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Sample records for addition confocal microscopy

  1. 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.

  2. [Artefacts of confocal microscopy].

    PubMed

    Vekshin, N L; Frolov, M S

    2014-01-01

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

  3. Confocal microscopy and exfoliative cytology

    PubMed Central

    Reddy, Shyam Prasad; Ramani, Pratibha; Nainani, Purshotam

    2013-01-01

    Context: Early detection of potentially malignant lesions and invasive squamous-cell carcinoma in the oral cavity could be greatly improved through techniques that permit visualization of subtle cellular changes indicative of the neoplastic transformation process. One such technique is confocal microscopy. Combining rapidity with reliability, an innovative idea has been put forward using confocal microscope in exfoliative cytology. Aims: The main objective of this study was to assess confocal microscopy for cytological diagnosis and the results were compared with that of the standard PAP stain. Settings and Design: Confocal microscope, acridine orange (AO) stain, PAP (Papanicolaou) stain. The study was designed to assess confocal microscopy for cytological diagnosis. In the process, smears of patients with (clinically diagnosed and/or suspected) oral squamous cell carcinoma as well as those of controls (normal people) were stained with acridine orange and observed under confocal microscope. The results were compared with those of the standard PAP method. Materials and Methods: Samples of buccal mucosa smears from normal patients and squamous cell carcinoma patients were made, fixed in 100% alcohol, followed by AO staining. The corresponding set of smears was stained with PAP stain using rapid PAP stain kit. The results obtained were compared with those obtained with AO confocal microscopy. Results: The study had shown nuclear changes (malignant cells) in the smears of squamous cell carcinoma patients as increased intensity of fluorescence of the nucleus, when observed under confocal microscope. Acridine orange confocal microscopy showed good amount of sensitivity and specificity (93%) in identifying malignant cells in exfoliative cytological smears. Conclusion: Confocal microscopy was found to have good sensitivity in the identification of cancer (malignant) cells in exfoliative cytology, at par with the PAP method. The rapidity of processing and screening a

  4. Confocal microscopy in transmitted light

    NASA Astrophysics Data System (ADS)

    Dodt, Hans-Ulrich; Becker, Klaus

    2003-10-01

    We developed a confocal microscope for transmitted light to visualize fine details in phase objects like unstained biological specimens. The main difficulty of confocal microscopy in transmission is the alignment of illumination and detector pinholes. This alignment was achieved by using "electronic pinholes" on the detector side. As a first step, we were able to image cells in onion skin at greater depths and with higher resolution than by using conventional microscopy.

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

    NASA Astrophysics Data System (ADS)

    Grimm, Tobias; Wiora, Georg; Witt, Gerd

    2015-03-01

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

  6. Confocal multiview light-sheet microscopy

    PubMed Central

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

    2015-01-01

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

  7. Confocal microscopy in microgravity research

    NASA Astrophysics Data System (ADS)

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

    We have studied the application and the feasibility of confocal scanning laser microscopy (CSLM) in microgravity research. Its superior spatial resolution and 3D imaging capabilities and its use of light as a probe, render this instrument ideally suited for the study of living biological material on a (sub-)cellular level. In this paper a number of pertinent biological microgravity experiments is listed, concentrating on the direct observation of developing cells and cellular structures under microgravity condition. A conceptual instrument design is also presented, aimed at sounding rocket application followed by Biorack/Biolab application at a later stage.

  8. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY

    EPA Science Inventory

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

  9. Immunofluorescence and Confocal Microscopy of Neutrophils

    PubMed Central

    Allen, Lee-Ann H.

    2015-01-01

    Rapid recruitment of neutrophils to sites of infection and their ability to phagocytose and kill microbes is an important aspect of the innate immune response. Challenges associated with imaging of these cells include their short lifespan and small size and the fact that unstimulated cells are nonadherent. In addition, although cytoplasmic granules are plentiful, the abundance of many other organelles is diminished. Here we reprise methods for analysis of resting and activated cells using immunofluorescence and confocal microscopy, including kinetic analysis of phagosome maturation and degranulation, and detection of intraphagosomal superoxide accumulation. We describe approaches for rapid cell fixation and permeabilization that maximize antigen detection and discuss other variables that also affect data interpretation and image quality (such as cell spreading, degranulation, and phagocytosis). Finally, we show that these methods are also applicable to studies of neutrophil interactions with the extracellular matrix. PMID:24504957

  10. Imaging white adipose tissue with confocal microscopy.

    PubMed

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

    2014-01-01

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

  11. Confocal microscopy imaging of solid tissue

    EPA Science Inventory

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

  12. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: AXIAL RESOLUTION

    EPA Science Inventory

    Abstract

    Confocal Microscopy System Performance: Axial resolution.
    Robert M. Zucker, PhD

    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Re...

