCLE (Cellvizio-GI®, Mauna Kea Technologies) was performed after intravenous injection of 5-10  mL fluorescein 1 % using a 1.8-mm probe (GastroFlex/ColoFlex Z-probe) at the deepest point of DBE insertion and in case of any pathological lesion. Primary outcome measure was technical success, defined as (i) successful advancement of the probe at the deepest DBE insertion and (ii) successful pCLE imaging of the intestinal mucosa. Secondary outcome was safety of the pCLE procedure. 27 DBE procedures (14 antegrade) were performed in 16 patients. The mean depth of small bowel insertion was 255  cm for antegrade and 130  cm for retrograde DBE. Technical success of pCLE was achieved in 96.3 % (antegrade 92.8 %, retrograde 100 %). One technical failure occurred (incomplete probe advancement). There were no adverse events related to the pCLE procedure. pCLE imaging of the small bowel mucosal architecture was possible in all cases. Pathological conditions within the small bowel such as loss of villi, crypt hyperplasia, advanced neoplasia, or increased blood flow due to inflammation tissue could be successful visualized. This study is the first to demonstrate successful and safe application of pCLE in the deep small bowel during double balloon enteroscopy. Further studies are needed to determine the clinical benefit of pCLE in the management of patients with small bowel diseases. © Georg Thieme Verlag KG Stuttgart · New York.

  10. 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

  11. 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. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

  12. 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

  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. 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

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

    PubMed

    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.

  16. Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy

    PubMed Central

    Siegel, Nisan; Brooker, Gary

    2014-01-01

    FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called “CINCH”. PMID:25321701

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

    PubMed

    Paddock, Stephen W; Eliceiri, Kevin W

    2014-01-01

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

  18. 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.

  19. 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.

  20. Confocal laser method for quantitative evaluation of critical optical properties of toric intraocular lenses.

    PubMed

    Walker, Bennett N; James, Robert H; Song, Samuel; Calogero, Don; Ilev, Ilko K

    2016-03-01

    To present a proof-of-concept study on the development and implementation of an innovative confocal laser method platform for precise quantitative evaluation of critical optical properties unique to toric intraocular lenses (IOLs). U.S. Food and Drug Administration, Optical Therapeutics and Medical Nanophotonics Laboratory, Silver Spring, Maryland, USA. Experimental study. The optical properties of hydrophobic toric IOLs were evaluated with a confocal laser method that was modified to isolate the 2 planes of focus that are observed with toric IOLs. The results show the confocal laser method has the potential to measure the orthogonally separated optical powers and then calculate them to the commonly referenced spherical equivalent and cylinder powers of toric IOLs with high accuracy (≤1 μm of focal length measurement). Furthermore, the proposed confocal laser method design includes a new component for precise differentiation of the 2 focal planes and isolation of the 2 focal points, and thus for accurate measurement of the anterior cylinder axis of toric IOLs. The modifications to the confocal laser method platform enabled the quantitative evaluation of optical properties attributed to toric IOLs. None of the authors has a financial or proprietary interest in any material or method mentioned. Published by Elsevier Inc.

  1. 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.

  2. Confocal Laser Endomicroscopy in Inflammatory Bowel Disease--A Systematic Review.

    PubMed

    Rasmussen, Ditlev Nytoft; Karstensen, John Gásdal; Riis, Lene Buhl; Brynskov, Jørn; Vilmann, Peter

    2015-12-01

    Confocal laser endomicroscopy is an endoscopic method that provides in vivo real-time imaging of the mucosa at a cellular level, elucidating mucosal changes that are undetectable by white light endoscopy. This paper systematically reviews current indications and perspectives of confocal laser endomicroscopy for inflammatory bowel disease. Available literature was searched systematically for studies applying confocal laser endomicroscopy in Crohn's disease or ulcerative colitis. Relevant literature was reviewed and only studies reporting original clinical data were included. Next, eligible studies were analysed with respect to several parameters, such as technique and clinical aim and definitions of outcomes. Confocal laser endomicroscopy has been used for a wide range of purposes in inflammatory bowel disease, covering assessment of inflammatory severity, prediction of therapeutic response and relapse and adenoma surveillance in patients with ulcerative colitis. Methods for measurement of the histological changes ranged from subjective grading to objective quantification analysed by computer-aided models. The studies derived their conclusions from assessment of histological features such as colonic crypts, epithelial gaps and epithelial leakiness to fluorescein. Confocal laser endomicroscopy remains an experimental but emerging tool for assessment of inflammatory bowel disease. It is the only method that enables in vivo functional assessment of intestinal barrier function. There is great heterogeneity in the literature and no single approach has been validated and reproduced to the level of general acceptance. Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  3. 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.

  4. 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.

  5. Adaptive optics loop for en-face optical coherence tomography and laser scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

    Tuohy, Simon; Bradu, Adrian; Harms, Fabrice; Chateau, Nicolas; Podoleanu, Adrian G.

    2008-09-01

    The capabilities of a novel deformable mirror and wave-front sensor combination to correct aberrations in microscopy are analyzed. The deformable mirror, (Mirao52-D, Imagine Eyes) is incorporated with a Shack-Hartmann sensor (HASO, Imagine Optic) within a complex imaging system able to produce simultaneous en-face Optical Coherence Tomography and Laser Scanning Confocal Microscopy images as well as B-scan OCT images. A large angle imaging along one of the scanning directions is demonstrated using the AO loop to correct for the interface optics aberration. The image is split into three panels, and each panel is imaged using its own set of corrections. The three images are subsequently collaged into a final image and preliminary promising results are presented.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

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

    PubMed

    Mowla, Alireza; Taimre, Thomas; Lim, Yah Leng; Bertling, Karl; Wilson, Stephen J; Prow, Tarl W; Soyer, H Peter; Rakić, Aleksandar D

    2016-09-01

    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.

  12. 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.

  13. 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.

  14. Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

    PubMed Central

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

    2013-01-01

    Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 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.7 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.11 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.12 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.10 Recent availability of a < 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence

  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. 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

  17. 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...

  18. 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...

  19. 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.

  20. Laser skin rejuvenation: epidermal changes and collagen remodeling evaluated by in vivo confocal microscopy.

    PubMed

    Longo, Caterina; Galimberti, Michela; De Pace, Barbara; Pellacani, Giovanni; Bencini, Pier Luca

    2013-05-01

    Fractionated carbon dioxide (CO2) laser resurfacing is an effective treatment of skin aging. Several studies investigated the morphologic changes due to this laser treatment by using skin biopsies or animal model. Recently, reflectance confocal microscopy (RCM) has emerged as a new tool that can "optically" scan the skin in vivo with a nearly histologic resolution and in a totally noninvasive modality. Our study aims to analyze the skin changes following the ablative fractional CO2 laser sessions by using RCM. Ten patients were subjected to ablative fractional CO2 laser sessions for skin aging. Confocal microscopic images were acquired at baseline (w0), 3 weeks (w3), 6 weeks (w6), and 12 weeks (w12) after laser session. Previously identified confocal parameters were used to assess the skin aging at baseline and after treatment. At w3, the epidermis showed a complete disappearance of the mottled pigmentation upon RCM along with the presence of few Langherans' cells. The collagen type as seen upon RCM observed at baseline was replaced by a newly formed collagen type of long, bright and straight fibers (collagen remodeling). These fibers were parallel arranged and observed throughout the entire RCM mosaic. At w6 and w12 the confocal aspects of the skin was unchanged compared to w3. RCM confirmed the presence of an intense collagen remodeling following laser resurfacing. In line with previous studies, this collagen showed a peculiar arrangement and distribution. The collagen remodeling was still present after 3 months and confirms the long-term effect of the treatment. This is the first time that the skin can be analyzed in vivo at patient's bedside. In the near future, RCM can be an essential adjunct for Clinicians to measure the effects of laser treatment and possibly to gain new insights into the development of side effects.

  1. Combined confocal Raman and quantitative phase microscopy system for biomedical diagnosis.

    PubMed

    Kang, Jeon Woong; Lue, Niyom; Kong, Chae-Ryon; Barman, Ishan; Dingari, Narahara C; Goldfless, Stephen J; Niles, Jacquin C; Dasari, Ramachandra R; Feld, Michael S

    2011-09-01

    We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exogenous staining. For validation and characterization, we have used this multi-modal system to investigate healthy and diseased blood samples. We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution. Further, in malaria infected RBCs, we successfully image the distribution of hemozoin (malaria pigment) inside the cell. Our observations lead us to propose morphological screening by QPM and subsequent chemical imaging by Raman for investigating blood disorders. This new approach allows monitoring cell development and cell-drug interactions with minimal perturbation of the biological system of interest.

  2. Confocal scanning laser ophthalmoscopy improvement by use of Mueller-matrix polarimetry.

    PubMed

    Bueno, Juan M; Campbell, Melanie C W

    2002-05-15

    A new technique for improving the signal-to-noise ratio and the contrast in images recorded with a confocal scanning laser system is presented. The method is based on the incorporation of a polarimeter into the setup. After the spatially resolved Mueller matrix of a sample was calculated, images for incident light with different states of polarization were reconstructed, and both the best and the worst images were computed. In both the microscope and the opthalmoscope modes, the best images are better than the originals. In contrast, the worst images are poorer. This technique may be useful in different fields such as confocal microscopy and retinal imaging.

  3. Confocal scanning laser ophthalmoscopy improvement by use of Mueller-matrix polarimetry

    NASA Astrophysics Data System (ADS)

    Bueno, Juan M.; Campbell, Melanie C. W.

    2002-05-01

    A new technique for improving the signal-to-noise ratio and the contrast in images recorded with a confocal scanning laser system is presented. The method is based on the incorporation of a polarimeter into the setup. After the spatially resolved Mueller matrix of a sample was calculated, images for incident light with different states of polarization were reconstructed, and both the best and the worst images were computed. In both the microscope and the opthalmoscope modes, the best images are better than the originals. In contrast, the worst images are poorer. This technique may be useful in different fields such as confocal microscopy and retinal imaging.

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

    NASA Astrophysics Data System (ADS)

    Sun, Hui; Kurtz, Ronald; Juhasz, Tibor

    2012-08-01

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

  5. 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.

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

    PubMed

    De Palma, Giovanni D

    2009-12-14

    Recent technological advances in miniaturization have allowed for a confocal scanning microscope to be integrated into a conventional flexible endoscope, or into trans-endoscopic probes, a technique now known as confocal endomicroscopy or confocal laser endomicroscopy. This newly-developed technology has enabled endoscopists to collect real-time in vivo histological images or "virtual biopsies" of the gastrointestinal mucosa during endoscopy, and has stimulated significant interest in the application of this technique in clinical gastroenterology. This review aims to evaluate the current data on the technical aspects and the utility of this new technology in clinical gastroenterology and its potential impact in the future, particularly in the screening or surveillance of gastrointestinal neoplasia.

  7. 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.

  8. 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.

  9. Correlated Biofilm Imaging, Transport and Metabolism Measurements via Combined Nuclear Magnetic Resonance and Confocal Microscopy

    SciTech Connect

    Mclean, Jeffrey S.; Ona, Ositadinma; Majors, Paul D.

    2008-02-18

    Bacterial biofilms are complex, three-dimensional, communities that are found nearly everywhere in nature1 and are being recognized as the cause of treatment-resistant infections1 2. Advanced methods are required to characterize their collective and spatial patterns of metabolism however most techniques are invasive or destructive. Here we describe the use of a combined confocal laser scanning microscopy (CLSM) and nuclear magnetic resonance (NMR) microscopy system to monitor structure, mass transport, and metabolism in active biofilms. Non-invasive NMR methods provide macroscopic structure along with spatially-resolved metabolite profiles and diffusion measurements. CLSM enables monitoring of cells by fluorescent protein reporters to investigate biofilm structure and gene expression concurrently. A planar sample chamber design facilitates depth-resolved measurements on 140 nL sample volumes under laminar flow conditions. The techniques and approaches described here are applicable to environmental and medically relevant microbial communities, thus providing key metabolic information for promoting beneficial biofilms and treating associated diseases.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Optical Design of Adaptive Optics Confocal Scanning Laser Ophthalmoscope with Two Deformable Mirrors.

    PubMed

    Yang, Jinsheng; Wang, Yuanyuan; Rao, Xuejun; Wei, Ling; Li, Xiqi; He, Yi

    2017-01-01

    We describe the optical design of a confocal scanning laser ophthalmoscope with two deformable mirrors. Spherical mirrors are used for pupil relay. Defocus aberration of the human eye is corrected by a Badal focusing structure and astigmatism aberration is corrected by a deformable mirror. The main optical system achieves a diffraction-limited performance through the entire scanning field (6 mm pupil, 3 degrees on pupil plane). The performance of the optical system, with correction of defocus and astigmatism, is also evaluated.

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

    SciTech Connect

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

    2011-04-30

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

  20. [In vivo imaging of the conjunctival epithelium using confocal laser scanning microscopy].

    PubMed

    Rath, R; Stave, J; Guthoff, R; Giebel, J; Tost, F

    2006-05-01

    In various ocular diseases, cytomorphological findings of the ocular surface are an essential component of clinical diagnostics. When evaluating the conjunctival epithelium, minimally invasive acquisition of biomaterial is necessary for lab and technical processing and in vitro histological examination. To examine corneal structures in vivo, confocal laser scanning microscopy is a successful standard method. Our aim was to employ in vivo confocal laser scanning microscopy also for examining the conjunctival epithelium. Results were analyzed and compared with cytomorphological findings of impression cytology. Accordingly, the basic features of conjunctival in vivo examination using RLSM were described and defined. In vivo images were analyzed and compared with impression cytological slide preparations (n=110) of 23 healthy test persons. Examination was standardized. Finally, the confocal laser scan images were compared to the impression cytological patterns. Due to the distribution of reflectors (pixel brightness), diagnostic analysis of important morphological structures (cell nucleus, cytoplasm, nucleus/plasma relation) of the conjunctiva is possible. Secretory cells of the epithelium (goblet cells) can be easily recognized by their size. Highly reflective pixels depict cell walls or wide intercellular spaces with high contrast. The in vivo investigation of important anatomical and morphological structures of the conjunctival epithelium is possible using RLSM. The distribution pattern of goblet cell pixel brightness may correlate with various secretion contents or suggest distinct, recognizable, functional conditions (hypo- or hypersecretion).

  1. 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.

  2. 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

  3. 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.

  4. Comparison of calcium imaging in dorsal root ganglion neurons by using laser scanning confocal and two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Yang, Hongqin; Chen, Jiangxu; Shen, Xiuqiu; Zheng, Liqin; Wang, Yuhua; Xie, Shusen

    2012-03-01

    As one of the most important second messengers, calcium in nerve cells plays a critical role in neuronal processes, including excitability, neurotransmitter release, synaptic plasticity. Modulation of the calcium concentration is an important means of regulating diverse neuronal functions. To evaluate the role of calcium, quantitative measurement of cytosolic free calcium concentrations is necessary. There are several optical techniques that are available for measurement of calcium in live cells. Laser scanning confocal microscopy and two-photon microscopy are two prevalent techniques for their advantage in spatial resolution. In this paper, calcium in dorsal root ganglion neurons was imaged by laser scanning confocal microscopy and two-photon microscopy with Fluo-3, a calcium specific fluorescence probe. Both of spatial resolution and photobleaching, two common limitations of optical image modality, were compared between laser scanning confocal microscopy and two-photon microscopy, respectively. Three dimension images showed that laser scanning confocal microscopy and two-photon microscopy had not only similar lateral resolution but also parallel vertical resolution. However, Laser scanning confocal microscopy had an advantage over the two-photon microcopy in photobleaching. These results indicated that laser scanning confocal microscopy was more suitable than two-photon microscopy to be applied in imaging calcium in dorsal root ganglion neurons with Fluo-3.

  5. 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.

  6. Three-dimensional imaging of porous media using confocal laser scanning microscopy.

    PubMed

    Shah, S M; Crawshaw, J P; Boek, E S

    2017-02-01

    In the last decade, imaging techniques capable of reconstructing three-dimensional (3-D) pore-scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO2 storage potential. CLSM has a unique capability of producing 3-D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z-dimension) that can be imaged in porous materials. In this study, we introduce a 'grind and slice' technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3-D confocal volumetric data into pores and grains. Finally, we use the resulting 3-D pore-scale binarized confocal data obtained to quantitatively determine petrophysical pore-scale properties such as total porosity, macro- and microporosity and single-phase permeability using lattice Boltzmann (LB) simulations, validated by experiments. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  7. 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.

  8. 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.

  9. 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.

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Large-aperture laser differential confocal ultra-long focal length measurement and its system.

    PubMed

    Zhao, Weiqian; Li, Zhigang; Qiu, Lirong; Ren, Huan; Shao, Rongjun

    2015-06-29

    A new laser differential confocal ultra-long focal length measurement (LDCFM) method is proposed with the capability to self-calibrate the reference lens (RL) focal length and the axial space between the test lens and RL. Using the property that the focus of laser differential confocal ultra-long focal length measurement system (LDCFS) precisely corresponds to the null point of the differential confocal axial intensity curve, the proposed LDCFM measures the RL focal length f(R)' by precisely identifying the positions of the focus and last surface of RL, measures the axial space d(0) between RL and test ultra-long focal length lens (UFL) by identifying the last surface of RL and the vertex of UFL last surface, and measures the variation l in focus position of LDCFS with and without test UFL, and then calculates the UFL focal length f(T)' by the above measured f(R)', d(0) and l. In addition, a LDCFS based on the proposed method is developed for a large aperture lens. The experimental results indicate that the relative uncertainty is less than 0.01% for the test UFL, which has an aperture of 610 mm and focal length of 31,000 mm. LDCFM provides a novel approach for the high-precision focal-length measurement of large-aperture UFL.

  12. Multimodal backside imaging of a microcontroller using confocal laser scanning and optical-beam-induced current imaging

    NASA Astrophysics Data System (ADS)

    Finkeldey, Markus; Göring, Lena; Schellenberg, Falk; Brenner, Carsten; Gerhardt, Nils C.; Hofmann, Martin

    2017-02-01

    Microscopy imaging with a single technology is usually restricted to a single contrast mechanism. Multimodal imaging is a promising technique to improve the structural information that could be obtained about a device under test (DUT). Due to the different contrast mechanisms of laser scanning microscopy (LSM), confocal laser scanning microscopy (CLSM) and optical beam induced current microscopy (OBICM), a combination could improve the detection of structures in integrated circuits (ICs) and helps to reveal their layout. While OBIC imaging is sensitive to the changes between differently doped areas and to semiconductor-metal transitions, CLSM imaging is mostly sensitive to changes in absorption and reflection. In this work we present the implementation of OBIC imaging into a CLSM. We show first results using industry standard Atmel microcontrollers (MCUs) with a feature size of about 250nm as DUTs. Analyzing these types of microcontrollers helps to improve in the field of side-channel attacks to find hardware Trojans, possible spots for laser fault attacks and for reverse engineering. For the experimental results the DUT is placed on a custom circuit board that allows us to measure the current while imaging it in our in-house built stage scanning microscope using a near infrared (NIR) laser diode as light source. The DUT is thinned and polished, allowing backside imaging through the Si-substrate. We demonstrate the possibilities using this optical setup by evaluating OBIC, LSM and CLSM images above and below the threshold of the laser source.

  13. Confocal scanning laser microscopy and its application in biomedical health sciences

    NASA Astrophysics Data System (ADS)

    Vardaxis, Nicholas J.

    1999-07-01

    The confocal scanning laser microscope (CSLM) is an exciting new tool in microscopy. It offers improved rejection of out- of-focus `noise' and greater resolution than conventional imaging. By integrating a computer into the system and generating digital image data files, a rapid way of storing, processing, and analyzing images is available to the user. The production of 3D reconstruction representations is easy and effective. The technique of optical sectioning and confocal optics has revolutionized epifluorescence microscopy, the CSLM providing a highly desirable link between conventional light microscopy and electron microscopy. The use of the CSLM in biomedical health sciences is considered in this paper and the functional basics of the instrument are discussed with reference to several important applications in research and diagnostic work, with illustrations from the numerous and continually increasing publications in the area. It is veritably a `solution in search of problems' as this short review demonstrates.

  14. Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy

    PubMed Central

    McLean, Jeffrey S; Ona, Ositadinma N; Majors, Paul D

    2015-01-01

    Bacterial biofilms are complex, three-dimensional communities found nearly everywhere in nature and are also associated with many human diseases. Detailed metabolic information is critical to understand and exploit beneficial biofilms as well as combat antibiotic-resistant, disease-associated forms. However, most current techniques used to measure temporal and spatial metabolite profiles in these delicate structures are invasive or destructive. Here, we describe imaging, transport and metabolite measurement methods and their correlation for live, non-invasive monitoring of biofilm processes. This novel combination of measurements is enabled by the use of an integrated nuclear magnetic resonance (NMR) and confocal laser scanning microscope (CLSM). NMR methods provide macroscopic structure, metabolic pathway and rate data, spatially resolved metabolite concentrations and water diffusion profiles within the biofilm. In particular, current depth-resolved spectroscopy methods are applied to detect metabolites in 140–190 nl volumes within biofilms of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1 and the oral bacterium implicated in caries disease, Streptococcus mutans strain UA159. The perfused sample chamber also contains a transparent optical window allowing for the collection of complementary fluorescence information using a unique, in-magnet CLSM. In this example, the entire three-dimensional biofilm structure was imaged using magnetic resonance imaging. This was then correlated to a fluorescent CLSM image by employing a green fluorescent protein reporter construct of S. oneidensis. Non-invasive techniques such as described here, which enable measurements of dynamic metabolic processes, especially in a depth-resolved fashion, are expected to advance our understanding of processes occurring within biofilm communities. PMID:18253132

  15. 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

  16. Added value of narrow band imaging and confocal laser endomicroscopy in detecting Barrett's esophagus neoplasia.

    PubMed

    Jayasekera, C; Taylor, A C F; Desmond, P V; Macrae, F; Williams, R

    2012-12-01

    Advances in endoscopic imaging techniques have enabled more accurate identification of subtle mucosal abnormalities. The aim of the study was to assess the accuracy of predicting high grade dysplasia (HGD) and intramucosal cancer (IMC) in mucosa predicted as being nondysplastic vs. dysplastic by high definition white light endoscopy (HD-WLE), narrow band imaging (NBI), and confocal laser endomicroscopy (CLE). A cross-sectional study was performed in a tertiary referral setting between February 2010 and September 2011. A total of 50 consecutive patients who were referred to St Vincent's Hospital for management of dysplastic Barrett's esophagus were included. A prediction of likely histology was made for each mucosal point (four-quadrant every 1 cm and any visible mucosal abnormality), first with HD-WLE, followed by NBI, and finally CLE. Biopsies were taken at all of these points. A total of 1190 individual biopsy points were assessed. At histology, 39 biopsy points were found to harbor HGD and 52 biopsy points harbored IMC. For the detection of HGD/IMC the sensitivity, specificity, and accuracy were: HD - WLE, 79.1 %, 83.1 %, and 82.8 %; NBI, 89.0 %, 80.1 %, and 81.4 %; and CLE, 75.7 %, 80.0 %, and 79.9 %, respectively. All mucosal points with IMC and all patients with HGD were detected by targeted biopsies guided by HD-WLE and NBI without the need for random Seattle protocol biopsies. HD-WLE in combination with NBI is highly accurate in the detection of HGD/IMC. Performing targeted biopsies in the surveillance of Barrett's esophagus is possible in expert centers. © Georg Thieme Verlag KG Stuttgart · New York.

  17. A novel approach to the diagnosis of pancreatic serous cystadenoma: needle-based confocal laser endomicroscopy.

    PubMed

    Napoléon, Bertrand; Lemaistre, Anne-Isabelle; Pujol, Bertrand; Caillol, Fabrice; Lucidarme, Damien; Bourdariat, Raphaël; Morellon-Mialhe, Blandine; Fumex, Fabien; Lefort, Christine; Lepilliez, Vincent; Palazzo, Laurent; Monges, Geneviève; Filoche, Bernard; Giovannini, Marc

    2015-01-01

    The differential diagnosis of solitary pancreatic cystic lesions is frequently difficult. Needle-based confocal laser endomicroscopy (nCLE) performed during endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is a new technology enabling real-time imaging of the internal structure of such cysts. The aim of this pilot study was to identify and validate new diagnostic criteria on nCLE for pancreatic cystic lesions. A total of 31 patients with a solitary pancreatic cystic lesion of unknown diagnosis were prospectively included at three centers. EUS-FNA was combined with nCLE. The final diagnosis was based on either a stringent gold standard (surgical specimen and/or positive cytopathology) or a committee consensus. Six nonblinded investigators reviewed nCLE sequences from patients with the most stringent final diagnosis, and identified a single feature that was only present in serous cystadenoma (SCA). The findings were correlated with the pathology of archived specimens. After a training session, four blinded independent observers reviewed a separate independent video set, and the yield and interobserver agreement for the criterion were assessed. A superficial vascular network pattern visualized on nCLE was identified as the criterion. It corresponded on pathological specimen to a dense and subepithelial capillary vascularization only seen in SCA. 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. Interobserver agreement was substantial (κ = 0.77). This new nCLE criterion seems highly specific for the diagnosis of SCA. The visualization of this criterion could have a direct impact on the management of patients by avoiding unnecessary surgery or follow-up.Clinicaltrials.gov NCT01563133. © Georg Thieme Verlag KG Stuttgart · New York.

  18. Endoscopic ultrasound-guided needle-based confocal laser endomicroscopy in solid pancreatic masses.

    PubMed

    Giovannini, Marc; Caillol, Fabrice; Monges, Geneviève; Poizat, Flora; Lemaistre, Anne-Isabelle; Pujol, Bertrand; Lucidarme, Damien; Palazzo, Laurent; Napoléon, Bertrand

    2016-10-01

    The differential diagnosis of solid pancreatic masses by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is currently suboptimal in centers that are not equipped with rapid on-site evaluation. Needle-based confocal laser endomicroscopy (nCLE) enables real-time in vivo microscopic imaging during endoscopy. This study aimed to describe nCLE interpretation criteria for the characterization of pancreatic masses, with histopathological correlation, and to perform the first validation of these criteria. A total of 40 patients were evaluated by EUS-FNA combined with nCLE for the diagnosis of pancreatic masses. Final diagnosis was based on EUS-FNA histology and follow-up at 1 year. Five unblinded examiners defined nCLE criteria for adenocarcinoma, chronic pancreatitis, and neuroendocrine tumor (NET) using a set of video sequences from 14 patients with confirmed pathology (Step 1). These criteria were retrospectively validated by four independent, blinded examiners using sequences from 32 patients (Step 2). nCLE criteria were described for adenocarcinoma (dark cell aggregates, irregular vessels with leakages of fluorescein), chronic pancreatitis (residual regular glandular pancreatic structures), and NET (black cell aggregates surrounded by vessels and fibrotic areas). These criteria correlated with the histological features of the corresponding lesions. In the validation review, a conclusive nCLE result was obtained in 75 % of cases (96 % correct). Statistical evaluation provided promising results, with high specificity, and negative and positive predictive values for all types of pancreatic masses. Considering the low negative predictive value of EUS-FNA, nCLE could help to rule out malignancy after a previous inconclusive EUS-FNA. Larger studies are required to confirm these findings and to establish the role of nCLE in the diagnosis of pancreatic masses. ClinicalTrials.gov (NCT01563133). © Georg Thieme Verlag KG Stuttgart · New York.

  19. 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.

  20. Monitoring Pancreatic Carcinogenesis by the Molecular Imaging of Cathepsin E In Vivo Using Confocal Laser Endomicroscopy

    PubMed Central

    Cui, Lei; Wang, Biyuan; Cui, Wenli; Li, Minghua; Cheng, Yingsheng

    2014-01-01

    The monitoring of pancreatic ductal adenocarcinoma (PDAC) in high-risk populations is essential. Cathepsin E (CTSE) is specifically and highly expressed in PDAC and pancreatic intraepithelial neoplasias (PanINs), and its expression gradually increases along with disease progression. In this study, we first established an in situ 7,12-dimethyl-1,2-benzanthracene (DMBA)-induced rat model for PanINs and PDAC and then confirmed that tumorigenesis properties in this model were consistent with those of human PDAC in that CTSE expression gradually increased with tumor development using histology and immunohistochemistry. Then, using in vivo imaging of heterotopically implanted tumors generated from CTSE- overexpressing cells (PANC-1-CTSE) in nude mice and in vitro imaging of PanINs and PDAC in DMBA-induced rats, the specificity of the synthesized CTSE-activatable probe was verified. Quantitative determination identified that the fluorescence signal ratio of pancreatic tumor to normal pancreas gradually increased in association with progressive pathological grades, with the exception of no significant difference between PanIN-II and PanIN-III grades. Finally, we monitored pancreatic carcinogenesis in vivo using confocal laser endomicroscopy (CLE) in combination with the CTSE-activatable probe. A prospective double-blind control study was performed to evaluate the accuracy of this method in diagnosing PDAC and PanINs of all grades (>82.7%). This allowed us to establish effective diagnostic criteria for CLE in PDAC and PanINs to facilitate the monitoring of PDAC in high-risk populations. PMID:25184278

  1. 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.

  2. Laser differential confocal interference multi-parameter comprehensive measurement method and its system for spherical lens.

    PubMed

    Zhao, Weiqian; Qiu, Lirong; Xiao, Yang; Yang, Jiamiao

    2016-10-03

    Different measurement methods have been used to achieve different parameter measurements of a spherical lens, and multi-parameter measurements of a spherical lens have low measurement accuracy and efficiency. This paper proposes a new, laser differential confocal interference multi-parameter measurement (DCIMPM) method for spherical lens. Based on this proposed DCIMPM, a multi-parameter comprehensive measurement system is developed for spherical lens, which uses the laser differential confocal parameter measurement technique to measure the radius of curvature, thickness, and refractivity of spherical lens, and uses the laser interference measurement technique to measure the surface figure of a spherical lens. Therefore, the DCIMPM system, for the first time, achieves high-accuracy multi-parameter comprehensive measurements of a spherical lens on a single instrument. Experiments indicate that the developed DCIMPM system can achieve a measurement accuracy of 5 × 10-6 for the lens radius, 2.5 × 10-4 for the lens thickness, 2.2 × 10-4 for the lens refractivity, and a peak to valley of λ/20 for the surface figure of the lens. The proposed DCIMPM principle and developed system provide a new approach to achieve multi-parameter comprehensive measurements for spherical lens.

  3. 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.

  4. 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.

  5. In vivo identification of pancreatic cystic neoplasms with needle-based confocal laser endomicroscopy.

    PubMed

    Tsujino, Takeshi; Yan-Lin Huang, Jason; Nakai, Yosuke; Samarasena, Jason B; Lee, John G; Chang, Kenneth J

    2015-08-01

    Pancreatic cystic lesions (PCLs) are increasingly identified with the widespread use of imaging modalities. The precise diagnosis of PCLs remains a challenge despite the use of CT, MRI, and EUS-FNA. Confocal laser endomicroscopy (CLE) is a new endoscopic imaging modality that provides real-time, very high magnification images. A smaller CLE probe, which can be passed through a 19-gauge FNA needle, is now available. Needle-based CLE during EUS has recently been examined to evaluate PLCs, and the specific criteria of nCLE for the diagnosis of PLCs have been proposed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 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.

  7. 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

  8. 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.

  9. [Confocal laser endomicroscopy principles and performing algorithm in gastric mucosa examination].

    PubMed

    Pirogov, S S; Sokolov, V V; Karpova, E S; Pavlov, P V; Volchenko, N N; Kaprin, A D

    2014-01-01

    Accuracy of endoscopic examination in early gastric cancer and precancerous conditions diagnostics for many years depended only on quality of biopsy. That's why, risk of overlooking gastric focal carcinoma, particularly-- multiple, was relatively high. Last couple of years new endoscopic method--confocal laser endomicroscopy (CLE) was released for commercial use. This approach provides real-time information about morphology of gastric mucosa during endoscopic examination. CLE is a variation of confocal microscopy--morphologic technique, providing examination of thick specimens or live tissue. CLE system is a single-channel fluorescence microscope, used in endoscopy, where confocal probe incorporated into endoscope or mounted into accessory channel. For proper results of CLE intravenous administration of fluorescence agent is needed. In our study in P.A. Herzen Moscow Cancer Research Institute we have used 10% fluorescein sodium, due to acriflavine use is prohibited in Russian Federation. In 157 patients with suspected early gastric cancer mean time of CLE was 24 ± 3.5 min. In all cases descriptive images were acquired. Mean amount of endomicrosocpic images in one patient was as high as 162 ± 8.3.

  10. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  12. Laser differential confocal ultra-large radius measurement for convex spherical surface.

    PubMed

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

    2016-08-22

    A new laser differential confocal ultra-large radius measurement (LDCRM) method is proposed for high-precision measurement of ultra-large radii. Based on the property that the zero point of a differential confocal axial intensity curve precisely corresponds to the focus points of focusing beam, LDCRM measures the vertex positions of the test lens and the last optical surface of objective lens to obtain position difference L1, and then measures the vertex positions of the reflector and the last optical surface of objective lens to obtain the position difference L2, finally uses the measured L1 and L2 to calculate the radius of test lens. This method does not require the identification of confocal position. Preliminary experimental results and theoretical analyses indicate that the relative uncertainty is 0.03% for a convex spherical lens with a radius of approximately 20 m. LDCRM provides a novel approach for high-precision ultra-large radius measurement.

  13. 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.

  14. Fiber-Optic Confocal Laser Endomicroscopy of Small Renal Masses: Toward Real-Time Optical Diagnostic Biopsy.

    PubMed

    Su, Li-Ming; Kuo, Jennifer; Allan, Robert W; Liao, Joseph C; Ritari, Kellie L; Tomeny, Patrick E; Carter, Christopher M

    2016-02-01

    The incidental detection of small renal masses is increasing. However, not all require aggressive treatments as up to 20% are benign and the majority of malignant tumors harbor indolent features. Improved preoperative diagnostics are needed to differentiate tumors requiring aggressive treatment from those more suitable for surveillance. We evaluated and compared confocal laser endomicroscopy with standard histopathology in ex vivo human kidney tumors as proof of principle towards diagnostic optical biopsy. Patients with a solitary small renal mass scheduled for partial or radical nephrectomy were enrolled in study. Two kidneys were infused with fluorescein via intraoperative intravenous injection and 18 tumors were bathed ex vivo in dilute fluorescein prior to confocal imaging. A 2.6 mm confocal laser endomicroscopy probe was used to image tumors and surrounding parenchyma from external and en face surfaces after specimen bisection. Confocal laser endomicroscopy images were compared to standard hematoxylin and eosin analysis of corresponding areas. Ex vivo confocal laser endomicroscopy imaging revealed normal renal structures that correlated well with histology findings. Tumor tissue was readily distinguishable from normal parenchyma, demonstrating features unique to benign and malignant tumor subtypes. Topical fluorescein administration provided more consistent confocal laser endomicroscopy imaging than the intravenous route. Additionally, en face tumor imaging was superior to external imaging. We report what is to our knowledge the first feasibility study using confocal laser endomicroscopy to evaluate small renal masses ex vivo and provide a preliminary atlas of images from various renal neoplasms with corresponding histology. These findings serve as an initial and promising step toward real-time diagnostic optical biopsy of small renal masses. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights

  15. 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%.

  16. 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

  17. 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.

  18. Lowest-order axial and ring mode lasing in confocal quasi-stadium laser diodes.

    PubMed

    Fukushima, Takehiro; Sunada, Satoshi; Harayama, Takahisa; Sakaguchi, Koichiro; Tokuda, Yasunori

    2012-05-10

    We investigated the lasing modes of quasi-stadium laser diodes that have confocal cavity geometries, with stripe electrode contacts formed either along the cavity axis or a diamond-shaped trajectory. It was clearly demonstrated that by using narrow electrode contact patterns of 2 μm width, the lowest-order axial and ring modes were excited selectively. On the other hand, the second-lowest-order axial and ring modes were excited by using broad electrode patterns of 14 μm width. Experimentally obtained far-field patterns for lasers with broad and narrow electrode contact patterns agree very well with the simulation results obtained using an extended Fox-Li mode calculation method.

  19. Emerging enhanced imaging technologies of the esophagus: spectroscopy, confocal laser endomicroscopy, and optical coherence tomography.

    PubMed

    Robles, Lourdes Y; Singh, Satish; Fisichella, Piero Marco

    2015-05-15

    Despite advances in diagnoses and therapy, esophageal adenocarcinoma remains a highly lethal neoplasm. Hence, a great interest has been placed in detecting early lesions and in the detection of Barrett esophagus (BE). Advanced imaging technologies of the esophagus have then been developed with the aim of improving biopsy sensitivity and detection of preplastic and neoplastic cells. The purpose of this article was to review emerging imaging technologies for esophageal pathology, spectroscopy, confocal laser endomicroscopy (CLE), and optical coherence tomography (OCT). We conducted a PubMed search using the search string "esophagus or esophageal or oesophageal or oesophagus" and "Barrett or esophageal neoplasm" and "spectroscopy or optical spectroscopy" and "confocal laser endomicroscopy" and "confocal microscopy" and "optical coherence tomography." The first and senior author separately reviewed all articles. Our search identified: 19 in vivo studies with spectroscopy that accounted for 1021 patients and 4 ex vivo studies; 14 clinical CLE in vivo studies that accounted for 941 patients and 1 ex vivo study with 13 patients; and 17 clinical OCT in vivo studies that accounted for 773 patients and 2 ex vivo studies. Human studies using spectroscopy had a very high sensitivity and specificity for the detection of BE. CLE showed a high interobserver agreement in diagnosing esophageal pathology and an accuracy of predicting neoplasia. We also found several clinical studies that reported excellent diagnostic sensitivity and specificity for the detection of BE using OCT. Advanced imaging technology for the detection of esophageal lesions is a promising field that aims to improve the detection of early esophageal lesions. Although advancing imaging techniques improve diagnostic sensitivities and specificities, their integration into diagnostic protocols has yet to be perfected. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. 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

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

    PubMed

    Ravi, S V; Nageswar, Rao; Swapna, Honwad; Sreekant, Puthalath; Ranjith, Madhavan; Mahidhar, Surabhi

    2014-03-01

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

  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. 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.

  4. Advances in confocal laser endomicroscopy for the diagnosis of gastrointestinal diseases.

    PubMed

    Goetz, Martin; Kiesslich, Ralf

    2009-09-01

    Confocal laser endomicroscopy (CLE) is a novel technique enabling in vivo microscopy of the human gastrointestinal mucosa. Cellular details even below the tissue surface can be visualized at high resolution during ongoing endoscopy. This review summarizes the current clinical data on the use of CLE in different disease states and discusses a perspective for future clinical and scientific application of CLE. Review on published literature and meeting abstracts. Confocal laser endomicroscopy covers a growing field of indications in both upper and lower gastrointestinal endoscopy and beyond. It has been shown to reliably predict the presence of neoplastic lesions and inflammatory changes of the gastrointestinal mucosa during endoscopy. With CLE, 'smart' biopsies can be targeted to regions with microscopic alterations rather than having to rely on blind, untargeted sampling. This results in a reduction in the number of biopsies and in an increase in their diagnostic yield at the same time. Dynamic imaging of microscopic events in their natural environment and molecular imaging by CLE will open a door for an advanced understanding of tissue function and microarchitecture in vivo.

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  6. 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.

  7. 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.

  8. Osteoclast Responses to Lipopolysaccharide, Parathyroid Hormone and Bisphosphonates in Neonatal Murine Calvaria Analyzed by Laser Scanning Confocal Microscopy

    PubMed Central

    Suzuki, Keiko; Takeyama, Sadaaki; Kikuchi, Takashi; Yamada, Shoji; Sodek, Jaro; Shinoda, Hisashi

    2005-01-01

    Because the development and activity of osteoclasts in bone remodeling is critically dependent on cell-cell and cell-matrix interactions, we used laser confocal microscopy to study the response of osteoclasts to lipopolysaccharide (LPS; 10 μg/ml), parathyroid hormone (PTH; 10−8 M), and bisphosphonates (BPs; 1–25 μM clodronate or 0.1–2.5 μM risedronate) in cultured neonatal calvaria. Following treatment with LPS or PTH (<48 hr), osteopontin (OPN) and the αvβ3 integrin were found colocalized with the actin ring in the sealing zone of actively resorbing osteoclasts. In contrast, non-resorbing osteoclasts in BP-treated cultures showed morphological abnormalities, including retraction of pseudopods and vacuolization of cytoplasm. In the combined presence of LPS and BP, bone-resorbing osteoclasts were smaller and the sealing zone diffuse, reflecting reduced actin, OPN, and β3 integrin staining. Depth analyses of calvaria showed that the area of resorbed bone was filled with proliferating osteoblastic cells that stained for alkaline phosphatase, collagen type I, and bone sialoprotein, regardless of the presence of BPs. These studies show that confocal microscopy of neonatal calvaria in culture can be used to assess the cytological relationships between osteoclasts and osteoblastic cells in response to agents that regulate bone remodeling in situ, avoiding systemic effects that can compromise in vivo studies and artifacts associated with studies of isolated osteoclasts. PMID:16087705

  9. 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

  10. Reflectance confocal microscopy-guided laser ablation of basal cell carcinomas: initial in vivo results

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

    Laser ablation offers a procedure for precise, fast and minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs). However, the lack of histopathological confirmation has been a limitation toward widespread use in the clinic. A reflectance confocal microscopy (RCM) imaging-guided laser ablation approach offers cellular-level histopathology-like feedback directly on the patient, which may guide and help improve the efficacy of this procedure. We performed an initial study on 44 BCCs on 21 patients in vivo (based in an ex vivo bench-top study reported in our earlier papers), using a pulsed erbium: ytterbium aluminum garnet laser and a contrast agent (aluminum chloride). Initial 10 lesions, the RCM imaging-guided detection of either presence of residual tumor or complete clearance was immediately confirmed with histopathology. Additionally, 34 BCCs on 15 patients were treated with RCM imaging-guided laser ablation, and the clearance of tumor is currently being monitored with follow-up imaging (i. e., no histopathology) at 3, 6 and 18 months. Thus far, the imaging resolution appears to be sufficient and consistent for monitoring efficacy in the wound, both immediately post-ablation and subsequently during recovery. The efficacy appears to be promising. However, further investigation and optimization to image over the entire wound (without missing any areas) need to be investigated.

  11. A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes

    PubMed Central

    Kang, Minchul; Day, Charles A.; Drake, Kimberly; Kenworthy, Anne K.; DiBenedetto, Emmanuele

    2009-01-01

    Abstract Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells. PMID:19720039

  12. 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

  13. Patent blue V and indocyanine green for fluorescence microimaging of human peritoneal carcinomatosis using probe-based confocal laser endomicroscopy.

    PubMed

    Abbaci, Muriel; Dartigues, Peggy; De Leeuw, Frederic; Soufan, Ranya; Fabre, Monique; Laplace-Builhé, Corinne

    2016-12-01

    Peritoneal carcinomatosis is a metastatic stage aggravating abdominal and pelvic cancer dissemination. The preoperative evaluation of lesions remains difficult today. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of tissue architecture and cellular details. This technology allows in vivo histological interpretation of tissue. The main limitation of pCLE for adoption in the clinic is the unavailability of fluorescent contrast agents. The aim of our study was to evaluate the staining performance of indocyanine green and patent blue V for histological diagnosis of pCLE images of pathological and non-pathological peritoneal tissue. We performed a correlative study with the histological gold standard on ex vivo human specimens from 25 patients operated for peritoneal carcinomatosis; 70 specimens were stained by topical application with ICG or patent blue V and then imaged with a probe-based confocal laser endomicroscope. A total of 350 pCLE images and 70 corresponding histological sections were randomly and blindly interpreted by two pathologists (PT1 and PT2). The images were first classified into two categories, tumoral versus non-tumoral, and a refined histological diagnosis was then given. All presented images were interpreted by PT1 (who received prior training on PCLE image reading) and PT2 (no training). 100 % sensitivity for PT1 and PT2 was noticed with tissues stained with ICG to differentiate tumoral and non-tumoral tissue. Global scores were always better for PT1 (major concordance between 86 and 94 %) than for PT2 (major concordance between 77 and 89 %) independently of the fluorescent dye when histological diagnosis was done on pCLE images. In conclusion, the pair ICG-pCLE offers the best combination for a non-trained pathologist for the interpretation of pCLE images from peritoneum.

  14. Dynamic Real-time Microscopy of the Urinary Tract Using Confocal Laser Endomicroscopy

    PubMed Central

    Wu, Katherine; Liu, Jen-Jane; Adams, Winifred; Sonn, Geoffrey A.; Mach, Kathleen E.; Pan, Ying; Beck, Andrew H.; Jensen, Kristin C.; Liao, Joseph C.

    2014-01-01

    OBJECTIVES To develop the diagnostic criteria for benign and neoplastic conditions of the urinary tract using probe-based confocal laser endomicroscopy (pCLE), a new technology for dynamic, in vivo imaging with micron-scale resolution. The suggested diagnostic criteria will formulate a guide for pCLE image interpretation in urology. METHODS Patients scheduled for transurethral resection of bladder tumor (TURBT) or nephrectomy were recruited. After white-light cystoscopy (WLC), fluorescein was administered as contrast. Different areas of the urinary tract were imaged with pCLE via direct contact between the confocal probe and the area of interest. Confocal images were subsequently compared with standard hematoxylin and eosin analysis. RESULTS pCLE images were collected from 66 participants, including 2 patients who underwent nephrectomy. We identified key features associated with different anatomic landmarks of the urinary tract, including the kidney, ureter, bladder, prostate, and urethra. In vivo pCLE of the bladder demonstrated distinct differences between normal mucosa and neoplastic tissue. Using mosaicing, a post hoc image-processing algorithm, individual image frames were juxtaposed to form wideangle views to better evaluate tissue microarchitecture. CONCLUSIONS In contrast to standard pathologic analysis of fixed tissue with hematoxylin and eosin, pCLE provides real time microscopy of the urinary tract to enable dynamic interrogation of benign and neoplastic tissues in vivo. The diagnostic criteria developed in this study will facilitate adaptation of pCLE for use in conjunction with WLC to expedite diagnosis of urinary tract pathology, particularly bladder cancer. PMID:21601243

  15. 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

  16. 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

  17. [In vivo study of normal human limbal and central corneas using laser confocal microscope].

    PubMed

    Rong, Bei; Yan, Xiao-ming

    2006-01-01

    To observe the microstructure and cellular populations of normal human limbal and central corneas using laser confocal microscope. The study was consisted of 28 eyes of 15 examiners (age distribution, 16-61 years). After examinations with slit lamp biomicroscopy and ophthalmoscope, the images of superior, inferior limbus and central cornea were acquired by laser confocal microscope. Corneal microstructure and cell morphology were analyzed, and cell densities were measured. Lateral (x, y axis) and axial (z axis) images of limbal and central cornea were clear and vivid. Limbal palisades of Vogt were visible at the superior and inferior limbus. The red blood cells in the vessel of limbus were visible. The arrangement of surface epitheliums was rather loose, with bright borders and dark cell bodies. The surface epithelial cell density was (812 +/- 297) cells/mm(2) at the superior limbus and (785 +/- 263) cells/mm(2) at the inferior limbus. There was no statistical difference between the two group (P > 0.05). The bright and dendriform Langerhans cells were readily visible just under the epithelial cells. The Langerhans cell density was (288 +/- 102) cells/mm(2) at the superior limbus and (254 +/- 127) cells/mm(2) at the inferior limbus. There was no statistical difference between the two group (P > 0.05). At central cornea, the surface epithelial cells were loose, the borders of the cell were clear and bright, and the cell bodies were dark. The cell density was (1098 +/- 315) cells/mm(2), there were more cells in central cornea than other area of superior and inferior limbus (P < 0.05). The arrangement of basal epithelial cells was tight. Nerve plexus at subepithelium and anterior stroma were prominent with strong brightness. Langerhans cells were occasionally visible, and cell density cannot be measured. Subepithelial nerve fibers were thin with high tortuosity and a lot of branches, and deeper ones were thick with low tortuosity and few branch. In the stroma

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

    PubMed Central

    Nikhil, Vineeta; Singh, Renuka

    2013-01-01

    Background: Sealers are used to attain an impervious seal between the core material and root canal walls. Aim: To compare the depth and percentage of sealer penetration with three different placement techniques using confocal laser scanning microscopy as the evaluative tool. Materials and Methods: Root canals of 30 single-rooted teeth were prepared to a size of F3 and AH plus sealer with Rhodamine B was applied with Ultlrasonic file (Gr-1), lentulospiral (Gr-2), and Endoactivator (Gr-3). Canals were obturated with gutta-percha. The roots were sectioned at the 3 and 6-mm levels from the apical foramen and were examined on a confocal microscope. Results: A statistical significant differences among Gr-1, Gr-2, and Gr-3 were found at the 3 and 6-mm level (P < 0.05; ANOVA-Tukey tests) for the depth and percentage of sealer penetration except for Gr-1 and Gr-2 at 3-mm level. Gr-1 showed maximum mean depth of penetration (810 μm) and maximum mean percentage of sealer penetration (64.5) while Gr-3 showed minimum mean depth of penetration (112.7 μm) and minimum mean percentage of sealer penetration (26.7). Conclusion: Depth and percentage of penetration of sealer is influenced by the type of placement technique and by the root canal level with penetration decreasing apically. PMID:23956528

  19. 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.

  20. Assessment of iontophoretic and passive ungual penetration by laser scanning confocal microscopy.

    PubMed

    Dutet, Julie; Delgado-Charro, M Begoña

    2012-12-01

    To estimate the in vitro ungual penetration depth of sodium fluorescein and nile blue chloride by laser scanning confocal microscopy. The depth, uniformity and pathways of penetration of both markers into human nail during passive and iontophoretic experiments were investigated. The penetration of sodium fluorescein into the dorsal, ventral and intermediate layers of the nail was also studied. Transversal images were used to estimate directly the relative penetration of the markers with respect to the complete thickness of the nail. "Exposed layer" images allowed estimating the depth of penetration by taking xy-plans, starting by the exposed layer, and following the z axis into the nail. The fluorescent markers penetrated 7-12% of the nail thickness. Iontophoresis increased penetration of both markers compared to passive diffusion. However, ungual penetration was not modified by the intensity of current applied. Penetration into the dorsal, ventral, and intermediate nail layers was similar. The method developed allowed inter- and intra- nail variability to be accounted for. Iontophoresis enhanced moderately the penetration of the two markers into the nail plate as compared to passive diffusion. The confocal images suggested the transcellular pathway to be predominant during both passive and iontophoretic experiments.

  1. 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.

  2. 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.

  3. 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.

  4. 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

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

    PubMed

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

    2009-10-01

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

  6. Confocal scanning laser ophthalmoscopy versus modified conventional fundus camera for fundus autofluorescence.

    PubMed

    Calvo-Maroto, Ana M; Esteve-Taboada, Jose J; Domínguez-Vicent, Alberto; Pérez-Cambrodí, Rafael J; Cerviño, Alejandro

    2016-10-01

    Fundus autofluorescence (FAF) is a noninvasive imaging method to detect fundus endogenous fluorophores, mainly lipofuscin located in the retinal pigment epithelium (RPE). The FAF provides information about lipofuscin distribution and RPE health, and consequently an increased accumulation of lipofuscin has been correlated with ageing and development of certain retinal conditions. Areas covered: An exhaustive literature search in MEDLINE (via OVID) and PUBMED for articles related to ocular FAF in retinal diseases and different devices used for acquiring FAF imaging was conducted. Expert commentary: This review aims to show an overview about autofluorescence in the RPE and the main devices used for acquiring these FAF images. The knowledge of differences in the optical principles, acquisition images and the image post-processing between confocal scanning laser ophthalmoscopy and modified conventional fundus camera will improve the FAF images interpretation when are used as a complementary diagnosis and monitoring tool of retinal diseases.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Localization of puroindoline-a and lipids in bread dough using confocal scanning laser microscopy.

    PubMed

    Dubreil, Laurence; Biswas, Samares C; Marion, Didier

    2002-10-09

    Puroindolines are lipid-binding proteins from wheat flour that play a significant role in bread crumb texture. The localization of wheat flour lipids and puroindoline-a (PIN-a) in bread dough was studied by confocal scanning laser microscopy (CSLM). Wheat lipids were located around gas cells (GC) and embedded within the protein-starch matrix (SPM) of the dough. PIN-a was mainly located in the matrix of dough, where it was associated with lipids. In contrast, in defatted dough, PIN-a was found around GC. Addition of puroindolines in bread dough induced a defatting of the gas bubble surface and a decrease of the lipid vesicles and/or droplet size embedded within the SPM. Therefore, puroindolines control the lipid partitioning within the different phases of dough, a phenomenon that should have important consequence on the gas bubble expansion and GC formation in the further stages (fermentation, baking) of the bread-making process.

  15. 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.

  16. Comparison of Three Different Sealer Placement Techniques: An In vitro Confocal Laser Microscopic Study

    PubMed Central

    Dash, Avoy Kumar; Farista, Shanin; Dash, Abhilasha; Bendre, Ajinkya; Farista, Sana

    2017-01-01

    Introduction: Three-dimensional obturation of the root canal system is the final objective of root canal therapy. Greater penetration of sealer in root dentine lesser will be the voids at the dentine–sealer interface. Hence, analysis of the dentin/sealer interface allows the determination of a filling technique which could obturate the root canals with least gaps and voids. Therefore, the aim of this study is to compare the depth and percentage of sealer penetration into root dentin using three different root canal sealer placement techniques under confocal laser scanning microscope. Materials and Methods: Thirty single-rooted teeth were selected and prepared. Adseal sealer (Meta Biomed, South Korea) was mixed with Rhodamine B dye and applied using lentulo spiral (Dentsply Maillefer, USA) as Group 1, bidirectional spiral (EZ-Fill– EDS, USA) as Group 2, and ultrasonic endodontic tip (Sonofile– Dentsply Tulsa, USA) as Group 3. Canals were then obturated with gutta-percha. The roots were sectioned at the 3 and 6-mm levels from the apical foramen and examined under confocal laser microscope. Results: Maximum mean depth and percentage of sealer penetration were observed for Group 1 and minimum for Group 3. Furthermore, statistical significant differences among Group 1 and Group 3 were found at 6-mm level and among Group 2 and Group 3 were found at 3-mm level (P < 0.05). Conclusion: The depth and percentage of sealer penetration of sealer are influenced by the type of placement technique and by the root canal level, with penetration decreasing apically. Lentulo spiral has shown better penetration of sealer than the bidirectional file and ultrasonics. PMID:28839420

  17. Confocal Laser Endomicroscopy for Diagnosis and Histomorphologic Imaging of Brain Tumors In Vivo

    PubMed Central

    Foersch, Sebastian; Heimann, Axel; Ayyad, Ali; Spoden, Gilles A.; Florin, Luise; Mpoukouvalas, Konstantin; Kiesslich, Ralf; Kempski, Oliver; Goetz, Martin; Charalampaki, Patra

    2012-01-01

    Early detection and evaluation of brain tumors during surgery is crucial for accurate resection. Currently cryosections during surgery are regularly performed. Confocal laser endomicroscopy (CLE) is a novel technique permitting in vivo histologic imaging with miniaturized endoscopic probes at excellent resolution. Aim of the current study was to evaluate CLE for in vivo diagnosis in different types and models of intracranial neoplasia. In vivo histomorphology of healthy brains and two different C6 glioma cell line allografts was evaluated in rats. One cell line expressed EYFP, the other cell line was used for staining with fluorescent dyes (fluorescein, acriflavine, FITC-dextran and Indocyanine green). To evaluate future application in patients, fresh surgical resection specimen of human intracranial tumors (n = 15) were examined (glioblastoma multiforme, meningioma, craniopharyngioma, acoustic neurinoma, brain metastasis, medulloblastoma, epidermoid tumor). Healthy brain tissue adjacent to the samples served as control. CLE yielded high-quality histomorphology of normal brain tissue and tumors. Different fluorescent agents revealed distinct aspects of tissue and cell structure (nuclear pattern, axonal pathways, hemorrhages). CLE discrimination of neoplastic from healthy brain tissue was easy to perform based on tissue and cellular architecture and resemblance with histopathology was excellent. Confocal laser endomicroscopy allows immediate in vivo imaging of normal and neoplastic brain tissue at high resolution. The technology might be transferred to scientific and clinical application in neurosurgery and neuropathology. It may become helpful to screen for tumor free margins and to improve the surgical resection of malignant brain tumors, and opens the door to in vivo molecular imaging of tumors and other neurologic disorders. PMID:22911853

  18. Viability and antibacterial efficacy of four root canal disinfection techniques evaluated using confocal laser scanning microscopy

    PubMed Central

    Mathew, Joan; Emil, Jonathan; Paulaian, Benin; John, Bejoy; Raja, Jacob; Mathew, Jean

    2014-01-01

    Background: Several disinfection techniques have been recently introduced with the main objective of improving root canal disinfection in the inaccessible areas of the root canal system. This in vitro study was done to evaluate the antimicrobial effect and viability of Enterococcus faecalis biofilms using conventional irrigation, EndoActivator (Dentsply, Tulsa Dental, USA), diode laser irradiation and photon-initiated photoacoustic streaming (PIPS). Materials and Methods: Root canals of 130 single rooted mandibular premolars, standardized to a uniform length of 20 mm were instrumented until finishing file, F1 (Universal Protaper Rotary System, Dentsply, Tulsa Dental Specialties, USA). After smear layer removal and sterilization, five teeth were randomly selected to assure sterility before bacterial inoculation. The remaining 125 samples were contaminated with E. faecalis suspension, incubated for 21 days and divided into five groups (n = 25). In Group 1; untreated group (positive control), the root canals were not subjected to any disinfection procedure. Sampling was performed within the canals and the colony-forming unit count was evaluated for 20 samples. Five samples were selected to visualize the pattern of colonization at Level 1 (4 mm from the apex) and Level 2 (1 mm from the apex) by confocal laser scanning microscopy. Samples in Groups 2-5 namely conventional needle irrigation, EndoActivator, diode laser and PIPS were subjected to their respective disinfection procedures. Postdisinfection sample evaluation criteria was followed for all groups as same as that for Group 1. Results: Diode laser displayed the highest antibacterial efficacy and least viable bacteria than the other three disinfection techniques. Conclusion: Diode laser group showed better antibacterial efficacy and least viable bacteria when compared to conventional needle irrigation, PIPS and EndoActivator groups in minimally instrumented, experimentally infected root canals. PMID:25298645

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

    PubMed

    Krause, Marina; Te Riet, Joost; Wolf, Katarina

    2013-12-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m(-1), force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

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

    NASA Astrophysics Data System (ADS)

    Krause, Marina; te Riet, Joost; Wolf, Katarina

    2013-12-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m-1, force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

  1. 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.