  13. TelePresence Confocal Laser Scanning Microscopy.

    PubMed

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

    2001-05-01

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

  14. Confocal filtering in cathodoluminescence microscopy of nanostructures

    SciTech Connect

    Narváez, Angela C. E-mail: j.p.hoogenboom@tudelft.nl; Weppelman, I. Gerward C.; Moerland, Robert J.; Hoogenboom, Jacob P. E-mail: j.p.hoogenboom@tudelft.nl; Kruit, Pieter

    2014-06-23

    Cathodoluminescence (CL) microscopy allows optical characterization of nanostructures at high spatial resolution. At the nanoscale, a main challenge of the technique is related to the background CL generated within the sample substrate. Here, we implement confocal detection of the CL signal to minimize the background contribution to the measurement. Nano-phosphors were used as point sources to evaluate the filtering capabilities of our confocal CL system, obtaining an axial intensity profile with 2.7 μm full width at half maximum for the central peak, in good correspondence with theoretical expectations. Considering the electron interaction volume, we found that the confocal filter becomes effective for electron energies above 20 keV, when using a 25 μm pinhole (0.86 Airy units). To illustrate our approach, we present confocal CL imaging of gold nanowires and triangular shaped plates deposited on an indium-tin oxide covered glass substrate, comparing the images with those obtained in standard unfiltered CL detection. The results show that confocal CL microscopy is a valuable tool for the investigation of nanostructures on highly cathodoluminescent substrates, widely used in biological and optical applications.

  15. High brightness LED in confocal microscopy

    NASA Astrophysics Data System (ADS)

    Vakili, Ali; Xiong, Daxi; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2015-03-01

    We have introduced a novel illumination system for line scanning confocal microscopy. Confocal microscopy is a popular imaging tool in many applications specifically in medical imaging. Line scanning confocal microscopes have been proven to provide images with resolution comparable to point scanning microscopes. In the point scanning microscopes, the light is focused onto a diffraction limited spot. A pinhole is placed conjugate to the diffraction limited spot, in front of the detector to reject the light coming from out-of-focus planes. Therefore, confocal microscopy can provide optical sectioning. The size of the pinhole determines the amount of light that reaches the detector. A large pinhole results in a blurry image since more of the out-of-focus light contribute to the image. On the other hand, a smaller pinhole rejects more of the light, leading to a lower signal-to-noise ratio. Ideally it is desired to deliver a larger amount of optical power to the diffraction limited spot to increase the signal-to-noise ratio and have a smaller pinhole to reject more of the out-of-focus light. This is the property of the illumination system. In order to get a good signal-to noise ratio in the image, the light source has to provide sufficient radiance. We have introduced a new illumination system utilizing a high brightness LED in the line scanning confocal microscope. High brightness LEDs provide more optical power compared to ordinary LEDs from a smaller area; they have higher radiance. Preliminary results from our line scanning confocal microscope show that the high brightness LED is able to provide enough radiance to obtain an image with resolution comparable with the same microscope utilizing the laser diode. However, in high frame-rate application higher radiance or lower-noise detection system is required.

  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. Exploring the living cochlea using confocal microscopy.

    PubMed

    Ulfendahl, Mats; Boutet de Monvel, Jacques; Le Calvez, Sophie

    2002-01-01

    To obtain a more integrated view of the cellular behaviour of the cochlea it is essential to observe not only wider regions of the exposed turns but also to visualize structures below the reticular lamina. Using confocal microscopy and in vitro preparations of guinea pig and mouse inner ears, cellular structures within the intact organ of Corti can be visualized at high resolution. The approach thus offers a means to investigate detailed cellular events, e.g. structural reorganization following acoustic overstimulation. Confocal microscope images, although sharper than images acquired using regular light microscopy, are still subject to problems related to light scattering within the optical system and low signal-to-noise ratio. Significant image restoration can, however, be obtained by applying a combination of wavelet denoising techniques and deconvolution algorithms. Future work will focus both on more dynamical cellular events and on new in vivo models where the inner ear is visualized at a better functional state.