  2. Urothelial Tumors and Dual-Band Imaging: A New Concept in Confocal Laser Endomicroscopy.

    PubMed

    Marien, Arnaud; Rock, Aurélien; Maadarani, Khaled El; Francois, Catherine; Gosset, Pierre; Mauroy, Brigitte; Bonnal, Jean-Louis

    2017-05-01

    Confocal laser endomicroscopy (CLE) uses a low-energy laser light source to obtain microscopic histology images of bladder tissue exposed to a fluorescent dye. To evaluate the feasibility of using CLE with two fluorophores: fluorescein (FLUO) and hexylaminolevulinate (HAL) to determine histologic and cytologic bladder cancer criteria. Patients eligible for HAL-photodynamic diagnosis-assisted transurethral resection of bladder tumor were included. The procedures were performed with the patient under regional or general anesthesia (60-90 minutes) after bladder instillation of HAL (50 mL, 8 mmol/L; Hexvix(®); Ipsen, France). Resected tissue was examined ex vivo using CLE either with Cellvizio(®) system (CVI) single laser (488 nm) or with Cellvizio Dual system (CVII) double laser (488, 660 nm). Twenty-one patients were included, 12 examined by CVI and 9 by CVII. Sample examination on CVI after HAL-CLE-only histologic analysis was not possible because HAL is mostly cytoplasmic and gives poor details on cellular architecture. On the contrary, FLUO-CLE gives good extracellular architecture and not clear information of nucleocytoplasmic abnormality. Samples on CVII for seven out of nine patients clearly showed cytoplasm of suspect cells and nuclei. In real time, fluorescence observed on bandwidth (673-800 nm) with HAL and FLUO was associated with the presence of cancer, with a sensibility and specificity of 80% and 100%, respectively. Real-time cytodetection was feasible using two fluorophores (FLUO and HAL) and the new system of CVII. This technology was useful to observe cytoplasm, nuclei, and nucleocytoplasmic abnormality, but an improved system is necessary (to overcome the overlapping of fluorescence) to increase the specificity.

  3. Adhesive improvement in optical coherence tomography combined with confocal microscopy for class V cavities investigations

    NASA Astrophysics Data System (ADS)

    Rominu, Mihai; Sinescu, Cosmin; Negrutiu, Meda L.; Rominu, Roxana O.; Pop, Daniela M.; Topala, Florin; Stoia, Adelina; Petrescu, Emanuela; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

    2010-03-01

    The purpose of this study is to present a non invasive method for the marginal adaptation evaluation in class V composite restorations. Standardized class V cavities prepared in human extracted teeth were filled with composite resin (Premise, Kerr). The specimens were thermocycled. The interfaces were examined by Optical Coherence Tomography (OCT) combined with confocal microscopy and fluorescence. The optical configuration uses two single mode directional couplers with a superluminiscent diode as the source at 1300 nm. The scanning procedure is similar to that used in any confocal microscope, where the fast scanning is en-face (line rate) and the depth scanning is much slower (at the frame rate). Gaps at the interfaces as well as on the inside of the composite resin were identified. OCT has numerous advantages that justify its in vivo and in vitro use compared to conventional techniques. One of the main concerns was the fact that at the adhesive layer site it was very hard to tell the adhesive apart from material defects. For this reason the adhesive was optimized in order to be more scattering. This way we could make a difference between the adhesive layer and the material defects that could lead to microleakages.

  4. 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.

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

    PubMed

    Peduzzi, Peter; Agis, Martin; Luef, Birgit

    2013-07-01

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

  6. 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.

  7. Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry.

    PubMed

    Pauly, John L; Allison, Erin M; Hurley, Edward L; Nwogu, Chukwumere E; Wallace, Paul K; Paszkiewicz, Geraldine M

    2005-06-01

    Numerous highly fluorescent macrophages (MPhi), designated "smoker cells," exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MPhi, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MPhi is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., "tar"). Studies reported herein were undertaken to test the hypothesis that the origin of the MPhi fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MPhi isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MPhi from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MPhi is due to tobacco tar. Copyright (c) 2005 Wiley-Liss, Inc.

  8. Simple Windows-based software for the control of laser scanning confocal microscopes.

    PubMed

    Hartell, Nicholas A

    2007-05-15

    Rapid advances in computer processing power and the appearance of low cost, high speed multifunction data acquisition hardware makes the control of confocal laser scanning microscopes (CLSMs) with standard laboratory hardware a potentially straightforward task. This paper describes software designed to control a Biorad MRC 600 scan head under Windows 2000 or XP. Using a single high speed, multifunction data acquisition board running under the Igor Pro software environment, waveforms required to drive the scan head galvanometers can be generated and up to two channels of images (768 x 512 pixels at 8 or 12 bit levels) captured live or at set intervals. Image averaging, zooming, panning and cropping are supported as is live region of interest measurements over time. The software can trigger or be triggered by external devices via TTL signals and, with the addition of a commercial focus controller, Z scans can also be made. Control of the original neutral density and emission filters of multiple laser-based systems is also supported via serial control. The software should be easily adaptable to control custom designed scanning systems or other older makes of CLSM and it can be integrated with additional acquisition boards for simultaneous electrophysiological recording.

  9. 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.

  10. 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.

  11. Needle-Based Confocal Laser Endomicroscopy for Evaluation of Cystic Neoplasms of the Pancreas.

    PubMed

    Kadayifci, Abdurrahman; Atar, Mustafa; Basar, Omer; Forcione, David G; Brugge, William R

    2017-05-01

    The accurate diagnosis of cystic neoplasms of the pancreas (CNP) with current diagnostic methods is limited. Endoscopic ultrasound (EUS)-guided needle-based confocal laser endomicroscopy (nCLE) is a new technique which can obtain images from the cyst wall during EUS-fine needle aspiration (EUS-FNA). The aim of this study was to assess the safety, feasibility, and diagnostic value of nCLE for CNP. Patients who underwent EUS-FNA to evaluate a CNP larger than 2 cm were enrolled. The cyst was punctured with 19-G FNA needle preloaded with an nCLE probe. The images from different areas of the cyst wall were recorded. Using the final diagnosis defined by surgery or EUS-FNA cyst fluid analysis, the accuracy of the confocal images was defined. The procedure and image acquisition was successful in 18 of the 20 patients. Predefined typical structures for mucinous cysts were visualized in 8 of 12 (66%) cysts but none of the non-mucinous cysts. The superficial vascular network which is a typical finding of serous cysts was observed in 2 of 3 patients. The sensitivity, specificity, and diagnostic accuracy of the findings of epithelial structures by nCLE were 66, 100, and 80%, respectively, for a mucinous cyst diagnosis. All patients tolerated the procedure well, and no adverse effects were determined. nCLE was found to be safe and feasible with high technical success, in this pilot study. With an overall accuracy of 80%, it has the potential to contribute to the diagnosis of CNP with specific imaging.

  12. High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy.

    PubMed

    Whitley, K D; Comstock, M J; Chemla, Y R

    2017-01-01

    We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument's capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme. © 2017 Elsevier Inc. All rights reserved.

  13. 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

  14. 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.

  15. Reflectance confocal microscopy-guided laser ablation of basal cell carcinomas: initial clinical experience

    NASA Astrophysics Data System (ADS)

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

    2017-08-01

    Laser ablation offers a procedure for precise, fast, and minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs). However, the lack of histopathological confirmation has been a limitation toward widespread use in the clinic. A reflectance confocal microscopy (RCM) imaging-guided approach offers cellular-level histopathology-like feedback directly on the patient, which may then guide and help improve the efficacy of the ablation procedure. Following an ex vivo benchtop study (reported in our earlier papers), we performed an initial study on 44 BCCs on 21 patients in vivo, using a pulsed erbium:ytterbium aluminum garnet laser and a contrast agent (aluminum chloride). In 10 lesions on six patients, the RCM imaging-guided detection of either presence of residual tumor or complete clearance was immediately confirmed with histopathology. Additionally, 34 BCCs on 15 patients were treated with RCM imaging-guided laser ablation, with immediate confirmation for clearance of tumor (no histopathology), followed by longer-term monitoring, currently in progress, with follow-up imaging (again, no histopathology) at 3, 6, and 18 months. Thus far, the imaging resolution appears to be sufficient and consistent for monitoring efficacy of ablation in the wound, both immediately postablation and subsequently during recovery. The efficacy results appear to be promising, with observed clearance in 19 cases of 22 cases with follow-ups ranging from 6 to 21 months. An additional 12 cases with 1 to 3 months of follow-ups has shown clearance of tumor but a longer follow-up time is required to establish conclusive results. Further instrumentation development will be necessary to cover larger areas with a more automatically controlled instrument for more uniform, faster, and deeper imaging of margins.

  16. Ultra-narrow linewidth DFB-laser with optical feedback from a monolithic confocal Fabry-Perot cavity.

    PubMed

    Lewoczko-Adamczyk, Wojciech; Pyrlik, Christoph; Häger, Johannes; Schwertfeger, Sven; Wicht, Andreas; Peters, Achim; Erbert, Götz; Tränkle, Günther

    2015-04-20

    We present a compact, ultra-narrow-linewidth semiconductor laser based on a 780 nm distributed feedback diode laser optically self-locked to a mode of an external monolithic confocal Fabry-Perot resonator. We characterize spectral properties of the laser by measuring its frequency noise power spectral density. The white frequency noise levels at 5 Hz(2)/Hz above a Fourier frequency as small as 20 kHz. This noise level is more than five orders of magnitude smaller than the noise level of the same solitary diode laser without resonant optical feedback, and it is three orders of magnitude smaller than the noise level of a narrow linewidth, grating-based, extended-cavity diode laser. The corresponding Lorentzian linewidth of the laser with resonant optical feedback is 15.7 Hz at an output power exceeding 50 mW.

  17. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope.

    PubMed

    Brown, C M; Dalal, R B; Hebert, B; Digman, M A; Horwitz, A R; Gratton, E

    2008-01-01

    Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as approximately 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin-enhanced-green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented to account for bright immobile structures within the cell that dominate spatial correlation functions; allowing the extraction of fast protein dynamics within and near these structures. A running average algorithm is also presented to address slow cellular movement or movement of cellular features such as adhesions. Finally, methods to determine protein concentration in the presence of immobile structures within the cell are presented. A table is presented giving guidelines for instrument and imaging setting when performing RICS on the Olympus FV300 confocal and these guidelines are a starting point for performing the analysis on other commercial confocal systems.

  18. 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.

  19. Optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy in retinal nerve fiber layer measurements of glaucoma patients.

    PubMed

    Fanihagh, Farsad; Kremmer, Stephan; Anastassiou, Gerasimos; Schallenberg, Maurice

    2015-01-01

    To determine the correlations and strength of association between different imaging systems in analyzing the retinal nerve fiber layer (RNFL) of glaucoma patients: optical coherence tomography (OCT), scanning laser polarimetry (SLP) and confocal scanning laser ophthalmoscopy (CSLO). 114 eyes of patients with moderate open angle glaucoma underwent spectral domain OCT (Topcon SD-OCT 2000 and Zeiss Cirrus HD-OCT), SLP (GDx VCC and GDx Pro) and CSLO (Heidelberg Retina Tomograph, HRT 3). Correlation coefficients were calculated between the structural parameters yielded by these examinations. The quantitative relationship between the measured RNFL thickness globally and for the four regions (superior, inferior, nasal, temporal) were evaluated with different regression models for all used imaging systems. The strongest correlation of RNFL measurements was found between devices using the same technology like GDx VCC and GDx Pro as well as Topcon OCT and Cirrus OCT. In glaucoma patients, the strongest associations (R²) were found between RNFL measurements of the two optical coherence tomography devices Topcon OCT and Cirrus OCT (R² = 0.513) and between GDx VCC and GDx Pro (R² = 0.451). The results of the OCTs and GDX Pro also had a strong quantitative relationship (Topcon OCT R² = 0.339 and Cirrus OCT R² = 0.347). GDx VCC and the OCTs showed a mild to moderate association (Topcon OCT R² = 0.207 and Cirrus OCT R² = 0.258). The confocal scanning laser ophthalmoscopy (HRT 3) had the lowest association to all other devices (Topcon OCT R² = 0.254, Cirrus OCT R² = 0.158, GDx Pro R² = 0.086 and GDx VCC R² = 0.1). The measurements of the RNFL in glaucoma patients reveal a high correlation of OCT and GDx devices because OCTs can measure all major retinal layers and SLP can detect nerve fibers allowing a comparison between the results of this devices. However, CSLO by means of HRT topography can only measure height values of the retinal surface but it cannot distinguish

  20. Real-time confocal laser endomicroscopic evaluation of primary liver cancer based on human liver autofluorescence.

    PubMed

    Maki, Harufumi; Kawaguchi, Yoshikuni; Arita, Junichi; Akamatsu, Nobuhisa; Kaneko, Junichi; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Harihara, Yasushi; Kokudo, Norihiro

    2017-02-01

    Confocal laser endomicroscopy (CLE) is available for real-time microscopic examination. This study aims to evaluate the usefulness of intraoperative CLE examination as a modality to evaluate surgical margins in surgery for primary liver cancer. A probe-based CLE system (Cellvizio 100, Mauna Kea Technologies, Paris, France) was used. The subjects comprised seven specimens obtained from six patients with primary liver cancer in November 2015. The probe was manually attached to the surfaces of specimens, and images were collected without external fluorophores. CLE images were compared with hematoxylin and eosin-stained slides. Fluorescence intensity (FI) values of the CLE images were assessed using luminance-analyzing software. CLE examination visualized non-cancerous regions in the background liver as regular structures with high fluorescence because of human liver autofluorescence. Conversely, hepatocellular carcinoma and intrahepatic cholangiocarcinoma were depicted as irregular structures with low fluorescence. The median FI values of the non-cancerous regions and the cancerous regions were 104 (79.8-156) and 74.9 (60.6-106), respectively, and were significantly different (P = 0.031). The probe-based CLE enables real-time differentiation of cancerous regions from non-cancerous tissues in surgical specimens because of human liver autofluorescence. CLE can be used to confirm negative surgical margins in the operating room. J. Surg. Oncol. 2017;115:151-157. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. Using Confocal Laser Scanning Microscopy to Probe the Milk Fat Globule Membrane and Associated Proteins

    PubMed Central

    Gallier, Sophie; Gragson, Derek; JiméNez-Flores, Rafael; Everett, David

    2010-01-01

    The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied but its structure, especially the lateral organization of its components, still remains unclear. We have used confocal laser scanning microscopy (CLSM) to investigate the surface structure of the MFGM in globules with different degree of processing using two types of fluorescently-labeled phospholipid probes and a protein dye. Using this technique, we have observed heterogeneities in the distribution of MFGM lipids and proteins relating to the processing and size of the globules. The effect of pre-treating the milk (centrifugation, pasteurization-homogenization and churning) was studied by double-staining the surface of the milk fat globules, followed by observation using CLSM, and by determining the phospholipid profile of raw milk, raw cream, processed milk and buttermilk powder. Our findings agree with other techniques by showing that the composition of the MFGM changes with processing through the loss of phospholipids and the adsorption of caseins and whey proteins onto the surface. PMID:20218614

  8. Investigation of sample behaviors inside on-chip electrophoresis microcapillary using confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Etoh, Shinichi; Higashi, Toshihito; Fujimura, Tsuyoshi; Hattori, Reiji; Kuroki, Yukinori

    2004-04-01

    We report the observation of sample behaviors using the confocal laser scanning microscopy (CLSM) in on-chip microcapillary. Sample loading by pinched valve injection is observed in a new cross injector shape, which has the structure added conventional cross injector to circle shape. In sample loading, because this structure causes a different electric field compared with that in conventional cross injector, high efficient sample plug injection was performed. It is important to investigate further the detailed sample profiles using the CLSM in sample loading for development of the on-chip microcapillary. We attempt the simulation of sample loading in the cross injector using the semiconductor device simulator MEDICI in order to investigate it in further detail. The sample movements in the channel turn along the Z-direction are observed using the CLSM. In order to miniaturize the microfluidic channel, it is necessarily needed to fold the channel, but then it is inevitable that sample dispersion occurs in the turn. We present sample flow profiles along the Z-direction in the turn using the CLSM and the influence on the electrophoretic separation. Also, we improve that fabrication of duct channel for exhaustion the vaporized xylene to outside the chip and the adhesion process

  9. 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.

  10. Endocrine organs and laser scanning confocal microscopy (LSCM) imaging: vascular bed in human spleen.

    PubMed

    Galfiova, P; Pospisilova, V; Varga, I; Sikuta, J; Kiss, A; Majesky, I; Jakubovsky, J; Polak, S

    2010-10-01

    This work was aimed to utilize the precise method of laser confocal microscopy (LSCM) to depict the image of spatial relationships of the vessel network in the tissue structures of the human spleen. With the use of serial paraffin or vibratome sections of more than 20 μm thickness infiltrated with eosin fluorescence dye the images of arterial and venous walls of different calibres, capillaries, and venous sinuses were morphologically revealed. Venous sinuses were frequently found to create mutually communicating branches and their lining projected into the lumen protruding cells with distinct spherically or ovally shaped nuclei, positioned on the brightly fluorescent and fragmented lamina basalis. The presence of lymphocytes was distinct in periarteriolar lymphoid sheath (PALS) and lymphatic follicles. Lining cells of the red pulp veins sporadically contained marked eosinophilic granules. The method of LSCM allowed: 1. to reveal two-dimensional and sharp image of the human spleen structures, 2. to investigate the vertical course of venous structures in the tissue, 3. to obtain serial optic sections in z axis to their maximum spatial projections. These data will also serve for the creation of three-dimensional images of vessel network in the human spleen in the future studies.

  11. 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.

  12. 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. © 2015 Institute of Food Technologists®

  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. Collagen and Elastic Fibers in Odontogenic Entities: Analysis Using Light and Confocal Laser Microscopic Methods

    PubMed Central

    Moure, Sabrina P; Carrard, Vinicius C; Lauxen, Isabel S; Manso, Pedro Paulo A; Oliveira, Marcia G; Martins, Manoela D; Sant´Ana Filho, Manoel

    2011-01-01

    Dentigerous cyst (DC) and keratocystic odontogenic tumor (KOT) are odontogenic lesions arising from epithelial elements, such as those observed in dental follicles (DF), that have been part of the tooth forming apparatus. These lesions show different clinical and histological characteristics, as well as distinct biological behavior. This study aimed to qualify and quantify collagen and elastic fibers by means of histochemical techniques with light and confocal laser microscopic methods in three odontogenic entities. Eleven DF, 13 DC (n=10 with inflammation, n=3 without inflammation) and 13 KOT were processed to the following techniques: Hematoxylin and Eosin, Masson’s Trichrome, Picrosirius, Direct Blue, and Orcein. DF and DC without inflammation exhibited collagen with similar characteristics: no parallel pattern of fiber orientation, thick fibers with dense arrangement, and absence of distinct layers. A comparison between DC with inflammation and KOT revealed similar collagen organization, showing distinct layers: thin collagen fibers with loose arrangement near the epithelium and thick fibers with dense arrangement in distant areas. The only difference found was that KOT exhibited a parallel collagen orientation in relation to the odontogenic epithelia. It may be suggested that the connective tissue of DC is a reactive tissue, inducing an expansive growth associated with fluid accumulation and inflammatory process, which in turn may be present as part of the lesion itself. In KOT, loosely arranged collagen may be associated with the behavior of the neoplastic epithelium. PMID:21760864

  15. Ocular fundus images with confocal scanning laser ophthalmoscopy in the dog, monkey and minipig.

    PubMed

    Rosolen, S G; Saint-MacAry, G; Gautier, V; Legargasson, J F

    2001-03-01

    Confocal scanning laser ophthalmoscopy (CSLO) is a new technique that enables ocular fundus image recording and retinal dynamic angiography to be performed. The ocular fundus image is acquired sequentially, point by point, and is reconstructed on a video monitor at the rate of 25 images per second. The feasibility of performing both ocular fundus image recordings and retinal angiography image recordings were tested on two dogs, two monkeys and two minipigs using a 40 degrees field I + Tech CSLO. Fundus area of each dog, monkey and minipig were examined without any additional optical devices. The ocular fundus and angiography images were recorded, stabilized and analyzed under the same conditions. For each species, all images were easily recorded without any additional optical device in a lighted room and the morphology of the retinal images generated was similar to those obtained with a camera or angiography of higher resolution. Capillary phase or venous times are presented. Image recording at 25 frames/second enabled more retinal dynamics to be demonstrated than with use of regular angiography. This technique is noninvasive and easy to perform if the eye is fixed and eyelids maintained open. It also allows exploration of retinal microvascularization and could be utilized for clinical, pharmacologic and toxicologic investigations as well.

  16. 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.

  17. 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.

  18. Enamel erosion and prevention efficacy characterized by confocal laser scanning microscope.

    PubMed

    Maia, Ana Marly Araújo; Longbottom, Christopher; Gomes, Anderson Stevens Leonidas; Girkin, John Michael

    2014-06-01

    The aim of this study was to evaluate the erosion-inhibiting effect of two toothpastes on the development of erosion-like lesions, by a confocal laser scanning microscope (CLSM). Forty human enamel blocks were divided into five groups (n = 8), in accordance to evaluate the GC MI Paste Plus and Oral B with stannous fluoride, applied as slurries and associated with toothbrush. Specimens were submitted to an erosion challenge from citric acid (0.5%, pH = 2.8), for 5 min, six times a day, alternating in artificial saliva immersions. Reference group was not exposed to treatment. Part of specimens (Groups 02 and 03) was exposed twice daily just to slurries, for 2 min, therefore specimens from Groups 04 and 05 were also abraded, for 30 s. The enamel surfaces were morphological characterized using CLSM images, with mineral loss being measured using the resulting 3D images referenced to an un-challenged portion of the sample. Step values were compared using the one-way ANOVA test. CLSM was shown to be a viable, noncontact, and simple technique to characterize eroded surfaces. The statistical difference in the step size was significant between the groups (P = 0.001) and using multiple comparisons a statistically significant protective effect of toothpastes was shown when these were applied as slurries. Although groups submitted to tooth brush showed mineral loss similar to reference control group, due to the damages of abrasion associated. © 2014 Wiley Periodicals, Inc.

  19. Histopathological, immunohistochemical criteria and confocal laser-scanning data of arthrofibrosis.

    PubMed

    Ruppert, M; Theiss, C; Knöß, P; Kendoff, D; Krukemeyer, M G; Schröder, N; Brand-Saberi, B; Gehrke, T; Krenn, V

    2013-11-01

    Arthrofibrosis (af) is defined as a fibrosing disease of the synovial membrane, after joint operations, with painful restricted range of motion. The aim of this paper was to describe the histopathological substrate of af, hitherto only defined by clinical criteria. Based on a group of 222 tissue samples, the characteristic changes to af were analyzed. The control group comprised 29 cases with neosynovialis of the indifferent type. Due to cytoplasmic SM-actin positivity and the absence of specific cytoplasmic reactivity in CD 68 representation, af fibroblasts were characterized as myofibroblasts. In confocal laser-scanning microscopy, β-catenin-positive aggregates were detected in the cytoplasm. Over and above this, unequivocal colocalization of β-catenin and the tight junction protein ZO-1 became manifest, particularly on the cell membrane and, partly, in the cytoplasm. A threshold value of 20 β-catenin-positive cells/HPF was determined. This enables the histopathological diagnosis of an af to be made (sensitivity: 0.733, specificity: 0.867). Af is a fibrosing disease of the synovial membrane with variable grade of fibrotization (fibroblast cellularity). A threshold value of 20 β-catenin-positive fibroblasts per HPF was defined, which enables the histopathological diagnosis of af. Copyright © 2013 Elsevier GmbH. All rights reserved.

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  1. 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..

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

    PubMed Central

    Wang, Peng; Ji, Rui; Yu, Tao; Zuo, Xiu-Li; Zhou, Cheng-Jun; Li, Chang-Qing; Li, Zhen; Li, Yan-Qing

    2010-01-01

    AIM: To classify the histological severity of Helicobacter pylori (H. pylori) infection-associated gastritis by confocal laser endomicroscopy (CLE). METHODS: Patients with upper gastrointestinal symptoms or individuals who were screened for gastric cancer were enrolled in this study. Histological severity of H. pylori infection-associated gastritis was graded according to the established CLE criteria. Diagnostic value of CLE for histological gastritis was investigated and compared with that of white light endoscopy (WLE). Targeted biopsies from the sites observed by CLE were performed. RESULTS: A total of 118 consecutive patients with H. pylori infection-associated gastritis were enrolled in this study. Receiver operating characteristic curve analysis showed that the sensitivity and specificity of CLE were 82.9% and 90.9% for the diagnosis of H. pylori infection, 94.6% and 97.4% for predicting gastric normal mucosa, 98.5% and 94.6% for predicting histological active inflammation, 92.9% and 95.2% for predicting glandular atrophy, 98.6% and 100% for diagnosing intestinal metaplasia, respectively. Post-CLE image analysis showed that goblet cells and absorptive cells were the two most common parameters on the CLE-diagnosed intestinal metaplasia (IM) images (P < 0.001). More histological lesions of the stomach could be found by CLE than by WLE (P < 0.001). CONCLUSION: CLE can accurately show the histological severity of H. pylori infection-associated gastritis. Mapping IM by CLE has a rather good diagnostic accuracy. PMID:21049554

  3. Interactions between soluble dietary fibers and wheat gluten in dough studied by confocal laser scanning microscopy.

    PubMed

    Li, Qian; Liu, Rui; Wu, Tao; Zhang, Min

    2017-05-01

    Four soluble dietary fiber (SDF) fractions characterized by major components of AXs, relatively narrow molecular weight distribution, different substituted ratio, and structure-sensitive parameter (ρ) were prepared from wheat bran. The fractions were added to wheat dough to determine the interactions between the dough's network and the SDF fractions relative to their physicochemical characteristics. Furthermore, a comprehensive study focusing on the dough texture characteristic, tensile properties, thermodynamic stability, and the microstructure was conducted by performing texture profile analysis (TPA), differential scanning calorimetry (DSC), and confocal laser scanning microscopy (CLSM) experiments. Additionally, an estimation function of the interactions parameters between the dough's network and the SDF fractions related to the factor molecular weight and ρ of the SDFs was established. The results indicated that the SDF fractions exhibiting a medium molecular weight, and a higher substitution degree and di-substituted ratio, were the most suitable fortifier providing benefits to the dough's qualities. Furthermore, the research methodology might support the high potential of SDF fractions as fortifier for flour-based products. Copyright © 2017. Published by Elsevier Ltd.

  4. 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

  5. Noninvasive histological imaging of head and neck squamous cell carcinomas using confocal laser endomicroscopy.

    PubMed

    Linxweiler, Maximilian; Kadah, Basel Al; Bozzato, Alessandro; Bozzato, Victoria; Hasenfus, Andrea; Kim, Yoo-Jin; Wagner, Mathias; Igressa, Alhadi; Schick, Bernhard; Charalampaki, Patra

    2016-12-01

    Confocal laser endomicroscopy (CLE) is an imaging technique that uses miniaturized fiberoptic probes to allow real-time histological imaging of human tissue. An application of CLE in otorhinolaryngology has hardly been investigated so far. In our study, we analyzed the applicability of CLE to visualize cancerous and healthy tissue of the head and neck region. Formalin-fixed tissue specimens from 135 head and neck squamous cell carcinoma (HNSCC) patients and 50 healthy controls were investigated using CLE with and without topical application of acriflavine. Four head and neck surgeons, four pathologists, and four laymen evaluated the CLE images of the HNSCC cases regarding the tumor localization and its border to healthy tissue. The tumor localization and the tumor border were correctly identified in 97 % by the pathologists, 85 % by the head and neck surgeons, and 70 % by the laymen. The main difference in evaluation results was seen in the correct identification of the tumor site (p < 0.05), while there was no significant difference in the identification of the tumor border. CLE is a valuable tool for real-time histological imaging of HNSCCs. It can help to visualize the tumor border and, thereby, facilitate a more precise tumor surgery.

  6. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1996-04-01

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

  8. IL-6 Adsorption Dynamics in Hemoadsorption Beads Studied Using Confocal Laser Scanning Microscopy

    PubMed Central

    Kimmel, Jeremy D.; Gibson, Gregory A.; Watkins, Simon C.; Kellum, John A.; Federspiel, William J.