  18. Confocal Raman Microscopy in Pharmaceutical Development

    NASA Astrophysics Data System (ADS)

    Haefele, Thomas F.; Paulus, Kurt

    There is a wide range of applications of confocal Raman microscopy in pharmaceutical development. It is a powerful tool to probe the distribution of components within a formulation, to characterize homogeneity of pharmaceutical samples, to determine solid state of drug substances and excipients and to characterize contaminations and foreign particulates. The information obtained by confocal Raman microscopy is extremely useful, sometimes even crucial, for drug substance design, for the development of solid and liquid formulations, as a tool for process analytics and for patent infringements and counterfeit analysis. In this chapter, those aspects and applications will be presented, focusing on solid drug formulations. This chapter will also reveal the advantages and demonstrate the synergies of Raman mapping as compared to similar imaging methods such as SEM/EDX, NIR and MIR imaging.

  19. Comprehensive volumetric confocal microscopy with adaptive focusing

    PubMed Central

    Kang, DongKyun; Yoo, Hongki; Jillella, Priyanka; Bouma, Brett E.; Tearney, Guillermo J.

    2011-01-01

    Comprehensive microscopy of distal esophagus could greatly improve the screening and surveillance of esophageal diseases such as Barrett’s esophagus by providing histomorphologic information over the entire region at risk. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that can be configured to image the entire distal esophagus by helically scanning the beam using optics within a balloon-centering probe. It is challenging to image the human esophagus in vivo with balloon-based SECM, however, because patient motion and anatomic tissue surface irregularities decenter the optics, making it difficult to keep the focus at a predetermined location within the tissue as the beam is scanned. In this paper, we present a SECM probe equipped with an adaptive focusing mechanism that can compensate for tissue surface irregularity and dynamic focal variation. A tilted arrangement of the objective lens is employed in the SECM probe to provide feedback signals to an adaptive focusing mechanism. The tilted configuration also allows the probe to obtain reflectance confocal data from multiple depth levels, enabling the acquisition of three-dimensional volumetric data during a single scan of the probe. A tissue phantom with a surface area of 12.6 cm2 was imaged using the new SECM probe, and 8 large-area reflectance confocal microscopy images were acquired over the depth range of 56 μm in 20 minutes. Large-area SECM images of excised swine small intestine tissue were also acquired, enabling the visualization of villous architecture, epithelium, and lamina propria. The adaptive focusing mechanism was demonstrated to enable acquisition of in-focus images even when the probe was not centered and the tissue surface was irregular. PMID:21698005

  20. 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.

  1. Digital confocal microscopy through a multimode fiber

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    Acquiring high-contrast optical images deep inside biological tissues is still a challenging problem. Confocal microscopy is an important tool for biomedical imaging since it improves image quality by rejecting background signals. However, it suffers from low sensitivity in deep tissues due to light scattering. Recently, multimode fibers have provided a new paradigm for minimally invasive endoscopic imaging by controlling light propagation through them. Here we introduce a combined imaging technique where confocal images are acquired through a multimode fiber. We achieve this by digitally engineering the excitation wavefront and then applying a virtual digital pinhole on the collected signal. In this way, we are able to acquire images through the fiber with significantly increased contrast. With a fiber of numerical aperture 0.22, we achieve a lateral resolution of 1.5um, and an axial resolution of 12.7um. The point-scanning rate is currently limited by our spatial light modulator (20Hz).

  2. Probing individual molecules with confocal fluorescence microscopy.

    PubMed

    Nie, S; Chiu, D T; Zare, R N

    1994-11-11

    Confocal fluorescence microscopy coupled with a diffraction-limited laser beam and a high-efficiency detection system has been used to study the diffusive movement and emission process of individual fluorescent molecules in the liquid phase at room temperature. The high detection sensitivity achieved at fast data acquisition speeds (greater than 1 kilohertz) allows real-time observation of single-molecule fluorescence without statistical analysis. The results show fluorescence-cycle saturation at the single-molecule level and multiple recrossings of a single molecule into and out of the probe volume as well as the triplet state.

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

    SciTech Connect

    Ramanathan, Nathan Muruganathan; Darling, Seth B.

    2015-01-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2014-11-01

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

  6. EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: PRETTY PICTURES OR CONFOCAL QA

    EPA Science Inventory

    Evaluation of confocal microscopy system performance: Pretty pictures or confocal QA?

    Robert M. Zucker

    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, N...

  7. Quantitative confocal microscopy: beyond a pretty picture.

    PubMed

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

    2014-01-01

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

  8. 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.