    2010-01-01

    Sepsis is characterized by a systemic inflammatory response caused by infection, and can result in organ failure and death. Removal of inflammatory mediators such as cytokines from the circulating blood is a promising treatment for severe sepsis. We are developing an extracorporeal hemoadsorption device to remove cytokines from the blood using biocompatible, polymer sorbent beads. In this study, we used confocal laser scanning microscopy (CLSM) to directly examine adsorption dynamics of a cytokine (IL-6) within hemoadsorption beads. Fluorescently labeled IL-6 was incubated with sorbent particles, and CLSM was used to quantify spatial adsorption profiles of IL-6 within the sorbent matrix. IL-6 adsorption was limited to the outer 15μm of the sorbent particle over a relevant clinical time period, and intraparticle adsorption dynamics was modeled using classical adsorption/diffusion mechanisms. A single model parameter, α = qmax K / D, was estimated by fitting CLSM intensity profiles to our mathematical model, where qmax and K are Langmuir adsorption isotherm parameters, and D is the effective diffusion coefficient of IL-6 within the sorbent matrix. Given the large diameter of our sorbent beads (450μm), less than 20% of available sorbent surface area participates in cytokine adsorption. Development of smaller beads may accelerate cytokine adsorption by maximizing available surface area per bead mass. PMID:19904819

  9. 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.

  10. 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.

  11. 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.

  12. Hybrid Laser Would Combine Power With Efficiency

    NASA Technical Reports Server (NTRS)

    Sipes, Donald L., Jr

    1986-01-01

    Efficient laser system constructed by using two semiconductor lasers to pump neodymium yttrium aluminum garnet (Nd:YAG) device. Hybrid concept allows digital transmission at data rates of several megabits per second with reasonably sized optical aperture of 20 cm. Beams from two GaAs lasers efficiently coupled for pumping Nd:YAG crystal. Combination of lasers exploits best features of each.

  13. 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.

  14. In-situ observation of recrystallization in an AlMgScZr alloy using confocal laser scanning microscopy

    SciTech Connect

    Taendl, J.; Nambu, S.; Orthacker, A.; Kothleitner, G.; Inoue, J.; Koseki, T.; Poletti, C.

    2015-10-15

    In this work we present a novel in-situ approach to study the recrystallization behavior of age hardening alloys. We use confocal laser scanning microscopy (CLSM) at 400 °C to investigate the static recrystallization of an AlMg4Sc0.4Zr0.12 alloy in-situ. The results are combined with electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) analyses. It was found that CLSM is a powerful tool to visualize both the local initiation and temporal sequence of recrystallization. After fast nucleation and initial growth, the grain growth rate decreases and the grain boundary migration stops after some minutes due to Zener pinning from Al{sub 3}(Sc,Zr) precipitates produced during the heat treatment. EBSD and TEM analyses confirm both the boundary movements and the particle-boundary interactions. - Highlights: • First time that CLSM is used to study recrystallization in-situ. • The start and end of recrystallization can be directly observed. • The procedure is easy to apply and requires only simple data interpretation. • In-situ observations on the surface correlate to modifications inside the bulk. • In-situ observations correlate to EBSD and EFTEM analyses.

  15. [Corneal changes after wearing orthokeratology contact lenses: an investigation using in vivo, confocal laser scanning microscopy].

    PubMed

    Knappe, S; Stachs, O; Guthoff, R

    2007-08-01

    Wearing orthokeratology contact lenses (OCL, Hecht-see free; Hecht, Germany) overnight can change corneal refraction by up to -4.5 dioptre (dpt) based on corneal adaptation to the double reverse surface of the OCL. This allows a temporary independence on glasses or contact lenses. It is known that the central corneal thickness decreases while the corneal thickness in the periphery probably increases. The aim of this study was to investigate the corneal changes of volunteers wearing OCL with in vivo confocal microscopy. Five young adults (mean 22.8 years, three female, two male) with low to moderate myopia (range -1.75 to -3.5 dpt; sphere equivalent -2.7+/-0.59 dpt) were fitted with OCL of reverse-geometry design in both eyes. Lenses were worn in both eyes overnight and were removed immediately in the morning. The volunteers were examined with in vivo confocal microscopy using a combination of Heidelberg retina tomograph II and the Rostock cornea module before wearing the OCL and after the 1(st), 3(rd), 5(th), 7(th), 13(th), 20(th) and 25(th) nights. The central and mid-peripheral total corneal thickness as well as the epithelial thickness were examined in the morning between 7.30 am and 9.30 am. The central and the mid-peripheral epithelial corneal thickness was reduced significantly (p<0.05) from day 1 to the 13(th) day. This stabilized later until the the examination was concluded. No significant changes (p>0.05) were found in the central or mid-peripheral total corneal thickness after 25 days of wearing the OCL. Wearing OCL leads to a reduction in the central corneal epithelial thickness. Our inability to find an increase in mid-peripheral total and epithelial corneal thickness may be because the expected increase of the mid-peripheral cornea is limited to a defined area, which makes repeated measurements at a particular point difficult.

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

    PubMed

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

    2009-01-01

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

  17. An instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography.

    PubMed

    Mochalov, Konstantin E; Chistyakov, Anton A; Solovyeva, Daria O; Mezin, Alexey V; Oleinikov, Vladimir A; Vaskan, Ivan S; Molinari, Michael; Agapov, Igor I; Nabiev, Igor; Efimov, Anton E

    2017-06-21

    In the past decade correlative microscopy, which combines the potentials of different types of high-resolution microscopies with a variety of optical microspectroscopy techniques, has been attracting increasing attention in material science and biological research. One of outstanding solutions in this area is the combination of scanning probe microscopy (SPM), which provides data on not only the topography, but also the spatial distribution of a wide range of physical properties (elasticity, conductivity, etc.), with ultramicrotomy, allowing 3D multiparametric examination of materials. The combination of SPM and ultramicrotomy (scanning probe nanotomography) is very appropriate for characterization of soft multicompound nanostructurized materials, such as polymer matrices and microstructures doped with different types of nanoparticles (magnetic nanoparticles, quantum dots, nanotubes, etc.), and biological materials. A serious problem of this technique is a lack of chemical and optical characterization tools, which may be solved by using optical microspectroscopy. Here, we report the development of an instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography in a single apparatus. This approach retains all the advantages of SPM and upright optical microspectroscopy and allows 3D multiparametric characterization using both techniques. As the first test of the system developed, we have performed correlative characterization of the morphology and the magnetic and fluorescent properties of fluorescent magnetic microspheres doped with a fluorescent dye and magnetic nanoparticles. The results of this study can be used to obtain 3D volume images of a specimen for most high-resolution near-field scanning probe microscopies: SNOM, TERS, AFM-IR, etc. This approach will result in development of unique techniques combining the advantages of SPM (nanoscale morphology and a wide range of physical parameters) and high-resolution optical

  18. 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.

  19. 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.

  20. Confocal laser tomographic analysis of the retina in eyes with macular hole formation and other focal macular diseases.

    PubMed

    Bartsch, D U; Intaglietta, M; Bille, J F; Dreher, A W; Gharib, M; Freeman, W R

    1989-09-15

    To study the retinal surface in the human eye in normal and diseased states we used laser scanning tomography. The confocal arrangement of the laser tomographic scanner permits examination of retinal topography in the axis perpendicular to the retinal surface. The eyes examined with the laser tomographic scanner included normal eyes, eyes with macular holes, impending macular holes, radiation retinopathy, macular edema, photocoagulation scars, subfoveal scars, and serous detachment of the fovea associated with subretinal neovascularization. The laser tomographic scanner is a new method that allows measurements of the topography of the internal limiting membrane in the macular area and may improve our understanding of the pathophysiologic characteristics and treatment of a variety of disorders of the macula.

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

    PubMed

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

    2016-01-01

    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. 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. 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. 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.

  2. 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.

  3. Combined spectrally encoded confocal microscopy and optical frequency domain imaging system

    NASA Astrophysics Data System (ADS)

    Kang, DongKyun; Suter, Melissa J.; Boudoux, Caroline; Yachimski, Patrick S.; Bouma, Brett E.; Nishioka, Norman S.; Tearney, Guillermo J.

    2009-02-01

    Spectrally encoded confocal microscopy (SECM) and optical frequency domain imaging (OFDI) are two reflectancebased imaging technologies that may be utilized for high-resolution microscopic screening of internal organs. SECM provides en face images of tissues with a high lateral resolution of 1-2 μm, and a penetration depth of up to 300 μm. OFDI generates cross-sectional images of tissue architecture with a resolution of 10-20 μm and a penetration depth of 1- 2 mm. Since the two technologies yield complementary microscopic information on two different size scales (SECM-cellular and OFDI-architectural) that are commonly used for histopathologic evaluation, their combination may allow for more accurate optical diagnosis. Here, we report the integration of these two imaging modalities in a single bench top system. SECM images of swine small intestine showed the presence of goblet cells, and OFDI images revealed the finger-shaped villous architecture. In clinical study of 9 gastroesophageal biopsies from 8 patients, a diverse set of architectural and cellular features was observed, including squamous mucosa with mild hyperplasia and gastric antral mucosa with gastric pits and crypts. The capability of this multimodality device to enable the visualization of microscopic features on these two size scales supports our hypothesis that improved diagnostic accuracy may be obtained by merging these two technologies into a single instrument.

  4. Evaluation of hypericin-mediated photodynamic therapy in combination with angiogenesis inhibitor bevacizumab using in vivo fluorescence confocal endomicroscopy

    NASA Astrophysics Data System (ADS)

    Bhuvaneswari, Ramaswamy; Thong, Patricia S. P.; Gan, Yik-Yuen; Soo, Khee; Olivo, Malini

    2010-01-01

    Photodynamic therapy (PDT) is an alternative cancer treatment modality that offers localized treatment using a photosensitizer and light. However, tumor angiogenesis is a major concern following PDT-induced hypoxia as it promotes recurrence. Bevacizumab is a monoclonal antibody that targets vascular endothelial growth factor (VEGF), thus preventing angiogenesis. The combination of PDT with antiangiogenic agents such as bevacizumab has shown promise in preclinical studies. We use confocal endomicroscopy to study the antiangiogenic effects of PDT in combination with bevacizumab. This technique offers in vivo surface and subsurface fluorescence imaging of tissue. Mice bearing xenograft bladder carcinoma tumors were treated with PDT, bevacizumab, or PDT and bevacizumab combination therapy. In tumor regression experiments, combination therapy treated tumors show the most regression. Confocal fluorescence endomicroscopy enables visualization of tumor blood vessels following treatment. Combination therapy treated tumors show the most posttreatment damage with reduced cross-sectional area of vessels. Immunohistochemistry and immunofluorescence studies show that VEGF expression is significantly downregulated in the tumors treated by combination therapy. Overall, combining PDT and bevacizumab is a promising cancer treatment approach. We also demonstrate that confocal endomicroscopy is useful for visualization of vasculature and evaluation of angiogenic response following therapeutic intervention.

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

    PubMed

    Kuiper, T; van den Broek, F J C; van Eeden, S; Wallace, M B; Buchner, A M; Meining, A; van Hee, K; Fockens, P; Dekker, E

    2011-12-01

    Probe-based confocal laser endomicroscopy (pCLE; Cellvizio, Mauna Kea Technologies, Paris, France) enables in vivo histology during colonoscopy and may allow endoscopists to make real-time diagnoses. A collaboration of five experts proposed a new pCLE classification for colonic use. The aim of this study was to assess interobserver agreement and accuracy of the new pCLE classification in the colon. Eligible patients were prospectively investigated by pCLE. A subset of 13 pCLE video sequences was reviewed post hoc for the establishment of a new classification, which comprised three vessel categories and seven crypt categories. All five blinded observers then scored another set of 102 video sequences, using the new classification. Histopathology was used as a reference standard. The interobserver agreements on vessel and crypt architecture were 'fair' with kappa values of 0.29 and 0.27, respectively. When the classification was reduced to neoplasia vs. non-neoplasia (i.e. vessel or crypt type 3), overall agreement became 'moderate' (κ = 0.56). Overall sensitivity and specificity for predicting neoplasia was 66 % and 83 %, respectively. When all observers agreed (69 % of videos), the corresponding figures became 80 % and 95 %. A new classification for pCLE in the colon had a 'moderate' interobserver agreement for differentiating neoplasia from non-neoplastic tissue in the colon. The overall accuracy (81 %) for predicting neoplasia was acceptable and became excellent (94 %) when all five observers agreed. Future research should focus on refinement and validation of the classification. © Georg Thieme Verlag KG Stuttgart · New York.

  6. 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).

  7. Confocal laser endomicroscopy: a new gold standard for the assessment of mucosal healing in ulcerative colitis.

    PubMed

    Macé, Vincent; Ahluwalia, Amrita; Coron, Emmanuel; Le Rhun, Marc; Boureille, Arnaud; Bossard, Céline; Mosnier, Jean-François; Matysiak-Budnik, Tamara; Tarnawski, Andrzej S

    2015-03-01

    Endoscopic assessment of mucosal healing in ulcerative colitis (UC) is increasingly accepted as a measure of disease activity, therapeutic goal, and the key prognostic indicator. While regular endoscopy evaluates appearance of the mucosal surface, confocal laser endomicroscopy (CLE) enables in vivo visualization of subepithelial mucosa at 1000× magnification during ongoing endoscopy. Our aims were to determine using CLE whether endoscopically normal appearing colonic mucosa in patients with UC in remission (UC-IR) has fully regenerated mucosal structures, resolved inflammation, and to identify the mechanisms. Twelve patients (six controls and six with UC-IR) underwent colonoscopy using CLE and intravenous fluorescein infusion. During colonoscopy, CLE images of colonic mucosa and conventional mucosal biopsies were obtained and evaluated using image-analysis systems. We quantified; (i) regeneration of colonic crypts and blood microvessels; (ii) cyclooxygenase 2 (COX2) expression; (iii) mitochondrial DNA (mtDNA) mutations; (iv) inflammatory infiltration; and (v) vascular permeability (VP). In control subjects, CLE demonstrated normal colonic crypts and microvasculature. COX2 expression was minimal, and < 7% crypts showed mtDNA mutations. Colonic mucosa of UC-IR patients had impaired and distorted crypt regeneration, increased COX2, 69% crypts with mtDNA mutations, persistent inflammation, and abnormal vascular architecture with increased VP (all P < 0.001 vs normal mucosa). (i) Endoscopically normal appearing colonic mucosa of patients with UC-IR remains abnormal: CLE demonstrates impaired crypt regeneration, persistent inflammation, distinct abnormalities in angioarchitecture and increased vascular permeability; molecular imaging showed increased COX2 and mtDNA mutations; (ii) CLE may serve as a new gold standard for the assessment of mucosal healing in UC. © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  9. 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.

  10. [Confocal laser scanning electron microscopy for assessment of vaginal Lactobacillus crispatus biofilm].

    PubMed

    Wu, Li-jie; Wang, Ben; Liao, Qin-ping; Zhang, Rui

    2015-12-18

    To investigate the female vaginal Lactobacillus crispatus biofilm by using confocal laser scanning microscopy (CLSM),thus revealing the formation of biofilm. The cover slide biofilm culture approach in vitro was employed for induction of the vaginal Lactobacillus crispatus biofilm formation. Following the culture for 2, 4, 8, 12, 16, 20, 24, 48, 72, 96 and 120 hours, the cover slide was removed for subsequent staining with the fluoresce in isothiocyanate-conjugated concanavalin A(FITC-ConA) and propidium (PI).This was followed by determination of the formation and characteristics of the vaginal Lactobacillus crispatus biofilm by using CLSM. The CLSM images of biofilm formation at different time points were captured, suggesting that the vaginal Lactobacillus crispatus adhesion occurred at h 4, which was in reversible attachment, then more and more Lactobacillus crispatus aggregated at h 8 to h 20, which was in irreversible attachment.Lactobacillus crispatus clustered at h 20, with early development of biofilm architecture.Then the biofilm with extracellular matrix around the bacteria was set up at h 24,with gradual matureation at h 24 to h 48.The biofilm dispersed at h 72. The biofilm density of cultivating for 20 hours was 42.7 × 10⁻³ ± 6.8 × 10⁻³ ,and for 24 hours increased to 102.5 × 10⁻³ ± 23.1 × 10⁻³, suggesting a significant difference, P<0.05. This meant that mature biofilm was formed at h 24. The vaginal Lactobacillus crispatus is able to form typical biofilm with distinct developmental phases and architecture characteristics.Mature biofilm is formed at h 24 to h 48, then the biofilm begins to disperse.

  11. 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

    2017-04-01

    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.

  12. 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

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

    PubMed

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

    2000-09-01

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

  14. 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

  15. 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.

  16. Near-Infrared Confocal Laser Reflectance Cytoarchitectural Imaging of the Substantia Nigra and Cerebellum in the Fresh Human Cadaver.

    PubMed

    Cheyuo, Cletus; Grand, Walter; Balos, Lucia L

    2017-01-01

    Cytoarchitectural neuroimaging remains critical for diagnosis of many brain diseases. Fluorescent dye-enhanced, near-infrared confocal in situ cellular imaging of the brain has been reported. However, impermeability of the blood-brain barrier to most fluorescent dyes limits clinical utility of this modality. The differential degree of reflectance from brain tissue with unenhanced near-infrared imaging may represent an alternative technique for in situ cytoarchitectural neuroimaging. We assessed the utility of unenhanced near-infrared confocal laser reflectance imaging of the cytoarchitecture of the cerebellum and substantia nigra in 2 fresh human cadaver brains using a confocal near-infrared laser probe. Cellular images based on near-infrared differential reflectance were captured at depths of 20-180 μm from the brain surface. Parts of the cerebellum and substantia nigra imaged using the probe were subsequently excised and stained with hematoxylin and eosin for histologic correlation. Near-infrared reflectance imaging revealed the 3-layered cytoarchitecture of the cerebellum, with Purkinje cells appearing hyperreflectant. In the substantia nigra, neurons appeared hyporeflectant with hyperreflectant neuromelanin cytoplasmic inclusions. Cytoarchitecture of the cerebellum and substantia nigra revealed on near-infrared imaging closely correlated with the histology on hematoxylin-eosin staining. We showed that unenhanced near-infrared reflectance imaging of fresh human cadaver brain can reliably identify and distinguish neurons and detailed cytoarchitecture of the cerebellum and substantia nigra. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. 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

  18. 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.

  19. Assessment of corneal thickness and keratocyte density in a rabbit model of laser in situ keratomileusis using scanning laser confocal microscopy.

    PubMed

    Twa, Michael D; Giese, Michael J

    2011-12-01

    To determine the repeatability of corneal thickness and keratocyte density using in vivo confocal scanning laser microscopy in a rabbit model of laser in situ keratomileusis. Prospective, experimental animal study. En face tomographic images of corneal tissue were captured from 5 New Zealand white rabbits. Central corneal thickness was compared with conventional ultrasonic pachymetry. Keratocyte density was measured as a function of stromal depth at baseline and 6 weeks after a 130-μm lamellar incision in the following regions: first countable stromal image (30 to 39 μm), anterior stroma (40 to 75 μm), incision zone (76 to 150 μm), mid stroma (151 to 250 μm), and deep stroma (251 to 400 μm). The mean residual difference between ultrasonic and confocal corneal thickness measurements was 2.1 μm (95% confidence interval [CI], -7.0 to 11.2 μm; P = .61). Before the lamellar incision, keratocyte density was highest in the first countable frame of the anterior stroma, 53 800 cells/mm(3) (95% CI, 35 000 to 72 000 cells/mm(3)) and was least in deep stroma, 27 100 cells/mm(3) (95% CI, 22 400 to 32 000 cells/mm(3)). Six weeks after stromal lamellar incision, keratocyte density was unchanged in the first countable frame of the anterior stroma, 43 700 cells/mm(3) (95% CI, 31 800 to 55 500 cells/mm(3); P = .29). There were no changes in cell density in deeper stromal regions. There was excellent agreement between ultrasonic and confocal microscopy measurements of corneal thickness. In vivo repeatability of keratocyte density estimation using scanning laser confocal microscopy is comparable with the results of previous reports using tandem-scanning confocal microscopy. Keratocyte density was more varied, but not significantly different, in the anterior-most corneal stroma 6 weeks after a lamellar incision. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. In Vivo Confocal Microscopic Imaging of the Cornea After Femtosecond and Excimer Laser-assisted Penetrating Keratoplasty.

    PubMed

    Resch, Miklós D; Zemova, Elena; Marsovszky, László; Szentmáry, Nóra; Bauer, Florian; Daas, Loay; Pattmöller, Max; El-Husseiny, Moatasem; Németh, János; Nagy, Zoltán Z; Seitz, Berthold

    2015-09-01

    To examine the microstructure of the cornea after excimer and femtosecond laser-assisted penetrating keratoplasty (ELAK and FLAK) in eyes with Fuchs' dystrophy and keratoconus. Fifty-seven patients were divided into four groups according to corneal disease and surgical technique: Fuchs' dystrophy and ELAK (n = 9; mean age: 70.4 ± 10.6 years); Fuchs' dystrophy and FLAK (n = 13; mean age: 64.3 ± 11.2 years); keratoconus and ELAK (n = 9; mean age: 47.4 ± 13.9 years); and keratoconus and FLAK (n = 9; mean age: 43.5 ± 13.8 years). The control group comprised individuals without ocular disease (n = 17; mean age: 39.9 ± 17.3 years). In vivo investigation of the corneal graft and graft-host junction zone was performed with confocal corneal microscopy. All corneal grafts were transparent and no rejection reaction could be observed during the follow-up period. Confocal microscopy revealed no difference in basal epithelial cell density compared to controls. Anterior keratocyte density was lower than in the control group (818 ± 131 cells/mm(2)) in all four treatment groups (596 ± 174, 586 ± 113, 529 ± 75, 552 ± 91 cells/mm(2)). Langerhans cells could barely be seen; there was no difference in the cutting edge configuration and wound integrity. In vivo confocal microscopy provided evidence that good alignment of graft-host junction could be created with both techniques. The excimer laser was not inferior to the femtosecond laser in performing corneal cuts. The low density of Langerhans cells revealed well-controlled cellular immunological response and sustained corneal integrity in both laser groups. Copyright 2015, SLACK Incorporated.

  1. Raman beam combining for laser brightness enhancement

    DOEpatents

    Dawson, Jay W.; Allen, Graham S.; Pax, Paul H.; Heebner, John E.; Sridharan, Arun K.; Rubenchik, Alexander M.; Barty, Chrisopher B. J.

    2015-10-27

    An optical source capable of enhanced scaling of pulse energy and brightness utilizes an ensemble of single-aperture fiber lasers as pump sources, with each such fiber laser operating at acceptable pulse energy levels. Beam combining involves stimulated Raman scattering using a Stokes' shifted seed beam, the latter of which is optimized in terms of its temporal and spectral properties. Beams from fiber lasers can thus be combined to attain pulses with peak energies in excess of the fiber laser self-focusing limit of 4 MW while retaining the advantages of a fiber laser system of high average power with good beam quality.

  2. Combination free electron and gaseous laser

    DOEpatents

    Brau, Charles A.; Rockwood, Stephen D.; Stein, William E.

    1980-01-01

    A multiple laser having one or more gaseous laser stages and one or more free electron stages. Each of the free electron laser stages is sequentially pumped by a microwave linear accelerator. Subsequently, the electron beam is directed through a gaseous laser, in the preferred embodiment, and in an alternative embodiment, through a microwave accelerator to lower the energy level of the electron beam to pump one or more gaseous lasers. The combination laser provides high pulse repetition frequencies, on the order of 1 kHz or greater, high power capability, high efficiency, and tunability in the synchronous production of multiple beams of coherent optical radiation.

  3. Probe-based confocal laser endomicroscopy in the pancreatic duct provides direct visualization of ductal structures and aids in clinical management.

    PubMed

    Kahaleh, Michel; Turner, Brian G; Bezak, Karl; Sharaiha, Reem Z; Sarkaria, Savreet; Lieberman, Michael; Jamal-Kabani, Armeen; Millman, Jennifer E; Sundararajan, Subha V; Chan, Ching; Mehta, Shivani; Widmer, Jessica L; Gaidhane, Monica; Giovannini, Marc

    2015-03-01

    Confocal endomicroscopy provides real-time evaluation of various sites and has been used to provide detailed endomicroscopic imaging of the biliary tree. We aimed to evaluate the feasibility and utility of probe-based confocal laser endomicroscopy of the pancreatic duct as compared to cytologic and histologic results in patients with indeterminate pancreatic duct strictures. Retrospective data on patients with indeterminate pancreatic strictures undergoing endoscopic retrograde cholangiopancreatography (ERCP) and confocal endomicroscopy were collected from two tertiary care centres. Real-time confocal endomicroscopy images were obtained during ERCP and immediate interpretation according to the Miami Classification was performed. 18 patients underwent confocal endomicroscopy for evaluation of pancreatic strictures from July 2011 to December 2012. Mean pancreatic duct size was 4.2mm (range 2.2-8mm). Eight cases were interpreted as benign, 4 as malignant, 4 suggestive of intraductal papillary mucinous neoplasms, and 2 appeared normal. Cytology/histopathology for 15/16 cases showed similar results to confocal endomicroscopy interpretation. Kappa coefficient of agreement between cyto/histopathology and confocal endomicroscopy was 0.8 (p=0.0001). Pancreatic confocal endomicroscopy changed management in four patients, changing the type of surgery from total pancreatectomy to whipple. Confocal endomicroscopy is effective in assisting with diagnosis of indeterminate pancreatic duct strictures as well as mapping of abnormal pancreatic ducts prior to surgery. Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  4. Surface emitting lasers with combined output

    NASA Technical Reports Server (NTRS)

    Carlin, Donald B. (Inventor)

    1990-01-01

    Surface emitting lasers are laterally aligned and coupled together and also have their light output signals combined. This results in greater phase and frequency coherency and narrower and reduced amplitude sidelobes. Preferably, not more than two lasers are longitudinally aligned along the same axis for still greater coherency compared with adding the light output signals of more than two longitudinally aligned lasers. The lasers can be of the DH-LOC type or of the QW type.

  5. LASER BEAMS. CAVITIES: Formation of a beam with a uniform intensity distribution in a CO2 laser with a generalised confocal cavity

    NASA Astrophysics Data System (ADS)

    Gurin, O. V.; Yepishin, V. A.; Maslov, Vyacheslav A.; Militinskii, I. M.; Svich, V. A.; Topkov, A. N.

    1998-05-01

    A description is given of a method of forming, in a composite confocal cavity, non-Gaussian light beams with a uniform intensity distribution. The method is based on the use of a reflector with discretely distributed absorbing or scattering inhomogeneities. Theoretical and experimental confirmations are reported of the generation of such beams at the output of a CO2 laser with a nonuniform amplitude-stepped mirror.