  9. Reflectance Confocal Microscopy for Inflammatory Skin Diseases.

    PubMed

    Agozzino, M; Gonzalez, S; Ardigò, M

    2016-10-01

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

  10. Reflectance Confocal Microscopy for Inflammatory Skin Diseases.

    PubMed

    Agozzino, M; Gonzalez, S; Ardigò, M

    2016-10-01

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

  11. Automated cellular pathology in noninvasive confocal microscopy

    NASA Astrophysics Data System (ADS)

    Ting, Monica; Krueger, James; Gareau, Daniel

    2014-03-01

    A computer algorithm was developed to automatically identify and count melanocytes and keratinocytes in 3D reflectance confocal microscopy (RCM) images of the skin. Computerized pathology increases our understanding and enables prevention of superficial spreading melanoma (SSM). Machine learning involved looking at the images to measure the size of cells through a 2-D Fourier transform and developing an appropriate mask with the erf() function to model the cells. Implementation involved processing the images to identify cells whose image segments provided the least difference when subtracted from the mask. With further simplification of the algorithm, the program may be directly implemented on the RCM images to indicate the presence of keratinocytes in seconds and to quantify the keratinocytes size in the en face plane as a function of depth. Using this system, the algorithm can identify any irregularities in maturation and differentiation of keratinocytes, thereby signaling the possible presence of cancer.

  12. Confocal reference free traction force microscopy

    PubMed Central

    Bergert, Martin; Lendenmann, Tobias; Zündel, Manuel; Ehret, Alexander E.; Panozzo, Daniele; Richner, Patrizia; Kim, David K.; Kress, Stephan J. P.; Norris, David J.; Sorkine-Hornung, Olga; Mazza, Edoardo; Poulikakos, Dimos; Ferrari, Aldo

    2016-01-01

    The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Traction force microscopy (TFM) enables determination of the actuating forces. Despite progress, important limitations with intrusion effects in low resolution 2D pillar-based methods or disruptive intermediate steps of cell removal and substrate relaxation in high-resolution continuum TFM methods need to be overcome. Here we introduce a novel method allowing a one-shot (live) acquisition of continuous in- and out-of-plane traction fields with high sensitivity. The method is based on electrohydrodynamic nanodrip-printing of quantum dots into confocal monocrystalline arrays, rendering individually identifiable point light sources on compliant substrates. We demonstrate the undisrupted reference-free acquisition and quantification of high-resolution continuous force fields, and the simultaneous capability of this method to correlatively overlap traction forces with spatial localization of proteins revealed using immunofluorescence methods. PMID:27681958

  13. Digital confocal microscopy through a multimode fiber

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    Confocal laser-scanning microscopy is a well-known optical imaging method where a pinhole is used in the illumination and detection pathways of a normal microscope, in order to selectively excite and detect a particular focal volume. The advantage of this method is a significant increase in contrast, due to the rejection of background contributions to the signal. Here, we propose to apply this method in the context of multimode fiber endoscopy. Due to modal scrambling, it is not possible to use a physical pinhole to filter light signals that have travel through multimode fibers. Instead, we use a transmission matrix approach to characterize the propagation of light through the fiber, and we apply the filtering operation in the digital domain.

  14. Deep stroma investigation by confocal microscopy

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  15. Biological applications of confocal fluorescence polarization microscopy

    NASA Astrophysics Data System (ADS)

    Bigelow, Chad E.

    Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine

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

    PubMed

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

    2013-01-01

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

  17. Reflectance confocal microscopy in infectious diseases.

    PubMed

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

    2015-10-01

    In vivo reflectance confocal microscope (RCM) is a high-resolution non-invasive imaging technique that was initially focused on the diagnosis of skin cancers. A rising number of other indications have been later described for the diagnosis and management of inflammatory and infectious dermatological disorders. RCM can identify cutaneous parasites that are not visible to naked eye such as Sarcoptes scabiei and Demodex folliculorum and it allows to better identify the different body parts of bigger parasites such as ticks. Fungal filaments can also be identified as elongated bright structures in the cutaneous upper layers. RCM cannot observe virus directly. However, the cytopathic effect associated with some virus can be recognized. In addition of being helpful for the diagnosis and follow-up after treatment, thanks to its non-invasiveness, RCM allows pathophysiological studies. PMID:26129682

  18. Reflectance confocal microscopy for inflammatory skin diseases.

    PubMed

    Ardigò, M; Prow, T; Agozzino, M; Soyer, P; Berardesca, E

    2015-10-01

    Reflectance confocal microscopy evaluation of inflammatory skin diseases represents a relatively new indication that, during the last 5 years, has shown an increasing interest with consequent progressive increment of publications in literature. The success of RCM in this filed of dermatology is directly related to the high needing of non-invasive techniques able to reduce the number of skin biopsies and support the clinical diagnosis and patient's management. RCM demonstrated to visualize microscopic descriptors of inflammatory and pigmentary skin conditions with good reproducibility between observer and high grade of correspondence with optical histology. Moreover, RCM has shown to provide sufficient data to support clinical diagnosis and differential diagnosis of inflammatory and pigmentary skin diseases. Recently, several works published in literature have opened the prospective to use RCM also for therapeutic follow-up in order to monitor the improvement of the microscopic parameters and help to prevent treatment side effects. In this review article we present some examples of RCM application in inflammatory and pigmentary diseases. PMID:26333554

  19. EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE

    EPA Science Inventory

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

  20. Parallel detection experiment of fluorescence confocal microscopy using DMD.

    PubMed

    Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

    2016-05-01

    Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. PMID:26331288

  1. Confocal Fluorescence Microscopy of Mung Beanleaves

    NASA Astrophysics Data System (ADS)

    Chen, Zhiwei; Liu, Dongwu

    Recently, confocal microscope has become a routine technique and indispensable tool for cell biological studies and molecular investigations. The light emitted from the point out-of-focus is blocked by the pinhole and can not reach the detector, which is one of the critical features of the confocal microscope. In present studies, the probes acridine orange (AO) and rhodamine-123 were used to research stoma and mitochondria of mung bean leaves, respectively. The results indicated that the stomatal guard cells and mitochondria were clearly seen in epidermic tissue of mung bean leaves. Taken together, it is a good method to research plant cells with confocal microscope and fluorescence probes.

  2. 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...

  3. 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...

  4. Confocal microscopy via multimode fibers: fluorescence bandwidth

    NASA Astrophysics Data System (ADS)

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe

    2016-03-01

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

  5. 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 ...

  6. Stripe-shaped apertures in confocal microscopy.

    PubMed

    Shen, Shuhao; Zhu, Bingzhao; Zheng, Yao; Gong, Wei; Si, Ke

    2016-09-20

    We have theoretically verified that, compared with the aperture shapes of previous research, combining two stripe-shaped apertures in a confocal microscope with a finite-sized pinhole improves the axial resolution to a certain extent. Because different stripe shapes cause different effects, we also investigated the relationships among resolution, shapes, pinhole size, and the signal-to-background ratio.

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

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto

    2001-11-01

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

  8. Fluorescence performance standards for confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rüttinger, Steffen; Kapusta, Peter; Völlkopf, Volker; Koberling, Felix; Erdmann, Rainer; Macdonald, Rainer

    2010-02-01

    State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics. The main quantities such performance standard must address are: • Spatial resolution • Sensitivity • Fluorescence lifetime To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the corresponding standards have to be ready made, easy to handle and to store. The measurement procedures necessary should be available on as many different setups as possible and the procedures involved in their deployment should be as easy as possible. To this end, we developed two performance standards to accomplish the mentioned goals: • Resolution reference • Combined molecular brightness and fluorescence lifetime reference The first one is based on sub-resolution sized Tetra-SpeckTM fluorescent beads or alternatively on single molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to ascertain durability and road capabilities.

  9. Detection limits of confocal surface plasmon microscopy

    PubMed Central

    Pechprasarn, Suejit; Somekh, Michael G.

    2014-01-01

    This paper applies rigorous diffraction theory to evaluate the minimum mass sensitivity of a confocal optical microscope designed to excite and detect surface plasmons operating on a planar metallic substrate. The diffraction model is compared with an intuitive ray picture which gives remarkably similar predictions. The combination of focusing the surface plasmons and accurate phase measurement mean that under favorable but achievable conditions detection of small numbers of molecules is possible, however, we argue that reliable detection of single molecules will benefit from the use of structured surfaces. System configurations needed to optimize performance are discussed. PMID:24940537

  10. Visualizing Cochlear Mechanics Using Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Ulfendahl, M.; Boutet de Monvel, J.; Fridberger, A.