  6. Software for automated classification of probe-based confocal laser endomicroscopy videos of colorectal polyps

    PubMed Central

    André, Barbara; Vercauteren, Tom; Buchner, Anna M; Krishna, Murli; Ayache, Nicholas; Wallace, Michael B

    2012-01-01

    AIM: To support probe-based confocal laser endomicroscopy (pCLE) diagnosis by designing software for the automated classification of colonic polyps. METHODS: Intravenous fluorescein pCLE imaging of colorectal lesions was performed on patients undergoing screening and surveillance colonoscopies, followed by polypectomies. All resected specimens were reviewed by a reference gastrointestinal pathologist blinded to pCLE information. Histopathology was used as the criterion standard for the differentiation between neoplastic and non-neoplastic lesions. The pCLE video sequences, recorded for each polyp, were analyzed off-line by 2 expert endoscopists who were blinded to the endoscopic characteristics and histopathology. These pCLE videos, along with their histopathology diagnosis, were used to train the automated classification software which is a content-based image retrieval technique followed by k-nearest neighbor classification. The performance of the off-line diagnosis of pCLE videos established by the 2 expert endoscopists was compared with that of automated pCLE software classification. All evaluations were performed using leave-one-patient-out cross-validation to avoid bias. RESULTS: Colorectal lesions (135) were imaged in 71 patients. Based on histopathology, 93 of these 135 lesions were neoplastic and 42 were non-neoplastic. The study found no statistical significance for the difference between the performance of automated pCLE software classification (accuracy 89.6%, sensitivity 92.5%, specificity 83.3%, using leave-one-patient-out cross-validation) and the performance of the off-line diagnosis of pCLE videos established by the 2 expert endoscopists (accuracy 89.6%, sensitivity 91.4%, specificity 85.7%). There was very low power (< 6%) to detect the observed differences. The 95% confidence intervals for equivalence testing were: -0.073 to 0.073 for accuracy, -0.068 to 0.089 for sensitivity and -0.18 to 0.13 for specificity. The classification software proposed

  7. A novel approach of optical biopsy using probe-based confocal laser endomicroscopy for peritoneal metastasis.

    PubMed

    Hara, Hisashi; Takahashi, Tsuyoshi; Nakatsuka, Rie; Higashi, Shigeyoshi; Naka, Tetsuji; Sumiyama, Kazuki; Miyazaki, Yasuhiro; Makino, Tomoki; Kurokawa, Yukinori; Yamasaki, Makoto; Takiguchi, Shuji; Mori, Masaki; Doki, Yuichiro; Nakajima, Kiyokazu

    2016-08-01

    In digestive cancers, it is mandatory to diagnose peritoneal metastasis prior to selecting therapy. Therefore, exploratory laparoscopy has gained wider clinical acceptance. In laparoscopy, the peritoneal metastasis is pathologically confirmed by excisional biopsy; however, there remain technical difficulties in performing precise diagnosis and adequate biopsy on small peritoneal lesions without damaging organs. We have focused on "optical biopsy" using probe-based confocal laser endomicroscopy (pCLE). The aims of this study were (1) to optimize current CLE system for real-time observation of peritoneal metastases and (2) to assess its potential usefulness as diagnostic modality in preclinical settings. To optimize condition and evaluate feasibility, we prepared peritoneal metastasis mice model with gastric cancer cell line (MKN-45). On Day 10 after seeding, the mice were laparotomized and performed pCLE observations with CellvizioLAB (LSU-F 400/488 nm, Mauna Kea Technologies, Paris, France). We evaluated two different CLE probes, three different dyes, and optimal interval time. The detected sites were excised and pathologically evaluated on its morphology. Next, the feasibility and safety were validated in porcine model for clinical usage. After injection of fluorescein, pCLE was applied for the observation of intra-abdominal organs. A miniature probe-type pCLE system with 60 μm focal depth (UltraMini O) and 1 % fluorescein dye was chosen for good visualization in mice model. The irregular microarchitecture images suspected to malignancy were obtained from the metastases. In the porcine model, observation of abdominal organs was feasible without any organ injury in the laparoscopic procedures. The dosage of 1 % fluorescein (3 ml/body) was appropriate in observing intra-abdominal organs, and each intra-abdominal organ was clearly observed with the same imaging quality we obtained in mice model. The pCLE was feasible and safe and potentially useful for the

  8. Exploring the optimal fluorescein dose in probe-based confocal laser endomicroscopy for colonic imaging

    PubMed Central

    Shahid, Muhammad W; Crook, Julia E; Meining, Alexander; Perchant, Aymeric; Buchner, Anna; Gomez, Victoria

    2011-01-01

    Background Probe-based confocal laser endomicroscopy (pCLE) is an emerging method for in-vivo imaging of the gastrointestinal tract and requires a contrast agent. Fluorescein is the most commonly used agent. The optimal dose of fluorescein for pCLE in colon is unknown. Objective Exploration of optimal dose of fluorescein for pCLE in colon. Design Comparative, prospective pilot trail. Setting Tertiary-care center. Patients 18 participants underwent colonoscopy without complications. Interventions pCLE videos were recorded in normal cecum, using 10% fluorescein intravenously. Main Outcome Measurements For subjective analysis, pCLE videos were scored for quality, by 2 observers, independently and blinded to fluorescein dose. For objective analysis, signal-to-noise ratios (SNR) were calculated for each video by an expert. Results 6 fluorescein doses were used, including 0.5 mL, 1 mL, 2.5 mL, 5 mL, 7.5 mL and 10 mL and each dose was used in three patients. For each dose, median image quality score was 2.5, 2.0, 3.25, 4.0, 4.0 and 3.5 by first observer and 2.0, 3.0, 4.0, 5.0, 4.0 and 4.0 by second observer, respectively. The subjective quality scores increased from 0.5 mL to 5.0 mL, with no evidence of further improved quality at 7.5 mL and 10 mL doses. SNR were not significantly different between doses but trended higher for higher doses. Limitations Small sample size. The results can not be applied to other parts of gastrointestinal tract i.e. duodenum, esophagus with different blood supply. Conclusion This preliminary study suggests that the optimal dose of fluorescein for high quality pCLE imaging in colon is approximately 5.0 mL. PMID:22586530

  9. Software for automated classification of probe-based confocal laser endomicroscopy videos of colorectal polyps.

    PubMed

    André, Barbara; Vercauteren, Tom; Buchner, Anna M; Krishna, Murli; Ayache, Nicholas; Wallace, Michael B

    2012-10-21

    To support probe-based confocal laser endomicroscopy (pCLE) diagnosis by designing software for the automated classification of colonic polyps. Intravenous fluorescein pCLE imaging of colorectal lesions was performed on patients undergoing screening and surveillance colonoscopies, followed by polypectomies. All resected specimens were reviewed by a reference gastrointestinal pathologist blinded to pCLE information. Histopathology was used as the criterion standard for the differentiation between neoplastic and non-neoplastic lesions. The pCLE video sequences, recorded for each polyp, were analyzed off-line by 2 expert endoscopists who were blinded to the endoscopic characteristics and histopathology. These pCLE videos, along with their histopathology diagnosis, were used to train the automated classification software which is a content-based image retrieval technique followed by k-nearest neighbor classification. The performance of the off-line diagnosis of pCLE videos established by the 2 expert endoscopists was compared with that of automated pCLE software classification. All evaluations were performed using leave-one-patient-out cross-validation to avoid bias. Colorectal lesions (135) were imaged in 71 patients. Based on histopathology, 93 of these 135 lesions were neoplastic and 42 were non-neoplastic. The study found no statistical significance for the difference between the performance of automated pCLE software classification (accuracy 89.6%, sensitivity 92.5%, specificity 83.3%, using leave-one-patient-out cross-validation) and the performance of the off-line diagnosis of pCLE videos established by the 2 expert endoscopists (accuracy 89.6%, sensitivity 91.4%, specificity 85.7%). There was very low power (< 6%) to detect the observed differences. The 95% confidence intervals for equivalence testing were: -0.073 to 0.073 for accuracy, -0.068 to 0.089 for sensitivity and -0.18 to 0.13 for specificity. The classification software proposed in this study is not a

  10. Evaluation of Corneal Microstructure in Pseudoexfoliation Syndrome and Glaucoma: In Vivo Scanning Laser Confocal Microscopic Study.

    PubMed

    Yüksel, Nurşen; Emre, Esra; Pirhan, Dilara

    2016-01-01

    To quantitatively evaluate corneas of patients with pseudoexfoliation syndrome (PXS) and pseudoexfoliation glaucoma (PXG) using in vivo scanning laser confocal microscopy (IVCM). The study population comprised 30 patients with PXS, 30 patients with PXG, and 30 normal control subjects. IVCM of the cornea was performed on all participants using the Rostock Cornea Module of the Heidelberg Retinal Tomograph (HRT). Mean outcome measures included density of basal epithelial cells, endothelial cells, and anterior and posterior keratocytes; and tortuosity and density of subbasal plexus nerves. Mean densities of basal epithelial cells, endothelial cells, anterior and posterior keratocytes, and subbasal nerves differed significantly among the three groups. Subbasal nerve densities were significantly diminished in PXS and PXG patients (12.36 ± 2.89 and 6.8 ± 3.42 mm/mm(2), respectively) compared with that of control subjects (16.13 ± 3.42 mm/mm(2)) (p < 0.05). Mean densities of anterior and posterior stromal keratocytes were significantly lower in PXS and PXG patients compared with control subjects (p < 0.05). Endothelial cell densities were 3073.63 ± 654.49, 2592.60 ± 276.36, and 2110.20 ± 620.53 cells/mm(2) for control, PXS, and PXG groups, respectively (p < 0.05). The percentages of endothelial cell polymegathism and pleomorphism were higher in PXS and PXG patients compared with control subjects. Endothelial cell polymegathism and pleomorphism were more frequently associated with PXG. Results of this study demonstrate the existence of alterations in the (i) density of cells in the various layers of the cornea, (ii) cellular configuration of corneal endothelial cells, and (iii) density/diameter of the subbasal nerve plexus in patients with PXS, and that such alterations are common in patients with PXG. It would be beneficial to employ IVCM to assess the severity of pseudoexfoliation keratopathy (PXK).

  11. 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

  12. The neuromuscular system of Pycnophyes kielensis (Kinorhyncha: Allomalorhagida) investigated by confocal laser scanning microscopy.

    PubMed

    Altenburger, Andreas

    2016-01-01

    Kinorhynchs are ecdysozoan animals with a phylogenetic position close to priapulids and loriciferans. To understand the nature of segmentation within Kinorhyncha and to infer a probable ancestry of segmentation within the last common ancestor of Ecdysozoa, the musculature and the nervous system of the allomalorhagid kinorhynch Pycnophyes kielensis were investigated by use of immunohistochemistry, confocal laser scanning microscopy, and 3D reconstruction software. The kinorhynch body plan comprises 11 trunk segments. Trunk musculature consists of paired ventral and dorsal longitudinal muscles in segments 1-10 as well as dorsoventral muscles in segments 1-11. Dorsal and ventral longitudinal muscles insert on apodemes of the cuticle inside the animal within each segment. Strands of longitudinal musculature extend over segment borders in segments 1-6. In segments 7-10, the trunk musculature is confined to the segments. Musculature of the digestive system comprises a strong pharyngeal bulb with attached mouth cone muscles as well as pharyngeal bulb protractors and retractors. The musculature of the digestive system shows no sign of segmentation. Judged by the size of the pharyngeal bulb protractors and retractors, the pharyngeal bulb, as well as the introvert, is moved passively by internal pressure caused by concerted action of the dorsoventral muscles. The nervous system comprises a neuropil ring anterior to the pharyngeal bulb. Associated with the neuropil ring are flask-shaped serotonergic somata extending anteriorly and posteriorly. A ventral nerve cord is connected to the neuropil ring and runs toward the anterior until an attachment point in segment 1, and from there toward the posterior with one ganglion in segment 6. Segmentation within Kinorhyncha likely evolved from an unsegmented ancestor. This conclusion is supported by continuous trunk musculature in the anterior segments 1-6, continuous pharyngeal bulb protractors and retractors throughout the anterior

  13. 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

  14. Three-dimensional visualization and quantitation of fibrin in solid tumors by confocal laser scanning microscopy.

    PubMed

    Biggerstaff, J; Amirkhosravi, A; Francis, J L

    1997-10-01

    Fibrin forms part of the stroma essential for growth of solid tumors. Anticoagulants reduce primary tumor growth and tumor metastasis in murine and some human tumors. These effects may be partly mediated by reduction of intra-tumor fibrin, although there are no quantitative data to support this hypothesis. We therefore evaluated the effect of warfarin on fibrin deposition in a subcutaneously (s.c.) implanted murine tumor using confocal laser scanning microscopy (CLSM). AJ mice received no treatment (n = 6) or sodium warfarin (3.5 mg/L in drinking water, n = 5). All animals received 2 x 10(6) syngeneic Neuro2a neuroblastoma cells s.c. After 14 days, primary tumors were excised and placed in liquid nitrogen. Warfarin treatment resulted in a small, but significant (P < 0.05), decrease in wet tumor weight. Frozen sections (20 microns) were incubated with goat anti-mouse fibrin(ogen) or normal goat serum (isotypic control) and stained with FITC-conjugated rabbit anti-goat antibody. Using a Multiprobe 2001 CLSM (Molecular Dynamics, Sunnyvale, CA), 20 serial optical sections were taken from five, randomly chosen, high power fields (60x objective) for each slide. A threshold excluded all fluorescence except that from structural components within the tumor stroma (fibrin). The volume of fibrin in each section series was determined, and the percentage of tumor volume occupied by fibrin calculated. Intra- and inter-assay variation were assessed on serial frozen tumor sections from an untreated animal. The percentage fibrin volume was not significantly different among or within experiments, indicating that the procedure was reproducible. In controls, the median (range) volume occupied by fibrin was 8.1% (2.4-22.3%), whereas in anticoagulated animals, this was reduced to 3.7% (0.4-14.0%; P < 0.001). This is the first quantitative demonstration that warfarin reduces fibrin deposition in solid tumors. We conclude that three-dimensional CLSM is useful for the quantitation of

  15. Comparing optic nerve head analysis between confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography.

    PubMed

    Roberti, Gloria; Centofanti, Marco; Oddone, Francesco; Tanga, Lucia; Michelessi, Manuele; Manni, Gianluca

    2014-10-01

    Confocal scanning laser ophthalmoscopy, HRT3, and spectral domain optical coherence tomography (OCT), RTVue-100, are able to give 3-dimensional (3D) topography images of optic nerve head (ONH) and to derive stereometric parameters and sectorial analysis. The purpose of the study is to evaluate the agreement of these two devices and their diagnostic accuracy to discriminate eyes with glaucoma from those without. Glaucoma patients and healthy control subjects were included. All of them underwent a complete ophthalmological examination, including slit lamp evaluation and visual field (VF) test. After pupil dilatation, HRT3 and RTVue-100 were performed. The following stereometric parameters were recorded: disc area, rim area, rim volume, cup volume, cup area, cup/disk ratio, and the following sectors, superotemporal, superonasal, inferotemporal, inferonasal. Forty-six eyes of 46 glaucoma patients and 58 eyes of 58 healthy subjects were included in the study. In both groups, HRT3 rim area and rim volume were statistically higher than RTVue-100 (glaucomas: 0.95 ± 0.38 versus 0.44 ± 0.33 and 0.19 ± 0.13 versus 0.02 ± 0.03, p < 0.01. controls: 1.41 ± 0.30 versus 1.08 ± 0.37 and 0.37 ± 0.13 versus 0.14 ± 0.11, p < 0.01), while cup area was statistically higher by RTVue-100 (glaucomas: 1.42 ± 0.57 versus 1.14 ± 0.58, p < 0.01. controls: 1.05 ± 1.35 versus 0.65 ± 0.48). Bland and Altman plots confirmed the presence of a fixed bias. The parameters with largest AUROC were rim volume, rim area and cup/disk ratio for both instruments. HRT3 inferotemporal sector had the highest sensitivity (80.43%, at 75.9% specificity), while for RTVue-100, the superotemporal sector had the highest sensitivity (76.1%, at 81% specificity). The agreement was moderate for inferotemporal sector and fair for the others. HRT3 and RTVue-100 are not interchangeable for ONH analysis. They both have good diagnostic accuracy, but RTVue

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

    PubMed

    Ştefănescu, Daniela; Streba, Costin; Cârţână, Elena Tatiana; Săftoiu, Adrian; Gruionu, Gabriel; Gruionu, Lucian Gheorghe

    2016-01-01

    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. 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. 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 operations were cross-entropy with the

  17. 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

  18. Stromal haze after combined riboflavin-UVA corneal collagen cross-linking in keratoconus: in vivo confocal microscopic evaluation.

    PubMed

    Mazzotta, Cosimo; Balestrazzi, Angelo; Baiocchi, Stefano; Traversi, Claudio; Caporossi, Aldo

    2007-08-01

    The technique of corneal collagen cross-linking consists of photopolymerization of stromal fibres by the combined action of a photosensitizing substance (riboflavin or vitamin B2) and ultraviolet light from a solid state UVA source. Photopolymerization increases the rigidity of corneal collagen and its resistance to keratectasia. In this report we present two cases, studied through in vivo confocal microscopy, with stage III keratoconus that developed stromal haze after the cross-linking treatment.

  19. 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

  20. 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.

  1. Basic technique and anatomically imposed limitations of confocal scanning laser Doppler flowmetry at the optic nerve head level.

    PubMed

    Sehi, Mitra

    2011-02-01

    Many studies have suggested an association between blood flow dysregulation and glaucomatous damage to the optic nerve. Confocal scanning laser Doppler flowmetry (CSLDF) is a technique that measures the capillary blood flow of the retina and optic nerve head and provides a two-dimensional map of ocular perfusion in these areas. This review discusses the anatomy of the anterior optic nerve vasculature and the capabilities and limitations of the CSLDF. Methods to minimize error and to acquire more reliable measurements of capillary blood flow are also outlined. © 2009 The Authors. Journal compilation © 2009 Acta Ophthalmol.

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

    NASA Astrophysics Data System (ADS)

    Risi, Matthew D.

    Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100 μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (˜1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography

  3. 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

  4. Identification of ex-vivo confocal laser scanning microscopic features of melanocytic lesions and their histological correlates.

    PubMed

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

    2017-01-01

    Ex-vivo confocal laser scanning microscopy (CLSM) offers rapid tissue examination. Current literature shows promising results in the evaluation of non-melanoma skin cancer but little is known about presentation of melanocytic lesions (ML). This study evaluates ML with ex-vivo CLSM in comparison to histology and offers an overview of ex-vivo CLSM characteristics. 31 ML were stained with acridine orange or fluorescein and examined using ex-vivo CLSM (Vivascope2500(®) ; Lucid Inc; Rochester NY) in reflectance and fluorescence mode. Confocal images were correlated to histopathology. Benign and malignant features of the ML were listed and results were presented. Sensitivity and specificity were calculated using contingency tables. The ML included junctional, compound, dermal, Spitz and dysplastic nevi, as well as various melanoma subtypes. The correlation of the confocal findings with histopathology allowed the identification of different types of ML and differentiation of benign and malignant features. The study offers an overview of confocal characteristics of ML in comparison to histology. Ex-vivo CLSM does not reproduce the typical in-vivo horizontal mosaics but rather reflects the vertical histological presentation. Not all typical in-vivo patterns are detectable here. These findings may help to evaluate the ex-vivo CLSM as an adjunctive tool in the immediate intraoperative diagnosis of ML. Superficial spreading malignant melanoma. Histopathology (H&E stain; 200×) correlated to the reflectance (RM; 830 nm) and fluorescence mode (FM; 488 nm) in the ex-vivo CLSM (Vivablock(®) by VivaScan(®) , acridine orange). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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)

  6. Preliminary identification of unicellular algal genus by using combined confocal resonance Raman spectroscopy with PCA and DPLS analysis.

    PubMed

    He, Shixuan; Xie, Wanyi; Zhang, Ping; Fang, Shaoxi; Li, Zhe; Tang, Peng; Gao, Xia; Guo, Jinsong; Tlili, Chaker; Wang, Deqiang

    2017-09-18

    The analysis of algae and dominant alga plays important roles in ecological and environmental fields since it can be used to forecast water bloom and control its potential deleterious effects. Herein, we combine in vivo confocal resonance Raman spectroscopy with multivariate analysis methods to preliminary identify the three algal genera in water blooms at unicellular scale. Statistical analysis of characteristic Raman peaks demonstrates that certain shifts and different normalized intensities, resulting from composition of different carotenoids, exist in Raman spectra of three algal cells. Principal component analysis (PCA) scores and corresponding loading weights show some differences from Raman spectral characteristics which are caused by vibrations of carotenoids in unicellular algae. Then, discriminant partial least squares (DPLS) classification method is used to verify the effectiveness of algal identification with confocal resonance Raman spectroscopy. Our results show that confocal resonance Raman spectroscopy combined with PCA and DPLS could handle the preliminary identification of dominant alga for forecasting and controlling of water blooms. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. 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.

  8. 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.

  9. Use of endoscopic distal attachment cap to enhance image stabilization in probe-based confocal laser endomicroscopy in colorectal lesions*

    PubMed Central

    Ussui, Vivian; Xu, Can; Crook, Julia E.; Diehl, Nancy N.; Hardee, Joy; Staggs, Estela G.; Shahid, Muhammad W.; Wallace, Michael B.

    2015-01-01

    Background and study aims: Colorectal cancer can be prevented through the use of colonoscopy with polypectomy. Most colon polyps are benign or low grade adenomas. However, currently all lesions need histopathologic analysis, which increases diagnostic costs and delays the final diagnosis. Confocal laser endomicroscopy (CLE) is a new technology that enables real-time endomicroscopy. However, there are challenges to maintaining a stable image with currently available systems. We conducted a small study to obtain a preliminary assessment of whether the use of an endoscopic distal attachment cap may enhance image quality of CLE in comparison with images obtained with free-hand acquisition. Patients and methods: Forty outpatients underwent colonoscopy for evaluation of colon polyps in a single academic medical center. Patients were assigned randomly to 1 of 2 study arms on the basis of whether an endoscopic distal attachment cap was used (n = 21, Cap Used) or not used (n = 19, No Cap) in the procedure. The quality of confocal images and probe stabilization was summarized. Results: A total of 81 polyps were identified. The proportion of polyps with images of high quality was 74 % (28/38) in the Cap Used group and 79 % (30/38) in the No Cap arm. Image stability was also similar with and without a cap. Diagnostic accuracy was estimated to be slightly higher in the Cap Used group for probe-based confocal laser endomicroscopy (pCLE; 78 % vs 70 %). This was also true for white-light and narrow-band imaging. Conclusions: This preliminary study did not yield any evidence to support that the use of an endoscopic distal attachment cap improves the quality of images obtained during CLE. PMID:26528511

  10. 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

  11. Use of endoscopic distal attachment cap to enhance image stabilization in probe-based confocal laser endomicroscopy in colorectal lesions.

    PubMed

    Ussui, Vivian; Xu, Can; Crook, Julia E; Diehl, Nancy N; Hardee, Joy; Staggs, Estela G; Shahid, Muhammad W; Wallace, Michael B

    2015-10-01

    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. 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. 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. 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.

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

    PubMed

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

    2006-11-05

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

  13. Purchase of a Laser Scanning Confocal Microscope at Xavier University of Louisiana

    DTIC Science & Technology

    2016-05-04

    proposal was taught this semester . Senior- level undergraduate students used the confocal microscope to perform immunofluorescence studies of...Principles and Techniques) that was proposed was indeed taught in the Spring 2016 semester . Xavier University’s 1. REPORT DATE (DD-MM-YYYY) 4. TITLE...undergraduate course (Pathology: Principles and Techniques) that was proposed was indeed taught in the Spring 2016 semester . Xavier University’s

  14. [Opportunities for confocal and laser biomicroscopy of corneal nerves in diabetic polyneuropathy].

    PubMed

    Surnina, Z V

    2015-01-01

    The review concerns corneal nerves involvement in diabetes mellitus (DM), a pressing issue for ophthalmology and endocrinology. The history of research in this field along with anatomical, physiological, and biochemical features of corneal nerves is provided. Corneal nerves anatomy is described in accordance with Soviet scientific school and contemporary foreign sources. The most part of the paper is devoted to technical description of a confocal microscope and Heidelberg Retina Tomograph with corneal module as well as the feasibility of corneal nerves visualization. Diabetic neuropathy, a threatening complication of DM that can result in lower limb amputations, is discussed. A number of authors suggest confocal biomicroscopy for early diagnosis of polyneuropathy, yet few relevant publications can be found. If effective, confocal biomicroscopy can be considered as a possible screening tool able to detect early signs of diabetes complications and thus to ensure the treatment initiated in a timely manner. The latter is crucial to prevent DM progression to its terminal stage--diabetic polyneuropathy, which is dangerous of lower limb amputations.

  15. 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

    2017-04-01

    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. © 2017 Anatomical Society.

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

    PubMed Central

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

    2015-01-01

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

  17. 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

  18. Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model.

    PubMed

    Klemm, Matthias; Blum, Johannes; Link, Dietmar; Hammer, Martin; Haueisen, Jens; Schweitzer, Dietrich

    2016-09-01

    Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes determined from the intensity decays. Here, we present a new technique to suppress the autofluorescence of the crystalline lens by introducing an annular stop into the detection light path, which we call Schweitzer's principle. The efficacy of annular stops with an outer diameter of 7 mm and inner diameters of 1 to 5 mm are analyzed in an experimental setup using a model eye based on fluorescent dyes. Compared to the confocal principle, Schweitzer's principle with an inner diameter of 3 mm is able to reduce the simulated crystalline lens fluorescence to 4%, while 42% of the simulated retina fluorescence is preserved. Thus, we recommend the implementation of Schweitzer's principle in scanning laser ophthalmoscopes used for fundus autofluorescence measurements, especially the FLIO device, for improved image quality.

  19. Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model

    PubMed Central

    Klemm, Matthias; Blum, Johannes; Link, Dietmar; Hammer, Martin; Haueisen, Jens; Schweitzer, Dietrich

    2016-01-01

    Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes determined from the intensity decays. Here, we present a new technique to suppress the autofluorescence of the crystalline lens by introducing an annular stop into the detection light path, which we call Schweitzer’s principle. The efficacy of annular stops with an outer diameter of 7 mm and inner diameters of 1 to 5 mm are analyzed in an experimental setup using a model eye based on fluorescent dyes. Compared to the confocal principle, Schweitzer’s principle with an inner diameter of 3 mm is able to reduce the simulated crystalline lens fluorescence to 4%, while 42% of the simulated retina fluorescence is preserved. Thus, we recommend the implementation of Schweitzer’s principle in scanning laser ophthalmoscopes used for fundus autofluorescence measurements, especially the FLIO device, for improved image quality. PMID:27699092

  20. In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

    PubMed Central

    Assmus, B.; Hutzler, P.; Kirchhof, G.; Amann, R.; Lawrence, J. R.; Hartmann, A.

    1995-01-01

    The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology. PMID:16534951

  1. Combined reflectance confocal microscopy-optical coherence tomography for delineation of basal cell carcinoma margins: an ex vivo study

    NASA Astrophysics Data System (ADS)

    Iftimia, Nicusor; Peterson, Gary; Chang, Ernest W.; Maguluri, Gopi; Fox, William; Rajadhyaksha, Milind

    2016-01-01

    We present a combined reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) approach, integrated within a single optical layout, for diagnosis of basal cell carcinomas (BCCs) and delineation of margins. While RCM imaging detects BCC presence (diagnoses) and its lateral spreading (margins) with measured resolution of ˜1 μm, OCT imaging delineates BCC depth spreading (margins) with resolution of ˜7 μm. When delineating margins in 20 specimens of superficial and nodular BCCs, depth could be reliably determined down to ˜600 μm, and agreement with histology was within about ±50 μm.

  2. Combined reflectance confocal microscopy-optical coherence tomography for delineation of basal cell carcinoma margins: an ex vivo study

    PubMed Central

    Iftimia, Nicusor; Peterson, Gary; Chang, Ernest W.; Maguluri, Gopi; Fox, William; Rajadhyaksha, Milind

    2016-01-01

    Abstract. We present a combined reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) approach, integrated within a single optical layout, for diagnosis of basal cell carcinomas (BCCs) and delineation of margins. While RCM imaging detects BCC presence (diagnoses) and its lateral spreading (margins) with measured resolution of ∼1  μm, OCT imaging delineates BCC depth spreading (margins) with resolution of ∼7  μm. When delineating margins in 20 specimens of superficial and nodular BCCs, depth could be reliably determined down to ∼600  μm, and agreement with histology was within about ±50  μm. PMID:26780224

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

    PubMed Central

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

    2016-01-01

    This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response. PMID:27806070

  4. Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

    PubMed

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

    2016-01-01

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

  5. Line FRAP with the Confocal Laser Scanning Microscope for Diffusion Measurements in Small Regions of 3-D Samples

    PubMed Central

    Braeckmans, Kevin; Remaut, Katrien; Vandenbroucke, Roosmarijn E.; Lucas, Bart; De Smedt, Stefaan C.; Demeester, Joseph

    2007-01-01

    We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells. PMID:17208970

  6. 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

  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. 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. © 2016 John Wiley & Sons Ltd.

  9. 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.

  10. Combined analysis of in situ hybridization, cell cycle and structural markers using reflectance and immunofluorescence confocal microscopy.

    PubMed

    Linares-Cruz, G; Millot, G; De Cremoux, P; Vassy, J; Olofsson, B; Rigaut, J P; Calvo, F

    1995-01-01

    A method for the simultaneous detection of mRNA by reflectance in situ hybridization (RISH), cell cycle and structural markers by immunofluorescence using confocal laser scanning microscopy is presented. The mRNA expression of two ras-related genes rhoB and rhoC was analysed in human breast cancer cell lines and human histological specimens (breast cancer tissues and skin biopsies). In breast cancer cell lines, the conditions were optimized to detect RNA-RNA hybrids and DNA synthesis after pulse-labelling with bromodeoxyuridine. Endonuclease-exonuclease digestion, which allows the accessibility to specific antibodies of halogenated pyrimidine molecules, was carried out following ISH. Finally, cytokeratin or vimentin staining was performed. The detection of signals, arising from 1-nm colloidal gold particles without silver enhancement, by reflectance confocal laser scanning microscopy is described. Bromodeoxybiridine DNA markers and cytokeratin/vimentin staining were detected concomitantly using different fluorochromes. To allow comparative expression of two related genes, the mRNA of rhoB and rhoC were detected using digoxigenin- or biotin-labelled riboprobes and, after 3-D imaging, a detailed analysis by optical horizontal (x, y) and vertical (x, z) sectioning was undertaken. The subsequent bromodeoxyuridine detection procedure permitted to us explore the specific transcription of these two genes during S and non-S phases. This method allows the identification and localization of several subcellular components in cells within a complex tissue structure and makes it possible to analyse further transcript localization in relation to the function of the encoded protein and to the cell cycle.

  11. 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

  12. [In vivo reflectance-mode confocal laser microscopy: basic principles and clinical and research employments in dermatology].

    PubMed

    Levi, Assi; Ingber, Arieh; Enk, David Claes

    2012-10-01

    Reflectance-mode confocal scanning laser microscopy is a novel, non-invasive imaging technique which permits real time visualization of cellular components in the skin at a resolution close to that of conventional histology. It has been widely used in the diagnosis of both benign and malignant tumors of the skin. In recent years it was also employed in the investigation of a variety of inflammatory and infectious skin conditions. The non-invasive nature of the procedure allows examination of multiple lesions and/ or repetitive sampling of one lesion over time, making it an excellent tool for followup and for monitoring treatment outcome in medical and cosmetic dermatology. This review summarizes the main indications for the use of this novel technique in clinical and experimental dermatology.

  13. 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.

  14. 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.