    2003-02-01

    The sound-evoked vibration pattern of the hearing organ is based on complex mechanical interactions between different cellular structures. To explore the structural changes occurring within the organ of Corti during basilar-membrane motion, stepwise alterations of the scala tympani pressure were applied in an in vitro preparation of the guinea-pig temporal bone. Confocal images were acquired at each pressure level. In this way, the motion of several structures could be simultaneously observed with high resolution in a nearly intact system. Images were analyzed using a novel wavelet-based optical-flow estimation algorithm. Under the present experimental conditions, the reticular lamina moved as a stiff plate with a center of rotation in the region of the inner hair cells. The outer hair cells appeared non-rigid and the basal, synaptic regions of these cells displayed significant radial motion indicative of cellular bending and internal shearing.

  11. Reflectance confocal microscopy for cutaneous infections and infestations.

    PubMed

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

    2016-05-01

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

  12. 3D imaging of neutron tracks using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

    Neutron detection and neutron flux assessment are important aspects in monitoring nuclear energy production. Neutron flux measurements can also provide information on potential biological damage from exposure. In addition to the applications for neutron measurement in nuclear energy, neutron detection has been proposed as a method of enhancing neutrino detectors and cosmic ray flux has also been assessed using ground-level neutron detectors. Solid State Nuclear Track Detectors (or SSNTDs) have been used extensively to examine cosmic rays, long-lived radioactive elements, radon concentrations in buildings and the age of geological samples. Passive SSNTDs consisting of a CR-39 plastic are commonly used to measure radon because they respond to incident charged particles such as alpha particles from radon gas in air. They have a large dynamic range and a linear flux response. We have previously applied confocal microscopy to obtain 3D images of alpha particle tracks in SSNTDs from radon track monitoring (1). As a charged particle traverses through the polymer it creates an ionisation trail along its path. The trail or track is normally enhanced by chemical etching to better expose radiation damage, as the damaged area is more sensitive to the etchant than the bulk material. Particle tracks in CR-39 are usually assessed using 2D optical microscopy. In this study 6 detectors were examined using an Olympus OLS4100 LEXT 3D laser scanning confocal microscope (Olympus Corporation, Japan). The detectors had been etched for 2 hours 50 minutes at 85 °C in 6.25M NaOH. Post etch the plastics had been treated with a 10 minute immersion in a 2% acetic acid stop bath, followed by rinsing in deionised water. The detectors examined had been irradiated with a 2mSv neutron dose from an Am(Be) neutron source (producing roughly 20 tracks per mm2). We were able to successfully acquire 3D images of neutron tracks in the detectors studied. The range of track diameter observed was between 4

  13. Confocal fluorescence microscopy and three-dimensional reconstruction.

    PubMed

    Wright, S J; Schatten, G

    1991-05-01

    Several recent technological advances have considerably improved the field of confocal fluorescence microscopy. Improvements in confocal microscope design, new fluorescent probes and indicators, more sensitive imaging devices, and computer advances which allow for data manipulation and storage provide a convenient method to acquire complex three-dimensional (3-D) architectural details which previously were difficult or impossible to obtain from biological specimens. Applications of the laser scanning and tandem scanning confocal microscopes offer the potential for gaining powerful insights into the complex relationship of cellular structure and function. Confocal microscopy generates optical sections free from out-of-focus blur. With the development of new visualization tools to render and display complex 3-D data, a set of optical sections taken at different focal planes can be three-dimensionally reconstructed to create an animated sequence which can reveal latent features of the specimen. The combination of confocal microscopy and 3-D reconstruction provides a powerful new imaging tool to advance knowledge about structural and functional cellular properties as they occur dynamically in three dimensions.

  14. Spinning-disk confocal microscopy: present technology and future trends.

    PubMed

    Oreopoulos, John; Berman, Richard; Browne, Mark

    2014-01-01

    Live-cell imaging requires not only high temporal resolution but also illumination powers low enough to minimize photodamage. Traditional single-point laser scanning confocal microscopy (LSCM) is generally limited by both the relatively slow speed at which it can acquire optical sections by serial raster scanning (a few Hz) and the higher potential for phototoxicity. These limitations have driven the development of rapid, parallel forms of confocal microscopy, the most popular of which is the spinning-disk confocal microscope (SDCM). Here, we briefly introduce the SDCM technique, discuss its strengths and weaknesses against LSCM, and update the reader on some recent developments in SDCM technology that improve its performance and expand its utility for life science research now and in the future.

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

    PubMed

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

    2016-09-01

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

  16. Measuring rotational diffusion of colloidal spheres with confocal microscopy.

    PubMed

    Liu, Bing; Böker, Alexander

    2016-07-13

    We report an experimental method to measure the translational and rotational dynamics of colloidal spheres in three dimensions with confocal microscopy and show that the experimental values reasonably agree with the theoretical values. This method can be extended to study rotational dynamics in concentrated colloidal systems and complex bio-systems. PMID:27353601

  17. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: QA TESTS, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

    Confocal Microscopy System Performance: QA tests, Quantitation and Spectroscopy.

    Robert M. Zucker 1 and Jeremy M. Lerner 2,
    1Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research Development, U.S. Environmen...

  18. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2016-09-01

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

  1. Reflectance confocal microscopy: an overview of technology and advances in telepathology.

    PubMed

    Batta, Mari M; Kessler, Stephen E; White, Peter F; Zhu, Weijian; Fox, Christi A

    2015-05-01

    The value of in vivo reflectance confocal microscopy (RCM) as a noninvasive adjunctive tool in dermatology has steadily advanced since its inception. With RCM, dermatologists can view horizontal sections of lesions in a resolution comparable to histology, observe dynamic processes in living skin, and monitor lesion evolution longitudinally. This article will compare RCM to dermoscopy and histology, review the general principles of the microscope, describe the findings seen on confocal images, and discuss the clinical applications of this noninvasive tool. Additionally, we describe a telepathology network dedicated to the transfer of confocal images to remote dermatopathologists for interpretation. Finally, we will discuss the adoption of RCM and the telepathology network in clinical practice.

  2. 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.

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

    NASA Astrophysics Data System (ADS)

    Shieh, Ian C.

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

  4. Imaging liver biology in vivo using conventional confocal microscopy.

    PubMed

    Marques, Pedro E; Antunes, Maísa M; David, Bruna A; Pereira, Rafaela V; Teixeira, Mauro M; Menezes, Gustavo B

    2015-02-01

    Imaging of live animals using intravital microscopy (IVM) has provided a substantial advance in our understanding of cell biology. Here we describe how to adapt a conventional, relatively low-cost laser-scanning microscope to operate as a versatile imaging station. We present the surgical procedures needed to perform liver confocal IVM in mice, thereby allowing one to image different cells in their native environment, including hepatocytes, endothelial cells and leukocytes, as well as to analyze their morphology and function under physiological or pathological conditions. In addition, we propose a plethora of working doses of antibodies and probes to stain multiple cells and molecules simultaneously in vivo. Considering the central role of the liver in metabolism and immunity and the growing interest in the relationship between immune and parenchymal cells, this protocol, in which 20 min of preparation yields up to 4 h of imaging, provides useful insights for various research fields. In addition, the protocol can be easily adapted to investigate adipose tissue, mesentery, intestines, spleen and virtually any abdominal organ.

  5. Confocal microscopy of skin cancers: Translational advances toward clinical utility

    PubMed Central

    Rajadhyaksha, Milind

    2014-01-01

    Recent advances in translational research in and technology for confocal microscopy of skin cancers, toward clinical applications, are described. Advances in translational research are in diagnosis of melanoma in vivo, pre-operative mapping of lentigo maligna melanoma margins to guide surgery and intra-operative imaging of residual basal cell carcinomas to guide shave-biopsy. Advances in technology include mosaicing microscopy for detection of basal cell carcinomas in large areas of excised tissue, toward rapid pathology-at-the-bedside, and development of small, simple and low-cost line-scanning confocal microscopes for worldwide use in diverse primary healthcare settings. Current limitations and future opportunities and challenges for both clinicians and technologists are discussed. PMID:19964286

  6. Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

    PubMed

    Cushway, T R; Lanzetta, M; Cox, G; Trickett, R; Owen, E R

    1996-01-01

    Fibre-optic technology combined with confocality produces a microscope capable of optical thin sectioning. In this original study, tibial nerves have been stained in a rat model with a vital dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide, and analysed by fibre-optic confocal microscopy to produce detailed images of nerve ultrastructure. Schwann cells, nodes of Ranvier and longitudinal myelinated sheaths enclosing axons were clearly visible. Single axons appeared as brightly staining longitudinal structures. This allowed easy tracing of multiple signal axons within the nerve tissue. An accurate measurement of internodal lengths was easily accomplished. This technique is comparable to current histological techniques, but does not require biopsy, thin sectioning or tissue fixing. This study offers a standard for further in vivo microscopy, including the possibility of monitoring the progression of nerve regeneration following microsurgical neurorraphy. PMID:9393664

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

    PubMed

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

    2016-01-01

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

  9. Intravital confocal Raman microscopy with multiplexed SERS contrast agents

    NASA Astrophysics Data System (ADS)

    McVeigh, Patrick Z.; Wilson, Brian C.