  15. A novel approach to pseudopodia proteomics: excimer laser etching, two-dimensional difference gel electrophoresis, and confocal imaging

    PubMed Central

    Mimae, Takahiro; Ito, Akihiko; Hagiyama, Man; Nakanishi, Jun; Hosokawa, Yoichiroh; Okada, Morihito; Murakami, Yoshinori; Kondo, Tadashi

    2014-01-01

    Pseudopodia are actin-rich ventral cellular protrusions shown to facilitate the migration and metastasis of tumor cells. Here, we present a novel approach to perform pseudopodia proteomics. Tumor cells growing on porous membranes extend pseudopodia into the membrane pores. In our method, cell bodies are removed by horizontal ablation at the basal cell surface with the excimer laser while pseudopodia are left in the membrane pores. For protein expression profiling, whole cell and pseudopodia proteins are extracted with a lysis buffer, labeled with highly sensitive fluorescent dyes, and separated by two-dimensional gel electrophoresis. Proteins with unique expression patterns in pseudopodia are identified by mass spectrometry. The effects of the identified proteins on pseudopodia formation are evaluated by measuring the pseudopodia length in cancer cells with genetically modified expression of target proteins using confocal imaging. This protocol allows global identification of pseudopodia proteins and evaluation of their functional significance in pseudopodia formation within one month. PMID:25309719

  16. 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.

  17. 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.

  18. 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.

  19. Prospective Study of the Diagnostic Accuracy of the In Vivo Laser Scanning Confocal Microscope for Severe Microbial Keratitis.

    PubMed

    Chidambaram, Jaya D; Prajna, Namperumalsamy V; Larke, Natasha L; Palepu, Srikanthi; Lanjewar, Shruti; Shah, Manisha; Elakkiya, Shanmugam; Lalitha, Prajna; Carnt, Nicole; Vesaluoma, Minna H; Mason, Melanie; Hau, Scott; Burton, Matthew J

    2016-11-01

    graders to have a specific organism present (10 fungus, 1 Acanthamoeba) but had negative results via culture and light microscopy. Laser scanning IVCM performed with experienced confocal graders has high sensitivity, specificity, and test reproducibility for detecting fungal filaments and Acanthamoeba cysts in moderate to large corneal ulcers in India. This imaging modality was particularly useful for detecting organisms in deep ulcers in which culture and light microscopy results were negative. Copyright © 2016 American Academy of Ophthalmology. All rights reserved.

  20. 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

  1. 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.

  2. 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.

  3. Beam combining of quantum cascade laser arrays.

    PubMed

    Lee, Benjamin G; Kansky, Jan; Goyal, Anish K; Pflügl, Christian; Diehl, Laurent; Belkin, Mikhail A; Sanchez, Antonio; Capasso, Federico A

    2009-08-31

    Wavelength beam combining was used to co-propagate beams from 28 elements in an array of distributed-feedback quantum cascade lasers (DFB-QCLs). The beam-quality product of the array, defined as the product of near-field spot size and far-field divergence for the entire array, was improved by a factor of 21 by using wavelength beam combining. To demonstrate the applicability of wavelength beam combined DFB-QCL arrays for remote sensing, we obtained the absorption spectrum of isopropanol at a distance of 6 m from the laser array.

  4. 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.

  5. Understanding indocyanine green angiography in polypoidal choroidal vasculopathy: the group experience with digital fundus photography and confocal scanning laser ophthalmoscopy.

    PubMed

    Cheung, Chui Ming Gemmy; Lai, Timothy Y Y; Chen, Shih-Jen; Chong, Victor; Lee, Won Ki; Htoon, Hla; Ng, Wei Yan; Ogura, Yuichiro; Wong, Tien Yin

    2014-12-01

    To evaluate the angiographic features in using fundus camera-based versus confocal scanning laser ophthalmoscope (cSLO)-based indocyanine green angiography in differentiating polypoidal choroidal vasculopathy (PCV) from typical age-related macular degeneration. Sixty-five eyes of 44 patients with exudative maculopathy due to PCV or typical age-related macular degeneration were prospectively imaged with indocyanine green angiography using fundus camera and cSLO. Images were graded independently by retinal specialists. The main outcome measure was agreement between cSLO and fundus camera for the diagnosis of PCV. The rate of detection and area under the receiver operating characteristic curve of 7 preselected individual features were also compared. The diagnosis of PCV was made with the cSLO system in 36 eyes (55.4%) and typical age-related macular degeneration in 29 eyes (44.6%), whereas the fundus camera diagnosed PCV in 39 eyes (60.0%) and typical age-related macular degeneration in 26 eyes (40.0%). There was moderate agreement between the two indocyanine green angiography systems (Kappa = 0.53). Using cSLO as the gold standard, fundus camera has a sensitivity and specificity of 83.3% and 69.0%, respectively. Typical nodular appearance was the most commonly detected feature (median, 88.9% for cSLO, 80.6% for fundus camera, P = 0.63) and had the highest area under the curve for the diagnosis of PCV in both systems (median, 80.2% for cSLO, 73.2% for fundus camera, P = 0.13). Confocal scanning laser ophthalmoscope was more sensitive in detecting branching vascular network and late hyperfluorescent plaque. Both systems detected >80% of PCV based on typical nodular appearance of polyps. However, the cSLO is superior in detecting additional features, particularly branching vascular network.

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  7. Morphological characteristics of the optic nerve evaluated by confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population from the city of Barcelona.

    PubMed

    Fallon, M; Pazos, M; Morilla, A; Sebastián, M A; Xancó, R; Mora, C; Calderón, B; Vega, Z; Antón, A

    2015-11-01

    To evaluate morphological parameters of optic disc and retinal nerve fiber layer (RNFL) examined with confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population, and analyze correlations of these parameters with demographic variables. Cross-sectional study in the context of a glaucoma screening campaign in the primary care center of Barcelona. The individuals selected were non-hypertensive Mediterranean Caucasians with risk for glaucoma development (individuals≥60 years old or≥40 years old with family history of glaucoma or intraocular pressure or myopia>3diopter). All subjects underwent a complete ophthalmic examination, confocal laser tomography (HRT3) and scanning laser polarimetry (GDX-VCC), subjects with results within normal limits only being included. Structural parameters were analyzed along with age, refraction, and pachymetry based on the Spearman rank correlation test. A total of 224 subjects included, with a mean age of 63.4±11.1 years. Disc areas, excavation and ring area were 2.14±0.52mm(2), 0.44±0.34mm (2) and 1.69±0.38mm(2), respectively. The mean RNFL (GDX) was 55.9±6.9μm. Age was correlated with lower ring volume, highest rate of cup shape measure, largest mean and maximum cup depth, lower nerve fiber index (NFI) and RNFL (all p-values below .05). The mean values and distribution of several parameters of the papilla and the RNFL in normal Mediterranean Caucasians population are presented. A loss of thickness of the RNFL, ring thinning, and enlarged cup was observed with increased age. Copyright © 2014 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.

  8. 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.

  9. 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

  10. 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.

  11. Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology, calcium imaging and confocal microscopy.

    PubMed

    Galizia, C G; Kimmerle, B

    2004-01-01

    The insect antennal lobe is the first brain structure to process olfactory information. Like the vertebrate olfactory bulb the antennal lobe is substructured in olfactory glomeruli. In insects, glomeruli can be morphologically identified, and have characteristic olfactory response profiles. Local neurons interconnect glomeruli, and output (projection) neurons project to higher-order brain centres. The relationship between their elaborate morphology and their physiology is not understood. We recorded electrophysiologically from antennal lobe neurons, and iontophoretically injected a calcium-sensitive dye. We then measured their spatio-temporal calcium responses to a variety of odours. Finally, we confocally reconstructed the neurons, and identified the innervated glomeruli. An increase or decrease in spiking frequency corresponded to an intracellular calcium increase or decrease in the cell. While intracellular recordings generally lasted between 10 and 30 min, calcium imaging was stable for up to 2 h, allowing a more detailed physiological analysis. The responses indicate that heterogeneous local neurons get input in the glomerulus in which they branch most strongly. In many cases, the physiological response properties of the cells corresponded to the known response profile of the innervated glomerulus. In other words, the large variety of response profiles generally found when comparing antennal lobe neurons is reduced to a more predictable response profile when the innervated glomerulus is known.

  12. 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.

  13. 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.

  14. 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.

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

    NASA Astrophysics Data System (ADS)

    Hefti, Ryan Alf

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

  16. 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

  17. Real-Time Demonstration of Split Skin Graft Inosculation and Integra Dermal Matrix Neovascularization Using Confocal Laser Scanning Microscopy

    PubMed Central

    Greenwood, John; Amjadi, Mahyar; Dearman, Bronwyn; Mackie, Ian

    2009-01-01

    Objectives: During the first 48 hours after placement, an autograft “drinks” nutrients and dissolved oxygen from fluid exuding from the underlying recipient bed (“plasmatic imbibition”). The theory of inosculation (that skin grafts subsequently obtain nourishment via blood vessel “anastomosis” between new vessels invading from the wound bed and existing graft vessels) was hotly debated from the late 19th to mid-20th century. This study aimed to noninvasively observe blood flow in split skin grafts and Integra™ dermal regeneration matrix to provide further proof of inosculation and to contrast the structure of vascularization in both materials, reflecting mechanism. Methods: Observations were made both clinically and using confocal microscopy on normal skin, split skin graft, and Integra™. The VivaScope™ allows noninvasive, real-time, in vivo images of tissue to be obtained. Results: Observations of blood flow and tissue architecture in autologous skin graft and Integra™ suggest that 2 very different processes are occurring in the establishment of circulation in each case. Inosculation provides rapid circulatory return to skin grafts whereas slower neovascularization creates an unusual initial Integra™ circulation. Conclusions: The advent of confocal laser microscopy like the VivaScope 1500™, together with “virtual” journals such as ePlasty, enables us to provide exciting images and distribute them widely to a “reading” audience. The development of the early Integra™ vasculature by neovascularization results in a large-vessel, high-volume, rapid flow circulation contrasting markedly from the inosculatory process in skin grafts and the capillary circulation in normal skin and merits further (planned) investigation. PMID:19787028

  18. 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.

  19. Demons registration for in vivo and deformable laser scanning confocal endomicroscopy.

    PubMed

    Chiew, Wei-Ming; Lin, Feng; Seah, Hock Soon

    2017-09-01

    A critical effect found in noninvasive in vivo endomicroscopic imaging modalities is image distortions due to sporadic movement exhibited by living organisms. In three-dimensional confocal imaging, this effect results in a dataset that is tilted across deeper slices. Apart from that, the sequential flow of the imaging-processing pipeline restricts real-time adjustments due to the unavailability of information obtainable only from subsequent stages. To solve these problems, we propose an approach to render Demons-registered datasets as they are being captured, focusing on the coupling between registration and visualization. To improve the acquisition process, we also propose a real-time visual analytics tool, which complements the imaging pipeline and the Demons registration pipeline with useful visual indicators to provide real-time feedback for immediate adjustments. We highlight the problem of deformation within the visualization pipeline for object-ordered and image-ordered rendering. Visualizations of critical information including registration forces and partial renderings of the captured data are also presented in the analytics system. We demonstrate the advantages of the algorithmic design through experimental results with both synthetically deformed datasets and actual in vivo, time-lapse tissue datasets expressing natural deformations. Remarkably, this algorithm design is for embedded implementation in intelligent biomedical imaging instrumentation with customizable circuitry. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

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

    PubMed

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

    2016-08-01

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

  1. 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

  2. Confocal Laser Endomicroscopy in the Management of Endoscopically Treated Upper Urinary Tract Transitional Cell Carcinoma: Preliminary Data.

    PubMed

    Villa, Luca; Cloutier, Jonathan; Cotè, Jean-Francois; Salonia, Andrea; Montorsi, Francesco; Traxer, Olivier

    2016-02-01

    To describe our initial experience with confocal laser endomicroscopy (CLE) for the evaluation and treatment of patients with upper urinary tract transitional cell carcinoma (UUT-TCC). Preliminary data were analyzed from 11 patients with suspicion of UUT-TCC scheduled for flexible ureteroscopy (f-URS) and consensual holmium-YAG laser tumor ablation. CLE was performed before endoscopic biopsy and laser photoablation of the suspected lesion using a 3F-diameter flexible probe UroFlex™ B (Cellvizio® system; Mauna Kea Technologies, Paris, France), which allows to obtain microscopic resolution imaging (3.5 μm), with a field of view of 325 μm and a depth of tissue imaging of 40 to 70 μm. Video sequences were analyzed offline and thereafter compared with histopathologic findings. CLE technique was feasible and showed good quality imaging in all patients. Overall, the Cellvizio system provided reliable images of healthy urothelium when the probe was pointed toward normal tissue, showing umbrella cells on the surface and vessels in the lamina propria. Moreover, CLE displayed the characteristic features of high-density cellular aggregates and fibrovascular stalks in four patients with pathologically confirmed low-grade UUT-TCC. In the patient with pathologically confirmed high-grade UUT-TCC, more distorted microarchitecture and tortuous vessels were clearly recognized with CLE. These preliminary data showed the feasibility of CLE technique when applied to the diagnosis of UUT-TCC. Further clinical studies are required to confirm CLE accuracy in distinguishing healthy urothelial tissue from malignant lesions, thus helping clinicians in targeting ureteroscopic biopsy and improving the conservative management of UUT-TCC patients.

  3. High-power direct green laser oscillation of 598 mW in Pr(3+)-doped waterproof fluoroaluminate glass fiber excited by two-polarization-combined GaN laser diodes.

    PubMed

    Nakanishi, Jun; Horiuchi, Yuya; Yamada, Tsuyoshi; Ishii, Osamu; Yamazaki, Masaaki; Yoshida, Minoru; Fujimoto, Yasushi

    2011-05-15

    We demonstrated a high-power and highly efficient Pr-doped waterproof fluoride glass fiber laser at 522.2 nm excited by two-polarization-combined GaN laser diodes and achieved a subwatt output power of 598 mW and slope efficiency of 43.0%. This system will enable us to make a vivid laser display, a photocoagulation laser for eye surgery, a color confocal scanning laser microscope, and an effective laser for material processing. Direct visible ultrashort pulse generation is also expected.

  4. 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.

  5. Cutting efficiency of apical preparation using ultrasonic tips with microprojections: confocal laser scanning microscopy study.

    PubMed

    Kwak, Sang-Won; Moon, Young-Mi; Yoo, Yeon-Jee; Baek, Seung-Ho; Lee, WooCheol; Kim, Hyeon-Cheol

    2014-11-01

    The purpose of this study was to compare the cutting efficiency of a newly developed microprojection tip and a diamond-coated tip under two different engine powers. The apical 3-mm of each root was resected, and root-end preparation was performed with upward and downward pressure using one of the ultrasonic tips, KIS-1D (Obtura Spartan) or JT-5B (B&L Biotech Ltd.). The ultrasonic engine was set to power-1 or -4. Forty teeth were randomly divided into four groups: K1 (KIS-1D / Power-1), J1 (JT-5B / Power-1), K4 (KIS-1D / Power-4), and J4 (JT-5B / Power-4). The total time required for root-end preparation was recorded. All teeth were resected and the apical parts were evaluated for the number and length of cracks using a confocal scanning micrscope. The size of the root-end cavity and the width of the remaining dentin were recorded. The data were statistically analyzed using two-way analysis of variance and a Mann-Whitney test. There was no significant difference in the time required between the instrument groups, but the power-4 groups showed reduced preparation time for both instrument groups (p < 0.05). The K4 and J4 groups with a power-4 showed a significantly higher crack formation and a longer crack irrespective of the instruments. There was no significant difference in the remaining dentin thickness or any of the parameters after preparation. Ultrasonic tips with microprojections would be an option to substitute for the conventional ultrasonic tips with a diamond coating with the same clinical efficiency.

  6. 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.

  7. 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.

  8. Laser scanning confocal microscopy coupled with hydraulic permeability measurements for elucidating fluid flow across porous materials: application to human dentine.

    PubMed

    Williams, Cara G; Macpherson, Julie V; Unwin, Patrick R; Parkinson, Charles

    2008-04-01

    Laser scanning confocal microscopy (LSCM) coupled to a constant volume flow-pressure measuring system is introduced as a new technique for the quantitative measurement of fluid flow across porous materials. Such processes are ubiquitous from the life sciences to materials science and the methodology herein could find widespread application. The methodology has been applied to the detection of fluid flow through human dentine, in-vitro, and in the assessment of occlusion actives. Dentine is a calcareous material sandwiched between the pulp and enamel in the tooth structure that contains tubules which traverse dentine in the pulp to enamel direction. The tubules become patent during enamel erosion or gum recession, leading to dentinal hypersensitivity. Understanding the nature of fluid flow is important, as a pressure gradient exists across dentine in-vivo and this has implications for the development of suitable treatments. The methodology described herein firstly allows a ready assessment of the general efficacy of treatments via hydraulic permeability measurements. Second, LSCM images allow the nature of the flow process and the mode of action of the treatments to be revealed at high spatial resolution. For the particular case of dentine, we demonstrate how the method allows candidate treatments to be compared and assessed.

  9. 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.

  10. 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

  11. Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy.

    PubMed

    Keevil, C W

    2003-01-01

    Knowledge of biofilm structure and function has changed significantly in the last few years due to advances in light microscopy. One pertinent example is the use of scanning confocal laser microscopy (SCLM) to visualise corrosion pits caused by the biofilm mosaic footprint on corroding metal surfaces. Nevertheless, SCLM has some limitations as to its widespread use, including cost, inability to observe motile bacteria and eukaryotic grazers within biofilms, and difficulty to scan a curved surface. By contrast, episcopic differential interference contrast (EDIC) microscopy has provided a rapid, real time analysis of biofilms on opaque, curved, natural or man-made surfaces without the need for cover slips and oil. EDIC, coupled with epi-fluorescence (EDIC/EF), microscopy has been used successfully to visualise the 3-D biofilm structure, physiological niches, protozoal grazing and iron biomineralization, and the location of specific pathogens such as Legionella pneumophila, Campylobacter jejuni and Cryptosporidium parvum. These species were identified using gold nanoparticles or fluorophores coupled to monoclonal antibodies or 16S rRNA probes, respectively. Among its many potential uses, the EDIC technique will provide a rapid procedure to facilitate the calibration of the modern generation of biofilm-sensing electrodes.

  12. Response of E. faecalis biofilms to different associations of auxiliary substances during root canal preparation: a confocal laser microscopy analysis.

    PubMed

    Hohscheidt, Gabriela Luisa; Böttcher, Daiana Elisabeth; Fatturi Parolo, Clarissa Cavalcanti; Montagner, Francisco; Grecca, Fabiana Soares

    2013-06-01

    This in vitro study evaluated the effect of different endodontic auxiliary chemical substances over Enterococcus faecalis (Ef) biofilm through confocal laser scanning microscopy (CLSM). Forty-five bovine incisors were infected with Ef for 21 days. Teeth were divided into five groups: group 1: 2.5% NaOCl + EDTA, group 2: 2% CHX gel + EDTA, group 3: 2% CHX liquid + EDTA, group 4: 2.5% NaOCl + 2% CHX gel + EDTA, group 5: 2.5% NaOCl + 2% CHX liquid + EDTA and a negative and a positive control group (NCG; PCG). The samples were stained with SYTO9 and propidium iodide and analyzed by CLSM. Bacterial viability was quantitatively analyzed by the proportions of dead and live bacteria in the biofilm remnants. Scores were standardized according to the total bacterial load (TBL)-1: ≤ 25%, 2: >25 ≤ 50%, 3: >50 ≤ 75%, 4: >75% and debris-1: absence of debris; 2: presence of debris. Statistical analysis was carried out through the Kruskal-Wallis and the Fischer exact tests (P = 0.05). No statistical differences were observed to CFU, debris and bacterial viability. None of the tested substances could completely eliminate Ef from the root canal space. Copyright © 2013 Wiley Periodicals, Inc.

  13. 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

  14. Sensitivity and specificity of confocal laser-scanning microscopy for in vivo diagnosis of malignant skin tumors.

    PubMed

    Gerger, Armin; Koller, Silvia; Weger, Wolfgang; Richtig, Erika; Kerl, Helmut; Samonigg, Hellmut; Krippl, Peter; Smolle, Josef

    2006-07-01

    Melanoma and nonmelanoma skin cancer are the most frequent malignant tumors by far among whites. Currently, early diagnosis is the most efficient method for preventing a fatal outcome. In vivo confocal laser-scanning microscopy (CLSM) is a recently developed potential diagnostic tool. One hundred seventeen melanocytic skin lesions and 45 nonmelanocytic skin lesions (90 benign nevi, 27 malignant melanomas, 15 basal cell carcinomas, and 30 seborrheic keratoses) were sampled consecutively and were examined using proprietary CLSM equipment. Stored images were rated by 4 independent observers. Differentiation between melanoma and all other lesions based solely on CLSM examination was achieved with a positive predictive value of 94.22%. Malignant lesions (melanoma and basal cell carcinoma) as a group were diagnosed with a positive predictive value of 96.34%. Assessment of distinct CLSM features showed a strong interobserver correlation (kappa >0.80 for 11 of 13 criteria). Classification and regression tree analysis yielded a 3-step algorithm based on only 3 criteria, facilitating a correct classification in 96.30% of melanomas, 98.89% of benign nevi, and 100% of basal cell carcinomas and seborrheic keratoses. In vivo CLSM examination appeared to be a promising method for the noninvasive assessment of melanoma and nonmelanoma skin tumors. Copyright 2006 American Cancer Society.

  15. Confocal laser endomicroscopy for brain tumor surgery: a milestone journey from microscopy to cellular surgery (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Charalampaki, Cleopatra

    2017-02-01

    The aim in brain tumor surgery is maximal tumor resection with minimal damage of normal neuronal tissue. Today diagnosis of tumor and definition of tumor borders intraoperatively is based on various visualization methods as well as on the histopathologic examination of a limited number of biopsy specimens via frozen sections. Unfortunately, intraoperative histopathology bears several shortcomings, and many biopsies are inconclusive. Therefore, the desirable treatment could be to have the ability to identify intraoperative cellular structures, and differentiate tumor from normal functional brain tissue on a cellular level. To achieve this goal new technological equipment integrated with new surgical concepts is needed.Confocal Laser Endomicroscopy (CLE) is an imaging technique which provides microscopic information of tissue in real-time. We are able to use these technique to perform intraoperative "optical biopsies" in bringing the microscope inside to the patients brain through miniaturized fiber-optic probes, and allow real-time histopathology. In our knowledge we are worldwide the only one neurosurgical group using CLE intraoperative for brain tumor surgery. We can detect and characterize intraoperative tumor cells, providing immediate online diagnosis without the need for frozen sections. It also provides delineation of borders between tumor and normal tissue on a cellular level, making surgical margins more accurate than ever before. The applications of CLE-assisted neurosurgery help to accurate the therapy by extending the resection borders and protecting the functionality of normal brain tissue in critical eloquent areas.

  16. 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.

  17. Efficiency of cytoplasmic delivery by non-cationic liposomes to cells in vitro: a confocal laser scanning microscopy study.

    PubMed

    Mady, Mohsen M; Ghannam, M M; Khalil, W A; Müller, R; Fahr, Alfred

    2009-06-01

    It is necessary to understand liposomal uptake mechanisms and intracellular distribution in order to design more efficient gene (drug) carrier systems. Until now, a few studies have been carried out using confocal laser scanning microscopy (CLSM) to investigate the cellular uptake and transfection mediated with liposomes. So, by CLSM, we demonstrated that artificial virus-like envelope (AVE) vesicles labeled with rhodamine-PE (Rh-PE), carbocyanine (DiI) and carboxyfluorescein (CF) were investigated into the cytoplasm of two human cell lines, Mewo (human melanoma cell line) and HepG2 (human hepatoma cell line) cells grown in DMEM medium supplemented with different percentages (0%, 30%, and 100%) fetal calf serum (FCS). The liposome uptake was dependent on the cell line, in view that the whole process of liposomes associated with cells (uptake) is a two-step process involving binding and endocytosis. Based upon the various assays used to measure cellular uptake of liposomes, we conclude the efficacy of cytoplasmic delivery by AVE-liposomes to cells in culture.

  18. Live cell confocal laser imaging studies on the nuclear behavior during meiosis and ascosporogenesis in Morchella importuna under artificial cultivation.

    PubMed

    He, Peixin; Wang, Ke; Cai, Yingli; Liu, Wei

    2017-10-01

    The commercial production of Morchella mushrooms, especially M. importuna, has been realized since 2012 in China, which facilitates the fundamental studies of Morchella spp. In this paper, the cytological characteristics at three stages of ascocarpic development and the nuclear behavior during meiosis and ascosporogenesis in cultivated strain M. importuna 1(#) was visualized by confocal laser scanning microscopy. The results suggested that the strain sporulated at sporulation stage of ascocarpic development. A total of six nuclear divisions typically took place during ascosporogenesis. The first and second divisions were meiotic in which the single diploid nucleus divided into four haploid nuclei. The subsequent mitosis gave rise to eight nuclei, and eight incipient ascospores with one nucleus in each spore were formed after spore delimitation in the clavate ascus. Then, the nucleus in most of the young ascospore underwent three successive mitoses producing 6-8 haploid nuclei in each mature spore, and thus the multinucleate ascospores in each ascus were all homokaryons. To the best of our knowledge, this is the first dynamic tracing study of nuclear behavior during meiosis and ascosporogenesis in cultivated morels, and the spore delimitation time is also the first report. The study will be beneficial for the genetics study and strain breeding of Morchella mushrooms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Quantifying migration and polarization of murine mesenchymal stem cells on different bone substitutes by confocal laser scanning microscopy.

    PubMed

    Roldán, J C; Chang, E; Kelantan, M; Jazayeri, L; Deisinger, U; Detsch, R; Reichert, T E; Gurtner, G C

    2010-12-01

    Cell migration is preceded by cell polarization. The aim of the present study was to evaluate the impact of the geometry of different bone substitutes on cell morphology and chemical responses in vitro. Cell polarization and migration were monitored temporally by using confocal laser scanning microscopy (CLSM) to follow green fluorescent protein (GFP)±mesenchymal stem cells (MSCs) on anorganic cancellous bovine bone (Bio-Oss(®)), β-tricalcium phosphate (β-TCP) (chronOS(®)) and highly porous calcium phosphate ceramics (Friedrich-Baur-Research-Institute for Biomaterials, Germany). Differentiation GFP±MSCs was observed using pro-angiogenic and pro-osteogenic biomarkers. At the third day of culture polarized vs. non-polarized cellular sub-populations were clearly established. Biomaterials that showed more than 40% of polarized cells at the 3rd day of culture, subsequently showed an enhanced cell migration compared to biomaterials, where non-polarized cells predominated (p<0.003). This trend continued untill the 7th day of culture (p<0.003). The expression of vascular endothelial growth factor was enhanced in biomaterials where cell polarization predominated at the 7th day of culture (p=0.001). This model opens an interesting approach to understand osteoconductivity at a cellular level. MSCs are promising in bone tissue engineering considering the strong angiogenic effect before differentiation occurs. Copyright © 2010 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  20. 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

  1. Imaging and cell count in cleared intact cochlea in the Mongolian gerbil using laser scanning confocal microscopy.

    PubMed

    Risoud, M; Sircoglou, J; Dedieu, G; Tardivel, M; Vincent, C; Bonne, N-X

    2017-09-01

    To draw up a clearing protocol for Mongolian gerbil cochlea, and to assess the feasibility of quantifying and analyzing 3D cell architecture in the transparent cochleae. Freshly dissected inner ears were prepared on a 13-day protocol: fixation, microdissection, post-fixation, decalcification, pretreatment (signal enhancement, permeabilization and blocking), fluorescent labeling (indirect immunolabeling and direct labeling), dehydration, clearing in Spalteholz solution (MSBB: methyl salicylate and benzyl benzoate) and mounting. Image acquisition used laser scanning confocal microscopy. ImageJ software was used to measure the length of the organ of Corti thus available for analysis and to count inner and outer hair cells. Four cochleas underwent imaging. 3D reconstruction enabled organ of Corti length to be measured, at a mean 1269±346μm. Mean inner and outer hair-cell count per organ of Corti length was 142±44 and 400±122, respectively. Cochlear clearing by MSBB was feasible in Mongolian gerbils and provided high-resolution immunofluorescence-labeled inner-ear images. To our knowledge, this was the first application of the technique in this species. Cell count could thus be performed along the organ of Corti length without traumatic dissection. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. 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.

  3. A comparative study of allergic contact dermatitis by patch test versus reflectance confocal laser microscopy, with nickel and cobalt.

    PubMed

    Sakanashi, Emi Nishijima; Matsumura, Mitsuaki; Kikuchi, Katsuko; Ikeda, Masaomi; Miura, Hiroyuki

    2010-01-01

    Few studies have reported on the accuracy of reflectance confocal laser microscopy (RCLM) in observing allergic contact dermatitis in vivo. However, distinction of skin reactions from different reagents is not well understood. We sought to diagnose allergic contact dermatitis by RCLM images and compare with routine, visual patch test (PT) reading for 2 major allergen metals in Japan. The PT was performed on the upper back skin with 5% nickel sulfate (Ni) and 2% cobalt chloride (Co) in eight healthy volunteers and eleven patients. RCLM was used to calculate the thickness of the suprabasal epidermis after visual assessment of PT. Comparison of clinical scoring versus suprabasal epidermal thickness was observed. RCLM images of positive PT showed increased suprabasal epidermal thickness on day 2 (D2), and D3 for Co, whereas there was vesicle formation and an overall increase suprabasal epidermal thickness for Ni. In two of 3 doubtful positive PT to Co, RCLM images presented characteristics of irritant reactions; and one characteristic of a positive reaction. The frequency of Co doubtful-positive PT was higher than that of Ni. We found advantages in using RCLM for visualizing features of allergic contact dermatitis and found it a useful tool as an objective parameter in grading severity and types of PT reaction.

  4. 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.

  5. 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.

  6. 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.

  7. Cascaded injection resonator for coherent beam combining of laser arrays

    DOEpatents

    Kireev, Vassili [Sunnyvale, CA; Liu, Yun; Protopopescu, Vladimir [Knoxville, TN; Braiman, Yehuda [Oak Ridge, TN

    2008-10-21

    The invention provides a cascaded injection resonator for coherent beam combining of laser arrays. The resonator comprises a plurality of laser emitters arranged along at least one plane and a beam sampler for reflecting at least a portion of each laser beam that impinges on the beam sampler, the portion of each laser beam from one of the laser emitters being reflected back to another one of the laser emitters to cause a beam to be generated from the other one of the laser emitters to the beam reflector. The beam sampler also transmits a portion of each laser beam to produce a laser output beam such that a plurality of laser output beams of the same frequency are produced. An injection laser beam is directed to a first laser emitter to begin a process of generating and reflecting a laser beam from one laser emitter to another laser emitter in the plurality. A method of practicing the invention is also disclosed.

  8. 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.

  9. Detection of colonic dysplasia in patients with ulcerative colitis using a targeted fluorescent peptide and confocal laser endomicroscopy: A pilot study.

    PubMed

    De Palma, Giovanni Domenico; Colavita, Irene; Zambrano, Gerardo; Giglio, Mariano Cesare; Maione, Francesco; Luglio, Gaetano; Sarnelli, Giovanni; Rispo, Antonio; Schettino, Pietro; D'Armiento, Francesco Paolo; De Palma, Fatima Domenica Elisa; D'Argenio, Valeria; Salvatore, Francesco

    2017-01-01

    Targeted molecular probes have been used to detect sporadic colonic dysplasia during confocal laser endomicroscopy (CLE) with promising results. This is a feasibility pilot study aiming to assess the potential role of CLE combined with a fluorescent-labeled peptide to stain and detect dysplasia associated with Ulcerative Colitis. A phage-derived heptapeptide with predicted high binding affinity for dysplastic tissue, was synthesized and labeled with fluorescein. Eleven lesions with suspected dysplasia at endoscopy were excised from nine patients with long-standing ulcerative colitis. Specimens were sprayed with the peptide and examined by CLE. The CLE images were then compared to the corresponding histological sections. At definitive histology, 4 lesions were diagnosed as inflammatory polyps, 6 as dysplastic lesions and one as invasive cancer. In inflammatory polyps, the fluorescence signal came from peri-cryptal spaces and crypt lumen due to passive accumulation of the peptide in these areas. Dysplasia was associated with active binding of the peptide to dysplastic colonocytes. Ex vivo staining of ulcerative colitis-associated dysplasia using a fluorescent labeled molecular probe and CLE is feasible. In vivo studies on larger populations are required to evaluate the safety and the effective contribution of molecular probes in cancer surveillance of ulcerative colitis.

  10. Combined impact features for laser plasma generation

    NASA Astrophysics Data System (ADS)

    Loktionov, E.; Protasov, Yu; Telekh, V.

    2017-05-01

    Laser-induced plasma has been considered for multiple applications by the moment, and its characteristics strongly depend on laser radiation parameters. Reaching demanded values for the latter might be rather costly, but, in certain cases, similar or even better results could be reached in case of additional impact (optical, electric, magnetic, corpuscular, mechanical etc.). Combined impact effects are mainly based on target properties or interaction mechanism change, and found to decrease plasma generation thresholds by orders of magnitude, improve energy efficiency significantly, and also broaden the range of plasma parameters. Application area, efficiency and optimal regimes for laser plasma generation at such combined impact have been considered. Analysis based on published data and own experiments was performed for both target material and induced plasma flows. Criterial parameters have been suggested to characterize both combined impact and response to it. The data on plasma generation thresholds, controlled parameters, working media supply systems and recovery rate of droplets are very important for technology setups, including those for material modification.

  11. Chromatic confocal spectral interferometry

    SciTech Connect

    Papastathopoulos, Evangelos; Koerner, Klaus; Osten, Wolfgang

    2006-11-10

    Chromatic confocal spectral interferomertry (CCSI) is a novel scheme for topography measurements that combines the techniques of spectral interferometry and chromatic confocal microscopy. This hybrid method allows for white-light interferometric detection with a high NA in a single-shot manner. To the best of our knowledge, CCSI is the first interferometric method that utilizes a confocally filtered and chromatically dispersed focus for detection and simultaneously allows for retrieval of the depth position of reflecting or scattering objects utilizing the phase (modulation frequency) of the interferometric signals acquired. With the chromatically dispersed focus, the depth range of the sensor is decoupled from the NA of the microscope objective.

  12. Confocal laser endomicroscopy is a new imaging modality for recognition of intramucosal bacteria in inflammatory bowel disease in vivo

    PubMed Central

    Moussata, Driffa; Goetz, Martin; Gloeckner, Annabel; Kerner, Marcus; Campbell, Barry; Hoffman, Arthur; Biesterfeld, Stephan; Flourie, Bernard; Saurin, Jean-Christophe; Galle, Peter R; Neurath, Markus F; Kiesslich, Ralf

    2010-01-01

    Background and objectives Interaction of bacteria with the immune system within the intestinal mucosa plays a key role in the pathogenesis of inflammatory bowel disease (IBD). The aim of the current study was to develop a fluorescein-aided confocal laser endomicroscopy (CLE) method to visualise intramucosal enteric bacteria in vivo and to determine the involved mucosal area in the colon and ileum in patients with ulcerative colitis (UC) and Crohn's disease (CD). Methods Initially, E coli strains expressing enhanced green fluorescent protein (pEGFP) were endomicroscopically imaged in mice. In addition, ex vivo and in vivo imaging of fluorescent human enteric bacteria was performed to specify the distinct endomicroscopic appearance of enteral bacteria. Targeted mucosal biopsies towards endomicroscopic identifiable intramucosal bacteria and negative mucosal areas were prospectively obtained during colonoscopy and correlated with bench-top fluorescence microscopy (FISH) to prove the endomicroscopic visualisation of intramucosal bacteria. Finally, a retrospective analysis as well as a prospective study was performed in patients with UC and CD to confirm the presence and distribution of intramucosal bacteria within the gut. Results Confocal endomicroscopy was able to identify intramucosal pEGFP E coli in mice and strains of enteric microflora in the mucosa of humans. Using FISH as the gold standard, evaluation of 21 patients showed that CLE had a sensitivity of 89% and specificity of 100% to identify intramucosal bacteria. In a retrospective study, 113 patients with CD and UC had intramucosal bacteria significantly more often than 50 control patients (66% vs 60% vs 14%, p<0.001). This result was confirmed in a prospective study in which 10 patients with CD and 10 with UC had a significantly wider distribution of involvement with intramucosal bacteria in the colon and terminal ileum compared with 10 controls (85.2% vs 75.9% vs 16.8%, p<0.0001). Conclusions CLE is a new

  13. A novel method to analyse in vivo the physiological state and cell viability of phototrophic microorganisms by confocal laser scanning microscopy using a dual laser.

    PubMed

    Millach, Laia; Obiol, Aleix; Solé, Antonio; Esteve, Isabel

    2017-10-01

    Phototrophic microorganisms are very abundant in extreme environments, where are subjected to frequent and strong changes in environmental parameters. Nevertheless, little is known about the physiological effects of these changing environmental conditions on viability of these microorganisms, which are difficult to grow in solid media and have the tendency to form aggregates. For that reason, it is essential to develop methodologies that provide data in short time consuming, in vivo and with minimal manipulating the samples, in response to distinct stress conditions. In this paper, we present a novel method using Confocal Laser Scanning Microscopy and a Dual Laser (CLSM-DL) for determining the cell viability of phototrophic microorganisms without the need of either staining or additional use of image treating software. In order to differentiate viable and nonviable Scenedesmus sp. DE2009 cells, a sequential scan in two different channels was carried out from each same xyz optical section. On the one hand, photosynthetic pigments fluorescence signal (living cells) was recorded at the red channel (625- to 785-nm fluorescence emission) exciting the samples with a 561-nm laser diode, and an acousto-optic tunable filter (AOTF) of 20%. On the other hand, nonphotosynthetic autofluorescence signal (dead cells) was recorded at the green channel (500- to 585-nm fluorescence emission) using a 405-nm UV laser, an AOTF of 15%. Both types of fluorescence signatures were captured with a hybrid detector. The validation of the CLSM-DL method was performed with SYTOX green fluorochrome and electron microscopic techniques, and it was also applied for studying the response of distinct light intensities, salinity doses and exposure times on a consortium of Scenedesmus sp. DE2009. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  14. Combining tape stripping and non-invasive reflectance confocal microscopy : an in vivo model to study skin damage.

    PubMed

    Peppelman, M; van den Eijnde, W A J; Jaspers, E J; Gerritsen, M-J P; van Erp, P E J

    2015-11-01

    Evaluation of (immuno)histological and cell biological changes in damaged skin requires often an invasive skin biopsy, making in vivo models inappropriate to study skin damage. Reflectance confocal microscopy (RCM) might overcome this limitation. Therefore, we evaluated the use of a tape-stripping model in combination with RCM to provide morphological data on skin damage and recovery. In 25 volunteers, a tape-stripping stimulus was applied. The skin was imaged with RCM during 1 week and 3 mm punch biopsies were obtained. Strong correlations between epidermal thickness determined by RCM and conventional histological measurements were found. RCM thickness measurements correlated well with epidermal proliferation. The 10× or 15× repeated tape-stripping resulted in skin damage similar to acute stripping. Mild repeated tape-stripping showed no skin damage. Overall, we demonstrated that non-invasive RCM in combination with tape-stripping could be used as model to obtain morphological and cell biological data on skin-material interactions. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  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. Investigating the correlation between white matter and microvasculature changes in aging using large scale optical coherence tomography and confocal fluorescence imaging combined with tissue sectioning

    NASA Astrophysics Data System (ADS)

    Castonguay, Alexandre; Avti, Pramod K.; Moeini, Mohammad; Pouliot, Philippe; Tabatabaei, Maryam S.; Bélanger, Samuel; Lesage, Frédéric

    2015-03-01

    Here, we present a serial OCT/confocal scanner for histological study of the mouse brain. Three axis linear stages combined with a sectioning vibratome allows to cut thru the entire biological tissue and to image every section at a microscopic resolution. After acquisition, each OCT volume and confocal image is re-stitched with adjacent acquisitions to obtain a reconstructed, digital volume of the imaged tissue. This imaging platform was used to investigate correlations between white matter and microvasculature changes in aging mice. Three age groups were used in this study (4, 12, 24 months). At sacrifice, mice were transcardially perfused with a FITC containing gel. The dual imaging capability of the system allowed to reveal different contrast information: OCT imaging reveals changes in refractive indices giving contrast between white and grey matter in the mouse brain, while transcardial perfusion of a FITC shows microsvasculature in the brain with confocal imaging.

  17. Confocal Raman imaging of optical waveguides in LiNbO3 fabricated by ultrafast high-repetition rate laser-writing.

    PubMed

    Ródenas, Airán; Nejadmalayeri, Amir H; Jaque, Daniel; Herman, Peter

    2008-09-01

    We report on the confocal Raman characterization of the micro-structural lattice changes induced during the high-repetition rate ultrafast laser writing of buried optical waveguides in lithium niobate (LiNbO(3)) crystals. While the laser beam focal volume is characterized by a significant lattice expansion together with a high defect concentration, the adjacent waveguide zone is largely free of defects, undergoing only slight rearrangement of the oxygen octahedron in the LiNbO(3) lattice. The close proximity of these two zones has been found responsible for the propagation losses of the guided light. Subjacent laser-induced periodic micro-structures have been also observed inside the laser focal volume, and identified with a strong periodic distribution of lattice defects.

  18. A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains.

    PubMed

    Pinto, Sandra N; Fernandes, Fábio; Fedorov, Alexander; Futerman, Anthony H; Silva, Liana C; Prieto, Manuel

    2013-09-01

    The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes.

  19. Real-time visualization of melanin granules in normal human skin using combined multiphoton and reflectance confocal microscopy.

    PubMed

    Majdzadeh, Ali; Lee, Anthony M D; Wang, Hequn; Lui, Harvey; McLean, David I; Crawford, Richard I; Zloty, David; Zeng, Haishan

    2015-05-01

    Recent advances in biomedical optics have enabled dermal and epidermal components to be visualized at subcellular resolution and assessed noninvasively. Multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) are noninvasive imaging modalities that have demonstrated promising results in imaging skin micromorphology, and which provide complementary information regarding skin components. This study assesses whether combined MPM/RCM can visualize intracellular and extracellular melanin granules in the epidermis and dermis of normal human skin. We perform MPM and RCM imaging of in vivo and ex vivo skin in the infrared domain. The inherent three-dimensional optical sectioning capability of MPM/RCM is used to image high-contrast granular features across skin depths ranging from 50 to 90 μm. The optical images thus obtained were correlated with conventional histologic examination including melanin-specific staining of ex vivo specimens. MPM revealed highly fluorescent granular structures below the dermal-epidermal junction (DEJ) region. Histochemical staining also demonstrated melanin-containing granules that correlate well in size and location with the granular fluorescent structures observed in MPM. Furthermore, the MPM fluorescence excitation wavelength and RCM reflectance of cell culture-derived melanin were equivalent to those of the granules. This study suggests that MPM can noninvasively visualize and quantify subepidermal melanin in situ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Optical coherence tomography and confocal fluorescence microscopy as a combined method for studying morphological changes in lung dynamics

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Cimalla, Peter; Knels, Lilla; Meissner, Sven; Schnabel, Christian; Kuebler, Wolfgang M.; Koch, Edmund

    2011-03-01

    Acute lung injury (ALI) is a severe pulmonary disease leading to hypoxemia accompanied by a reduced compliance and partial edema of the lung. Most of the patients have to be ventilated to compensate for the lack of oxygen. The treatment is strongly connected with ventilator induced lung injury (VILI), which is believed to introduce further stress to the lung and changes in its elastic performance. A thorough understanding of the organs micro-structure is crucial to gain more insight into the course of the disease. Due to backscattering of near-infrared light, detailed description of lung morphology can be obtained using optical coherence tomography (OCT), a non-invasive, non-contact, high resolution and fast three-dimensional imaging technique. One of its drawbacks lies in the non-specificity of light distribution in relation to defined substances, like elastic biomolecules. Using fluorescence detection, these chemical components can be visualized by introducing specifically binding fluorophores. This study presents a combined setup for studying alveolar compliance depending on volume changes and elastic fiber distributions. Simultaneously acquired OCT and confocal fluorescence images allow an entire view into morphological rearrangements during ventilation for an ex vivo mouse model using continuous pulmonary airway pressure at different values.

  1. 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

  2. A reproducible automated segmentation algorithm for corneal epithelium cell images from in vivo laser scanning confocal microscopy.

    PubMed

    Bullet, Julien; Gaujoux, Thomas; Borderie, Vincent; Bloch, Isabelle; Laroche, Laurent

    2014-06-01

    To evaluate an automated process to find borders of corneal basal epithelial cells in pictures obtained from in vivo laser scanning confocal microscopy (Heidelberg Retina Tomograph III with Rostock corneal module). On a sample of 20 normal corneal epithelial pictures, images were segmented through an automated four-step segmentation algorithm. Steps of the algorithm included noise reduction through a fast Fourier transform (FFT) band-pass filter, image binarization with a mean value threshold, watershed segmentation algorithm on distance map to separate fused cells and Voronoi diagram segmentation algorithm (which gives a final mask of cell borders). Cells were then automatically counted using this border mask. On the original image either with contrast enhancement or noise reduction, cells were manually counted by a trained operator. The average cell density was 7722.5 cells/mm(2) as assessed by automated analysis and 7732.5 cells/mm(2) as assessed by manual analysis (p = 0.93). Correlation between automated and manual analysis was strong (r = 0.974 [0.934-0.990], p < 0.001). Bland-Altman method gives a mean difference in density of 10 cells/mm(2) and a limits of agreement ranging from -971 to +991 cells/mm(2) . Visually, the algorithm correctly found almost all borders. This automated segmentation algorithm is worth for assessing corneal epithelial basal cell density and morphometry. This procedure is fully reproducible, with no operator-induced variability. © 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  3. Poor agreement between endoscopists and gastrointestinal pathologists for the interpretation of probe-based confocal laser endomicroscopy findings

    PubMed Central

    Peter, Shajan; Council, Leona; Bang, Ji Young; Neumann, Helmut; Mönkemüller, Klaus; Varadarajulu, Shyam; Wilcox, Charles Melbern

    2014-01-01

    AIM: To compare the interpretation of probe-based confocal laser endomicroscopy (pCLE) findings between endoscopists and gastrointestinal (GI)-pathologists. METHODS: All pCLE procedures were undertaken and the endoscopist rendered assessment. The same pCLE videos were then viewed offline by an expert GI pathologist. Histopathology was considered the gold standard for definitive diagnosis. The sensitivity, specificity and accuracy for diagnosis of dysplastic/ neoplastic GI lesions and interobserver agreement between endoscopists and experienced gastrointestinal pathologist for pCLE findings were analyzed. RESULTS: Of the 66 included patients, 40 (60.6%) had lesions in the esophagus, 7 (10.6%) in the stomach, 15 (22.7%) in the biliary tract, 3 (4.5%) in the ampulla and 1 (1.5%) in the colon. The overall sensitivity, specificity and accuracy for diagnosing dysplastic/neoplastic lesions using pCLE were higher for endoscopists than pathologist at 87.0% vs 69.6%, 80.0% vs 40.0% and 84.8% vs 60.6% (P = 0.0003), respectively. Area under the ROC curve (AUC) was greater for endoscopists than the pathologist (0.83 vs 0.55, P = 0.0001). Overall agreement between endoscopists and pathologist was moderate for all GI lesions (K = 0.43; 95%CI: 0.26-0.61), luminal lesions (K = 0.40; 95%CI: 0.20-0.60) and those of dysplastic/neoplastic pathology (K = 0.55; 95%CI: 0.37-0.72), the agreement was poor for benign (K = 0.13; 95%CI: -0.097-0.36) and pancreaticobiliary lesions (K = 0.19; 95%CI: -0.26-0.63). CONCLUSION: There is a wide discrepancy in the interpretation of pCLE findings between endoscopists and pathologist, particularly for benign and malignant pancreaticobiliary lesions. Further studies are needed to identify the cause of this poor agreement. PMID:25548499

  4. 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.

  5. Improved classification of indeterminate biliary strictures by probe-based confocal laser endomicroscopy using the Paris Criteria following biliary stenting.

    PubMed

    Taunk, Pushpak; Singh, Satish; Lichtenstein, David; Joshi, Virendra; Gold, Jason; Sharma, Ashish

    2017-10-01

    Probe-based confocal laser endomicroscopy (pCLE) using the Miami Criteria has improved classification of indeterminate biliary strictures. However, previous biliary stenting may result in their misclassification as malignant strictures. Inflammatory criteria were added to form the Paris Classification to prevent this misclassification and reduce false positives. The aim of this study was to assess if the Paris Classification was more accurate than the Miami Classification in classifying indeterminate biliary strictures after biliary stenting. This was a retrospective observational study involving 21 patients with indeterminate biliary strictures from whom 27 pCLE video sequences were obtained (20 benign and seven malignant). Patients with and without prior biliary stenting underwent pCLE. Two investigators classified the strictures as malignant or benign using the Miami and Paris Classifications. Diagnostic accuracy, sensitivity (Se), and specificity (Sp) of endoscopic retrograde-guided pCLE were compared with final histopathology. In those without biliary stenting, the Miami Criteria resulted in Se 88%, Sp 75%, positive predictive value (PPV) 64%, negative predictive value (NPV) 92%, and accuracy 79%, while the Paris Classification resulted in Se 63%, Sp 88%, PPV 71%, NPV 82%, and accuracy 79%. In those with prior biliary stenting, the Miami Criteria resulted in Se 88%, Sp 36%, PPV 23%, NPV 93%, and accuracy 45%, while the Paris Classification resulted in Se 63%, Sp 73%, PPV 31%, NPV 91%, and accuracy 71%. The kappa statistic was 0.56. The Paris Classification improved specificity and accuracy of biliary stricture classification in those who had been previously stented and decreased the rate of misclassification of benign strictures as malignant. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  6. 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.

  7. Reversibility of gastric mucosal lesions induced by sodium phosphate tablets and characterized by probe-based confocal laser endomicroscopy

    PubMed Central

    Coron, Emmanuel; Dewitte, Marie; Aubert, Philippe; Musquer, Nicolas; Neunlist, Michel; Bruley des Varannes, Stanislas

    2015-01-01

    Background and study aims: Adequate bowel preparation is key for the optimal quality of colonoscopy. The sodium phosphate laxatives used for preparation may induce gastric injuries. However, in vivo studies monitoring the effects of sodium phosphate on the gastric mucosa are currently lacking. We aimed to characterize the effects of sodium phosphate tablets (Colokit®; Mayoly Spindler, Chatou, France) on the gastric mucosa in a large-animal model. Methods: Fourteen anesthetized pigs were used for this study. Fundic mucosal sites were analyzed at 1.5, 24, and 72 hours after the endoscopically guided application of sodium phosphate tablets (NaPT) and placebo tablets (PlaT) and were compared with unexposed sites. Different mucosal parameters were assessed with white light endoscopy, probe-based confocal laser endomicroscopy (pCLE), histology, and ex vivo permeability measurements. Results: At 90 minutes after the application of NaPT, significant increases in epithelial irregularity and crypt pit intensity were observed with pCLE. These microscopic lesions persisted at 24 hours but were resolved at 72 hours. In addition, white light endoscopy revealed local exanthema in 57 % of the animals at 1.5 hours after NaPT application. Such lesions were observed in 22 % of the pigs at 24 hours and disappeared at 72 hours after application. After 1.5 hours, PlaT induced a slight but significant increase in epithelial irregularity, as well as architectural scores that were significantly lower than the ones induced by NaPT and that disappeared after 72 hours. Conclusions: The direct and prolonged gastric application of NaPT in pigs can induce acute superficial macroscopic and microscopic injuries that are reversible within 72 hours. PMID:26134776

  8. Improving utility of brain tumor confocal laser endomicroscopy: objective value assessment and diagnostic frame detection with convolutional neural networks

    NASA Astrophysics Data System (ADS)

    Izadyyazdanabadi, Mohammadhassan; Belykh, Evgenii; Martirosyan, Nikolay; Eschbacher, Jennifer; Nakaji, Peter; Yang, Yezhou; Preul, Mark C.

    2017-03-01

    Confocal laser endomicroscopy (CLE), although capable of obtaining images at cellular resolution during surgery of brain tumors in real time, creates as many non-diagnostic as diagnostic images. Non-useful images are often distorted due to relative motion between probe and brain or blood artifacts. Many images, however, simply lack diagnostic features immediately informative to the physician. Examining all the hundreds or thousands of images from a single case to discriminate diagnostic images from nondiagnostic ones can be tedious. Providing a real time diagnostic value assessment of images (fast enough to be used during the surgical acquisition process and accurate enough for the pathologist to rely on) to automatically detect diagnostic frames would streamline the analysis of images and filter useful images for the pathologist/surgeon. We sought to automatically classify images as diagnostic or non-diagnostic. AlexNet, a deep-learning architecture, was used in a 4-fold cross validation manner. Our dataset includes 16,795 images (8572 nondiagnostic and 8223 diagnostic) from 74 CLE-aided brain tumor surgery patients. The ground truth for all the images is provided by the pathologist. Average model accuracy on test data was 91% overall (90.79 % accuracy, 90.94 % sensitivity and 90.87 % specificity). To evaluate the model reliability we also performed receiver operating characteristic (ROC) analysis yielding 0.958 average for area under ROC curve (AUC). These results demonstrate that a deeply trained AlexNet network can achieve a model that reliably and quickly recognizes diagnostic CLE images.

  9. Fellow Eye Comparison of Nerve Fiber Regeneration After SMILE and Femtosecond Laser-assisted LASIK: A Confocal Microscopy Study.

    PubMed

    Agca, Alper; Cankaya, Kadir I; Yilmaz, Ihsan; Yildirim, Yusuf; Yasa, Dilek; Olcucu, Onur; Demircan, Ali; Demirok, Ahmet; Yilmaz, Omer F

    2015-09-01

    To compare postoperative corneal nerve fiber regeneration after small incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK). The medical records of patients who underwent SMILE were retrospectively reviewed. Patients with SMILE in one eye (the SMILE group) and FS-LASIK in the fellow eye (the FS-LASIK group) and who had undergone a confocal microscopy study were retrospectively identified. Subbasal nerve fiber density was measured and compared between the groups preoperatively and at 1 week and 1, 3, and 6 months postoperatively. Thirty eyes of 15 patients were included in the study. Preoperative characteristics of the eyes were similar between the groups. However, there was a statistically significant difference in subbasal nerve fiber density between the groups at 1 month (SMILE group: 1,505 ± 627 µm/mm(2) [range: 625 to 2,540 µm/mm(2)], FS-LASIK group: 1,107 ± 509 µm/mm(2) [range: 595 to 2,313 µm/mm(2)], P = .003) and 3 months (SMILE group: 1,534 ± 503 µm/mm(2) [range: 650 to 2,255 µm/mm(2)], FS-LASIK group: 1,194 ± 485 µm/mm(2) [range: 530 to 1,695 µm/mm(2)], P = .004) postoperatively. No statistically significant difference was detected at 1 week and 6 months postoperatively. Eyes treated with SMILE have a higher density of subbasal nerve fibers than eyes treated with FS-LASIK in the early postoperative period (1 week to 3 months) but the two procedures are equivalent by 6 months postoperatively. Copyright 2015, SLACK Incorporated.

  10. Comparative study of ex vivo probe-based confocal laser endomicroscopy and light microscopy in lung cancer diagnostics.

    PubMed

    Sorokina, Anastasia; Danilevskaya, Olesya; Averyanov, Alexander; Zabozlaev, Fedor; Sazonov, Dmitry; Yarmus, Lonny; Lee, Hans J

    2014-08-01

    Probe-based confocal laser endoscopy (pCLE) allows for real-time non-invasive histological imaging via bronchoscopy. Interpreting CLE images and correlating with traditional histopathology remains challenging. We performed an ex vivo study to evaluate the correlation between light microscopy findings and pCLE imaging of primary lung carcinoma. Post-lobectomy specimens for lung cancer nodules were examined ex vivo by pCLE. The examined areas were marked with brilliant green dye, and the surrounding tissues were stained by methylene blue dye. Lung tissue segments were resected and histopathological specimens were generated with 50-μm thickness from the marked areas and stained with haematoxylin and eosin. Pathologists and pulmonologists reviewed the images for correlating features. Eighteen lobectomy specimens from 18 different patients were collected. Three primary features were observed in all samples using pCLE in the cancer surroundings: alveolar dystelectasis with thickening of alveolar walls, alveolar edema and a large amount of macrophages. The stromal and parenchymal components of the studied subtypes of non-small-cell lung cancer differed from each other. The stromal component for all nine adenocarcinoma specimens had a highly fluorescent field penetrated by dark hollows. All six squamous cell carcinoma specimens had the stromal component appeared as 'biparously' branching, highly fluorescent fibres. No stromal component was observed in any small-cell carcinoma specimen, and at low power field, the cellular component was dominant with an observed light scattering pattern. pCLE can identify lung carcinoma in ex vivo samples. Certain light microscopy features of lung carcinoma can be visualized with pCLE. © 2014 Asian Pacific Society of Respirology.

  11. 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.

  12. Comparison of bacterial leakage resistance of various root canal filling materials and methods: Confocal laser-scanning microscope study.

    PubMed

    Hwang, Ji Hee; Chung, Jin; Na, Hee-Sam; Park, Eunjoo; Kwak, Sangwon; Kim, Hyeon-Cheol

    2015-01-01

    This study evaluated the bacterial leakage resistance and root canal lining efficacy of various root canal filling materials and methods by using confocal laser-scanning microscope (CLSM). Sixty extracted human premolars with mature apex and single root canal were randomly divided into 2 control groups and 4 experimental groups. Group CW was filled with continuous wave technique using gutta-percha and AH Plus sealer. Group GC was coated with AH-Plus sealer and then obturated with soften GuttaCore. Group GF was obturated using GuttaFlow and gutta-percha. Group EM was filled with EndoSeal MTA and gutta-percha using ultrasonic vibration. The AH-Plus, GuttaFlow, and EndoSeal were labeled with Hoechst 33342 to facilitate fluorescence. The obturated root tip was incubated with Carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained E. faecalis for 14 days. CLSM was performed to evaluate the sealer distribution and bacterial leakage for the apical 1-, 2-, 3-mm specimens. Statistically significant differences were determined by 1-way ANOVA with Tukey's post-hoc test and Pearson's correlation analysis. Group EM showed the better sealer distribution score than the other groups (p < 0.05). Group CW and group GC exhibited the less bacterial leakage than the group GF, while group EM showed the similar bacterial leakage score with the groups CW and GC. There was no significant correlation between the sealer distribution and bacterial leakage (p > 0.05). Under the conditions of this study, different root canal filling materials and methods showed different efficacy for canal distribution and bacterial leakage resistance. © Wiley Periodicals, Inc.

  13. Penetrability of AH plus and MTA fillapex after endodontic treatment and retreatment: a confocal laser scanning microscopy study.

    PubMed

    Kok, Daniela; Rosa, Ricardo Abreu da; Barreto, Mirela Sangoi; Busanello, Fernanda Hoffmann; Santini, Manuela Favarin; Pereira, Jefferson Ricardo; Só, Marcus Vinícius Reis

    2014-06-01

    The aim of the study was to assess the penetrability of two endodontic sealers (AH Plus and MTA Fillapex) into dentinal tubules, submitted to endodontic treatment and subsequently to endodontic retreatment. Thirty ex vivo incisors were prepared using ProTaper rotary system up to F3 instrument and divided in three groups according to the endodontic sealer used for root canal filling: AH Plus (AHP), MTA Fillapex (MTAF), and control group (CG) without using EDTA previously to the root canal filling. Rhodamine B dye (red) was incorporated to the sealers in order to provide the fluorescence which will enable confocal laser scanning microscopy (CLSM) assessment. All specimens were filled with gutta-percha cones using the lateral compaction technique. The specimens were submitted to endodontic retreatment using ProTaper Retreatment system, re-prepared up to F5 instruments and filled with gutta-percha cones and the same sealer used during endodontic retreatment. Fluorescein dye (green) was incorporated to the sealer in order to distinguish from the first filling. The roots were sectioned 2 mm from the apex and assessed by CLSM. No difference was found between the two experimental groups (P > 0.05). On the other hand, in the control group the sealers were not capable to penetrate into dentinal tubules after endodontic treatment (P > 0.05). In retreatment cases, none of the sealers were able to penetrate into dentin tubules. It can be concluded that sealer penetrability is high during endodontic treatment. However, MTA Fillapex and AH Plus do not penetrate into dentinal tubules after endodontic retreatment. © 2014 Wiley Periodicals, Inc.

  14. Peri-implant bone organization surrounding zirconia-microgrooved surfaces circularly polarized light and confocal laser scanning microscopy study.

    PubMed

    Delgado-Ruiz, Rafael Arcesio; Abboud, Marcus; Romanos, Georgios; Aguilar-Salvatierra, Antonio; Gomez-Moreno, Gerardo; Calvo-Guirado, Jose Luis

    2015-11-01

    To study the peri-implant bone organization pattern of immediately loaded (IL) zirconia implants with microgrooved surfaces. Forty-eight dental implants of 4 mm diameter and 10 mm length were inserted after two months postextraction healing in the edentulous mandible of six dogs. Three groups of sixteen implants were used, titanium implants (Control), zirconia implants (test A), and zirconia-microgrooved implants (test B), which were loaded immediately. After 4-month healing period, implant-bone samples were processed and analyzed by circularly polarized light (CPL) and confocal laser scanning microscopy (CLSM) in two regions of interest ROI1 (to evaluate the interthread bone) and ROI2 (to evaluate the bone adjacent to the threads) of 1 mm thickness × 10 mm length each one. Bone organization differs near to the test B, compared with test A and control surfaces, active remodeling was detected surrounding test B implants, with alternancy of organized zones, meanwhile controls and test A areas showed organized areas mainly at 2 mm of implant surfaces. Transverse collagen fibers were significatively higher at ROI1 for test B implants (60.34 ± 4.34%), compared with controls (47.25 ± 3.51%) and test A (43.78 ± 2.78%) groups (P < 0.05). Meanwhile, it was not found any significant difference between groups in ROI2 (P > 0.05). CLSM confirmed the presence of collagen mineralized matrix inside microgrooves of test B groups. 3D reconstruction showed blood vessels in direct contact with the implant surfaces of all groups and bone and blood vessels penetration inside the microgrooves in test B group. The organized pattern of the microgrooved surfaces is able to induce transverse collagen fiber microenvironment reaction to the load, being positive to promote and to maintain the bone remodeling; in addition blood vessels and bone cells are able to penetrate microgrooved surfaces. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. 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.

  16. Evaluation of the therapeutic results of actinic keratosis treated with topical 5% fluorouracil by reflectance confocal laser microscopy: preliminary study*

    PubMed Central

    Ishioka, Priscila; Maia, Marcus; Rodrigues, Sarita Bartholomei; Marta, Alessandra Cristina; Hirata, Sérgio Henrique

    2015-01-01

    Topical treatment for actinic keratosis with 5% fluorouracil has a recurrence rate of 54% in 12 months of follow-up. This study analyzed thirteen actinic keratoses on the upper limbs through confocal microscopy, at the time of clinical diagnosis and after 4 weeks of treatment with fluorouracil. After the treatment was established and evidence of clinical cure was achieved, in two of the nine actinic keratoses, confocal microscopy enabled visualization of focal areas of atypical honeycomb pattern in the epidermis indicating therapeutic failure. Preliminary data suggest the use of confocal microscopy as a tool for diagnosis and therapeutic control of actinic keratosis. PMID:26131881

  17. Glycoconjugate profiles of the lancelet (Branchiostoma lanceolatum) ovary: a lectin histochemical study by laser confocal microscopy.

    PubMed

    Del Buono, Francesca; Candiani, Simona; Pestarino, Mario; Focarelli, Riccardo

    2004-08-01

    The presence and the distribution of carbohydrate moieties in ripe lancelet (Branchiostoma lanceolatum) oocytes (mean diameter 130 microm) was studied by lectin histochemistry in combination with enzyme and chemical treatments. Binding sites for eight lectins with specificities towards different glycan moieties were studied on sections of the whole body of mature female lancelets. Only three of the lectins tested reacted positively. Concanavalin-A (ConA)-binding glycoconjugates were localized in the cytoplasm, namely in yolk granules, whereas Artocarpus integrifolia (AIA) and Ricinus communis (RCA) agglutinins bound strongly to extracellular coats of the oocyte identified as the jelly coat and vitelline layer. No other tissues of the lancelet body were found to be positive to any lectin tested, except gut enterocytes which reacted strongly with AIA. Reactivity to ConA was abolished by pretreatment of sections with N-glycosidase F but not by mild alkaline hydrolysis, confirming that the glycoconjugates were of the N-linked type. On the contrary, chemical removal of O-linked chains by mild alkaline hydrolysis abolished AIA and RCA reactivity but had no effect on ConA positivity.

  18. A study of vascular response to thermal injury on hairless mice by fibre optic confocal imaging, laser doppler flowmetry and conventional histology.

    PubMed

    Vo, L T; Papworth, G D; Delaney, P M; Barkla, D H; King, R G

    1998-06-01

    Burn injury causes vascular thrombosis and occlusion by thermal damage to the vascular network in the dermis. In this study, fibre optic confocal imaging (FOCI) and laser doppler flowmetry were used to detect changes in vascular morphology and local dermal blood flux over 4 h, in three defined zones after a thermal burn (50 degrees C, 20 s duration, 3 mm in diameter) was induced on fully anaesthetised hairless mice. FITC-dextran (i.v.) was used to enable FOCI of vascular morphology including three-dimensional imaging of the burn site and its surrounding areas. Samples of the affected areas were collected for conventional histology, including Masson's trichrome. There was vascular damage in the zone of coagulation which showed no change during the 4 h period. The zone of stasis showed an initial reduction in blood flux and confocal imaging of the area indicated significant vessel leakage during the first 2 h which later improved. The zone of hyperaemia showed an initial increase in total blood flux and confocal imaging of the area showed initial blood vessel dilatation. This study demonstrates that FOCI is a useful non-invasive tool in the assessment of vascular changes in thermal burns in vivo, and compares the findings of FOCI with those from laser doppler flowmetry and histology.

  19. Confocal laser endomicroscopy and narrow-band imaging-aided endoscopy for in vivo imaging of colitis and colon cancer in mice.

    PubMed

    Waldner, Maximilian J; Wirtz, Stefan; Neufert, Clemens; Becker, Christoph; Neurath, Markus F

    2011-09-01

    New endoscopic techniques such as narrow-band imaging (NBI) and confocal laser endomicroscopy (CLE) have improved the in vivo diagnosis of human gastrointestinal diseases in the colon. Whereas NBI may facilitate the identification of neoplastic lesions, CLE permits real-time histology of the inflamed or neoplastic colonic mucosa through the use of fluorescent dyes. These techniques have been recently adopted for use during ongoing endoscopy in mice. This protocol, which can be completed in 2 h, provides a detailed description of NBI and CLE in the mouse colon. In contrast to other techniques, this approach does not require laparotomy, and it allows direct CLE analysis of lesions identified by NBI. Mice exposed to models of colitis or colorectal cancer are anesthetized and examined with a miniaturized NBI endoscope, which provides an increased contrast of the vasculature. Upon identification of suspicious areas by NBI and the administration of fluorescent dyes, a confocal laser probe can be directed to the area of interest through the endoscope and confocal images can be obtained. Through the use of various fluorescent dyes, different aspects of the mucosa can be assessed. In addition, fluorescence-labeled antibodies can be used for molecular imaging of mice in vivo. Mouse NBI endoscopy and CLE represent reliable and fast high-quality techniques for the endoscopic characterization and molecular imaging of the mucosa in colitis and colon cancer.

  20. 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.

  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. Atmospheric propagation and combining of high power lasers: comment.

    PubMed

    Goodno, Gregory D; Rothenberg, Joshua E

    2016-10-10

    Nelson et al. [Appl. Opt.55, 1757 (2016)APOPAI0003-693510.1364/AO.55.001757] recently concluded that coherent beam combining and remote phase locking of high-power lasers are fundamentally limited by the laser source linewidth. These conclusions are incorrect and not relevant to practical high-power coherently combined laser architectures.

  3. 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.

  4. Early detection of acute graft-versus-host disease by wireless capsule endoscopy and probe-based confocal laser endomicroscopy: results of a pilot study

    PubMed Central

    Laurent, Valerie; Malard, Florent; Le Rhun, Marc; Chevallier, Patrice; Guillaume, Thierry; Mosnier, Jean-François; Galmiche, Jean-Paul; Mohty, Mohamad

    2014-01-01

    Objective Acute gastrointestinal graft-versus-host disease (GI-GVHD) is usually diagnosed using endoscopic examinations and biopsies for conventional histology. The aim of this pilot study was to determine whether mini-invasive techniques such as probe-based confocal laser endomicroscopy (pCLE) combined with wireless capsule endoscopy (WCE) could detect early lesions of GI-GVHD prior to symptoms. Design Fifteen patients undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT) were prospectively examined with a small bowel WCE, duodenal and colorectal pCLE, and standard biopsies. Per study protocol, all these examinations were scheduled between day 21 and day 28 after allo-HSCT, independently of the presence or absence of digestive symptoms. Results During follow up, eight patients developed acute GI-GVHD. Sensitivity of WCE, pCLE, and histology were 50, 87.5, and 50%, respectively. Specificity of WCE, pCLE, and histology were 80, 71.5, and 80%, respectively. We showed a positive correlation between the Glücksberg scoring system and WCE (rho = 0.543, p = 0.036) and pCLE (rho = 0.727, p = 0.002) but not with standard histology (rho = 0.481, p = 0.069). Conclusions The results from this pilot study suggest that novel methods such as pCLE and WCE could be part of a mini-invasive algorithm for early detection of GI-GVHD. PMID:25360304

  5. 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

  6. 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

  7. Highly efficient phase locking and extracavity coherent combination of two diode-pumped Nd:YAG laser beams

    NASA Astrophysics Data System (ADS)

    Ménard, S.; Vampouille, M.; Colombeau, B.; Froehly, C.

    1996-12-01

    Intracavity phase locking and extracavity combination of the two emitted beams generated by a Nd:YAG laser longitudinally pumped by two fiber-coupled diode lasers are investigated. Phase locking is performed by a diffractive component located inside a confocal Fabry-Perot resonator. The coherent addition of the two synchronized beams in a single TEM00 wave is made by a birefringent interferometer, with 96.8% energy yield. 610-mW output power was obtained in a single-lobed beam profile with 30.5% optical-to-optical conversion efficiency in the continuous-wave regime.

  8. In vivo visualization of epidermal growth factor receptor and survivin expression in porcine pancreas using endoscopic ultrasound guided fine needle imaging with confocal laser-induced endomicroscopy.

    PubMed

    Nakai, Y; Shinoura, S; Ahluwalia, A; Tarnawski, A S; Chang, K J

    2012-12-01

    The aims of this pilot study were to establish a principle of molecular imaging of the pancreas and determine in vivo expression of epidermal growth factor receptor (EGF-R) and survivin using a novel endoscopic ultrasound-guided fine needle imaging (EUS-FNI) technique, which incorporates needle based confocal laser-induced endomicroscopy (nCLE) after intrapancreatic injection of FTIC-labeled antibodies. Studies were performed in anesthetized pigs. FITC-labeled specific antibodies against EGF-R and survivin were injected into the tail and neck of the pancreas using a 19 gauge needle introduced under EUS guidance. Thirty minutes later, nCLE was performed using a prototype needle-based confocal laser-induced endomicroscopy probe (Cellvizio AQ-Flex-19, Mauna Kea Technologies, Paris, France) to determine cellular and tissue localization of EGF-R and survivin in the pancreas. Then pigs were euthanized and specimens of pancreas from areas injected with antibodies were obtained for histologic examination under epifluorescence microscope. EUS-guided nCLE enabled visualization of EGF-R and survivin in pancreatic tissue. Expression of EGF-R and survivin in pancreas was confirmed by histology. EGF-R immunoreactivity was localized to majority of duct-lining cells and to the surface and cytoplasm of many acinar cells. Survivin was localized mainly to the acinar cells. This study demonstrated the feasibility of in vivo, real time visualization of EGF-R and survivin in the pancreas by local injection of FITC-labeled antibodies via EUS-guided fine needle injection, followed by EUS-guided needle based confocal laser-induced endomicroscopy.

  9. The applicability of hematoxylin-eosin staining plus fluorescence or confocal laser scanning microscopy to the study of elastic fibers in cartilages.

    PubMed

    de Carvalho, H F; Taboga, S R

    1996-11-01

    This study focuses on the use of hematoxylin-eosin staining plus fluorescence microscopy for the investigation of elastic fibers in some elastic cartilages. We have observed that elastic fibers are consistently imaged by the proposed procedure and the resolution attained is similar to that obtained with the classical Weigert's fuchsin-resorcin. The results also demonstrate that elastin autofluorescence gives little or no contribution to the final fluorescence and that the use of the confocal laser scanning microscope adds to the resolution, permits the use of thicker sections and reveals of minute structural at features. We conclude that this is a relevant tool in elastin research.

  10. Confocal laser endomicroscopy for in vivo diagnosis of Barrett's oesophagus and associated neoplasia: a pilot study conducted in a single Italian centre.

    PubMed

    Trovato, Cristina; Sonzogni, Angelica; Ravizza, Davide; Fiori, Giancarla; Tamayo, Darina; De Roberto, Giuseppe; de Leone, Annalisa; De Lisi, Stefania; Crosta, Cristiano

    2013-05-01

    Diagnosis and management of Barrett's oesophagus are controversial. Technical improvements in real-time recognition of intestinal metaplasia and neoplastic foci provide the chance for more effective target biopsies. Confocal laser endomicroscopy allows to analyze living cells during endoscopy. To assess the diagnostic accuracy, inter- and intra-observer variability of endomicroscopy for detecting in vivo neoplasia (dysplasia and/or early neoplasia) in Barrett's oesophagus. Prospective pilot study. Patients referred for known Barrett's oesophagus were screened. Endomicroscopy was carried out in a circular fashion, every 1-2 cm, on the whole columnar-lined distal oesophagus. Visible lesions, when present, were analyzed first. Targeted biopsies were taken. Confocal images were classified according to confocal Barrett classification. Endomicroscopic and histological findings were compared. Forty-eight out of 50 screened patients underwent endomicroscopy. Visible lesions were observed in 3 patients. In a per-biopsy analysis, Barrett's-oesophagus-associated neoplasia could be predicted with an accuracy of 98.1%. The agreement between endomicroscopic and histological results was substantial (κ=0.76). This study suggests that endomicroscopy can provide in vivo diagnosis of Barrett's oesophagus-associated neoplasia. Because it allows for the study of larger surface areas of the mucosa, endomicroscopy may lead to significant improvements in the in vivo screening and surveillance of Barrett's oesophagus. Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  11. Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier.

    PubMed

    Ilev, Ilko; Waynant, Ronald; Gannot, Israel; Gandjbakhche, Amir

    2007-09-01

    A novel fiber-optic confocal approach for ultrahigh depth-resolution (confocal microscope approach that is compatible with a differential confocal microscope technique. To improve the dynamic range of the resolving laser power and to achieve a high resolution in the nanometric range, we have designed a simple apertureless reflection confocal microscope with a highly sensitive single-mode-fiber confocal output. The fiber-optic design is an effective alternative to conventional pinhole-based confocal systems and offers a number of advantages in terms of spatial resolution, flexibility, miniaturization, and scanning potential. Furthermore, the design is compatible with the differential confocal pinhole microscope based on the use of the sharp diffraction-free slope of the axial confocal response curve rather than the area around the maximum of that curve. Combining the advantages of ultrahigh-resolution fiber-optic confocal microscopy, we can work beyond the diffraction barrier in the subwavelength (below 200 nm) nanometric range exploiting confocal nanobioimaging of single cell and intracellular analytes.

  12. Corneal healing after riboflavin ultraviolet-A collagen cross-linking determined by confocal laser scanning microscopy in vivo: early and late modifications.

    PubMed

    Mazzotta, Cosimo; Traversi, Claudio; Baiocchi, Stefano; Caporossi, Orsola; Bovone, Cristina; Sparano, Maria Caterina; Balestrazzi, Angelo; Caporossi, Aldo

    2008-10-01

    To assess early and late micromorphological modifications of cross-linked corneas in vivo by means of Heidelberg Retinal Tomography (HRT) II confocal microscopy. Prospective nonrandomized open trial. Micromorphological examination of 44 cross-linked keratoconic corneas was performed in vivo by HRT II confocal laser scanning microscopy. Riboflavin ultraviolet (UV)-A-induced corneal collagen cross-linking (CXL) was performed according to the Siena protocol: pilocarpin 1% drops 30 minutes before, topical anesthesia with lidocaine 4% drops 15 minutes before irradiation, mechanical scraping of epithelium (9-mm-diameter area), preirradiation soaking for 10 minutes in riboflavin solution 0.1% (Ricrolin, Sooft, Italy) applied every 2.5 minutes for 30 minutes, 30 minutes exposure to solid-state UVA illuminator (Caporossi; Baiocchi; Mazzotta, X-linker, CSO, Italy), 8-mm-diameter irradiated area, energy delivered 3 mW/cm(2). All patients were examined by confocal scans preoperatively and at the following times after treatment: one, three, and six months, and one, two, and three years. No damage to the limbal region was observed. Epithelial regrowth was complete after four days of soft contact lens bandage. The anatomy of the subepithelial plexus was restored one year after the operation with full corneal sensitivity. Increased density of extracellular matrix in late postoperative period indicated cross-linked collagen to a depth of 340 microm expressed by a late demarcation line. In vivo confocal microscopy showed early and late modification of corneal microstructure after the treatment. The three-year stability of CXL recorded could be related to increased cross-links formation, synthesis of well-structured collagen and new lamellar interconnections.

  13. Array confocal microscopy

    NASA Astrophysics Data System (ADS)

    Pacheco, Shaun

    -splitting gratings to efficiently split one laser beam into a number of equal energy outgoing beams, so this dissertation explores design methods and analyses of beam-splitting gratings to fabrication errors. In this dissertation, an optimization method to design single layer beam-splitting gratings with reduced sensitivity to fabrication errors is proposed. Beam-spitting gratings are typically only designed for a single wavelength, so achromatic beam-splitting grating doublets are also analyzed for possible use in array confocal microscopes with multiple excitation wavelengths. An analysis of the lateral shift between grating layers in the achromatic grating doublet proves grating profiles with constant first spatial derivatives are significantly less sensitive than continuous phase profiles. These achromatic grating doublets have designed performance at two wavelengths, but the diffraction angles at the two wavelengths differ. To overcome that limitation, scale-invariant achromatic gratings are designed, which not only provide designed performance at two wavelengths, but also equal diffraction angles at two wavelengths.

  14. A laser scanning confocal microscopy method. Simultaneous detection of intracellular Ca2+ and apoptosis using Fluo-3 and Hoechst 33342.

    PubMed

    Zhang, T; Cao, E H; Li, J F

    2000-04-01

    To develop a simple and direct method to simultaneously determine apoptotic cells from a treated population of cells and detect the changes of intracellular Ca2+ in these apoptotic cells, in particular single ones, by confocal microscopy. MGC-803 cells treated with As2O3 were used as the double-staining cell model with Hoechst 33342 as a DNA probe and Fluo-3AM as a Ca2+ indicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainability with Hoechst 33342 and corresponding fluorescence intensity between live and apoptotic cells, apoptotic cells and the changes in intracellular Ca2+ were detected at the same time by confocal microscopy. No necrotic cells in the group treated with As2O3 were found by the trypan blue exclusion test. The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intracellular Ca2+ in apoptotic and intact cells were simultaneously detected in the same sample. We provided a useful method to exactly detect changes in intracellular Ca2+ in apoptotic cells, especially in single ones, by confocal microscopy and to exclude the artifact effect of necrotic and intact cells.

  15. 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.

  16. Intraoperative Probe-Based Confocal Laser Endomicroscopy in Surgery and Stereotactic Biopsy of Low-Grade and High-Grade Gliomas: A Feasibility Study in Humans.

    PubMed

    Pavlov, Vladislav; Meyronet, David; Meyer-Bisch, Vincent; Armoiry, Xavier; Pikul, Brian; Dumot, Chloé; Beuriat, Pierre-Aurelien; Signorelli, Francesco; Guyotat, Jacques

    2016-10-01

    The management of gliomas is based on precise histologic diagnosis. The tumor tissue can be obtained during open surgery or via stereotactic biopsy. Intraoperative tissue imaging could substantially improve biopsy precision and, ultimately, the extent of resection. To show the feasibility of intraoperative in vivo probe-based confocal laser endomicroscopy (pCLE) in surgery and biopsy of gliomas. In our prospective observational study, 9 adult patients were enrolled between September 2014 and January 2015. Two contrast agents were used: 5-aminolevulinic acid (3 cases) or intravenous fluorescein (6 cases). Intraoperative imaging was performed with the Cellvizio system (Mauna Kea Technologies, Paris). A 0.85-mm probe was used for stereotactic procedures, with the biopsy needle modified to have a distal opening. During open brain surgery, a 2.36-mm probe was used. Each series corresponds to a separate histologic fragment. The diagnoses of the lesions were glioblastoma (4 cases), low-grade glioma (2), grade III oligoastrocytoma (2), and lymphoma (1). Autofluorescence of neurons in cortex was observed. Cellvizio images enabled differentiation of healthy "normal" tissue from pathological tissue in open surgery and stereotactic biopsy using fluorescein. 5-Aminolevulinic acid confocal patterns were difficult to establish. No intraoperative complications related to pCLE or to use of either contrast agent were observed. We report the initial feasibility and safety of intraoperative pCLE during primary brain tumor resection and stereotactic biopsy procedures. Pending further investigation, pCLE of brain tissue could be utilized for intraoperative surgical guidance, improvement in brain biopsy yield, and optimization of glioma resection via analysis of tumor margins. 5-ALA, 5-aminolevulinic acidpCLE, probe-based confocal laser endomicroscopyPpIX, protoporphyrin IX.

  17. Evaluation of baseline structural factors for predicting glaucomatous visual-field progression using optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy.

    PubMed

    Sehi, M; Bhardwaj, N; Chung, Y S; Greenfield, D S

    2012-12-01

    The objective of this study is to assess whether baseline optic nerve head (ONH) topography and retinal nerve fiber layer thickness (RNFLT) are predictive of glaucomatous visual-field progression in glaucoma suspect (GS) and glaucomatous eyes, and to calculate the level of risk associated with each of these parameters. Participants with ≥28 months of follow-up were recruited from the longitudinal Advanced Imaging for Glaucoma Study. All eyes underwent standard automated perimetry (SAP), confocal scanning laser ophthalmoscopy (CSLO), time-domain optical coherence tomography (TDOCT), and scanning laser polarimetry using enhanced corneal compensation (SLPECC) every 6 months. Visual-field progression was assessed using pointwise linear-regression analysis of SAP sensitivity values (progressor) and defined as significant sensitivity loss of >1 dB/year at ≥2 adjacent test locations in the same hemifield at P<0.01. Cox proportional hazard ratios (HR) were calculated to determine the predictive ability of baseline ONH and RNFL parameters for SAP progression using univariate and multivariate models. Seventy-three eyes of 73 patients (43 GS and 30 glaucoma, mean age 63.2±9.5 years) were enrolled (mean follow-up 51.5±11.3 months). Four of 43 GS (9.3%) and 6 of 30 (20%) glaucomatous eyes demonstrated progression. Mean time to progression was 50.8±11.4 months. Using multivariate models, abnormal CSLO temporal-inferior Moorfields classification (HR=3.76, 95% confidence interval (CI): 1.02-6.80, P=0.04), SLPECC inferior RNFLT (per -1 μm, HR=1.38, 95% CI: 1.02-2.2, P=0.02), and TDOCT inferior RNFLT (per -1 μm, HR=1.11, 95% CI: 1.04-1.2, P=0.001) had significant HRs for SAP progression. Abnormal baseline ONH topography and reduced inferior RNFL are predictive of SAP progression in GS and glaucomatous eyes.

  18. Evaluation of baseline structural factors for predicting glaucomatous visual-field progression using optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy

    PubMed Central

    Sehi, M; Bhardwaj, N; Chung, Y S; Greenfield, D S

    2012-01-01

    Purpose The objective of this study is to assess whether baseline optic nerve head (ONH) topography and retinal nerve fiber layer thickness (RNFLT) are predictive of glaucomatous visual-field progression in glaucoma suspect (GS) and glaucomatous eyes, and to calculate the level of risk associated with each of these parameters. Methods Participants with ≥28 months of follow-up were recruited from the longitudinal Advanced Imaging for Glaucoma Study. All eyes underwent standard automated perimetry (SAP), confocal scanning laser ophthalmoscopy (CSLO), time-domain optical coherence tomography (TDOCT), and scanning laser polarimetry using enhanced corneal compensation (SLPECC) every 6 months. Visual-field progression was assessed using pointwise linear-regression analysis of SAP sensitivity values (progressor) and defined as significant sensitivity loss of >1 dB/year at ≥2 adjacent test locations in the same hemifield at P<0.01. Cox proportional hazard ratios (HR) were calculated to determine the predictive ability of baseline ONH and RNFL parameters for SAP progression using univariate and multivariate models. Results Seventy-three eyes of 73 patients (43 GS and 30 glaucoma, mean age 63.2±9.5 years) were enrolled (mean follow-up 51.5±11.3 months). Four of 43 GS (9.3%) and 6 of 30 (20%) glaucomatous eyes demonstrated progression. Mean time to progression was 50.8±11.4 months. Using multivariate models, abnormal CSLO temporal-inferior Moorfields classification (HR=3.76, 95% confidence interval (CI): 1.02–6.80, P=0.04), SLPECC inferior RNFLT (per −1 μm, HR=1.38, 95% CI: 1.02–2.2, P=0.02), and TDOCT inferior RNFLT (per −1 μm, HR=1.11, 95% CI: 1.04–1.2, P=0.001) had significant HRs for SAP progression. Conclusion Abnormal baseline ONH topography and reduced inferior RNFL are predictive of SAP progression in GS and glaucomatous eyes. PMID:23060026

  19. 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.

  20. Development of a coculture system and use of confocal laser fluorescent microscopy to study human microvascular endothelial cell and mural cell interaction.

    PubMed

    Burch, Marcia G; Pepe, Gerald J; Dobrian, Anca D; Lattanzio, Frank A; Albrecht, Eugene D

    2005-07-01

    In the present study, human myometrial microvascular endothelial cells (HMMEC) were cocultured with human vascular smooth muscle cells (VSMC) labeled with fluorescent dyes to examine their morphological interaction using confocal laser fluorescent microscopy. HMMEC and VSMC labeled with fluorescent green and red dyes, respectively, attached to opposite sides of polyethyleneterephthalate membranes and remained viable for up to 96 h. In defined medium, 5%+/-3% of the VSMC cytoplasmic processes and 71%+/-17% of the HMMEC processes extended completely across the 13 microm thickness of the transmembrane. However, 41%+/-21% of the VSMC projections and 10%+/-3% of the HMMEC processes that traversed the membrane made contact with the opposing cell type. In cocultures incubated with angiopoietin-1 (Ang-1), although the number of VSMC or HMMEC projections was not significantly increased, the number of VSMC extending across the membrane and making contact with HMMEC was increased (P<0.05) to 88%+/-2%. The results of the current study demonstrate that coculture of fluorescent-labeled HMMEC and VSMC on a semipermeable transmembrane coupled with confocal laser fluorescent microscopy provides an in vitro experimental model to study morphological association of microvascular endothelial cells with mural cells. We propose that this system will greatly facilitate study of remodeling of the microvasculature in various organ systems.

  1. 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

  2. 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.

  3. 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