    2012-03-01

    Intravital microscopy has been demonstrated to be a powerful technique for studying the delivery of contrast or therapeutic agents to tumours growing in a realistic 3D environment at high resolution. Surface enhanced Raman scattering (SERS)-active nanoparticle contrast agents provide the ability to improve in-vivo detection of tumour tissue through multiplex detection of their uniquely bright spectral lines. However, most work to date has been carried out in-vitro or in ex-vivo tissues. Here we present the results from confocal Raman microscopy in a dorsal skinfold window chamber in mice using SERS-active gold nanoparticle contrast agents directed towards an overexpressed tumour receptor tyrosine kinase.

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

    PubMed

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

    2015-02-01

    Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive-index mismatch for high-aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high-angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point-scanning confocal microscope and showed that a nearly index-matched, micron-sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.

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

    PubMed Central

    BESSELING, TH; JOSE, J; BLAADEREN, A VAN

    2015-01-01

    Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive-index mismatch for high-aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high-angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point-scanning confocal microscope and showed that a nearly index-matched, micron-sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles. PMID:25444358

  12. Study of liquid jet instability by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Lisong; Adamson, Leanne J.; Bain, Colin D.

    2012-07-01

    The instability of a liquid microjet was used to measure the dynamic surface tension of liquids at the surface ages of ≤1 ms using confocal microscopy. The reflected light from a laser beam at normal incidence to the jet surface is linear in the displacement of the surface near the confocal position, leading to a radial resolution of 4 nm and a dynamic range of 4 μm in the surface position, thus permitting the measurement of amplitude of oscillation at the very early stage of jet instability. For larger oscillations outside the linear region of the confocal response, the swell and neck position of the jet can be located separately and the amplitude of oscillation determined with an accuracy of 0.2 μm. The growth rate of periodically perturbed water and ethanol/water mixture jets with a 100-μm diameter nozzle and mean velocity of 5.7 m s-1 has been measured. The dynamic surface tension was determined from the growth rate of the instability with a linear, axisymmetric, constant property model. Synchronisation of the confocal imaging system with the perturbation applied to the jet permitted a detailed study of the temporal evolution of the neck into a ligament and eventually into a satellite drop.

  13. Confocal microscopy patterns in nonmelanoma skin cancer and clinical applications.

    PubMed

    González, S; Sánchez, V; González-Rodríguez, A; Parrado, C; Ullrich, M

    2014-06-01

    Reflectance confocal microscopy is currently the most promising noninvasive diagnostic tool for studying cutaneous structures between the stratum corneum and the superficial reticular dermis. This tool gives real-time images parallel to the skin surface; the microscopic resolution is similar to that of conventional histology. Numerous studies have identified the main confocal features of various inflammatory skin diseases and tumors, demonstrating the good correlation of these features with certain dermatoscopic patterns and histologic findings. Confocal patterns and diagnostic algorithms have been shown to have high sensitivity and specificity in melanoma and nonmelanoma skin cancer. Possible present and future applications of this noninvasive technology are wide ranging and reach beyond its use in noninvasive diagnosis. This tool can also be used, for example, to evaluate dynamic skin processes that occur after UV exposure or to assess tumor response to noninvasive treatments such as photodynamic therapy. We explain the characteristic confocal features found in the main nonmelanoma skin tumors and discuss possible applications for this novel diagnostic technique in routine dermatology practice. PMID:24002008

  14. Spectral confocal reflection microscopy using a white light source

    NASA Astrophysics Data System (ADS)

    Booth, M.; Juškaitis, R.; Wilson, T.

    2008-08-01

    We present a reflection confocal microscope incorporating a white light supercontinuum source and spectral detection. The microscope provides images resolved spatially in three-dimensions, in addition to spectral resolution covering the wavelength range 450-650nm. Images and reflection spectra of artificial and natural specimens are presented, showing features that are not normally revealed in conventional microscopes or confocal microscopes using discrete line lasers. The specimens include thin film structures on semiconductor chips, iridescent structures in Papilio blumei butterfly scales, nacre from abalone shells and opal gemstones. Quantitative size and refractive index measurements of transparent beads are derived from spectral interference bands.

  15. Simultaneous multiplane confocal microscopy using acoustic tunable lenses.

    PubMed

    Duocastella, Martí; Vicidomini, Giuseppe; Diaspro, Alberto

    2014-08-11

    Maximizing the amount of spatiotemporal information retrieved in confocal laser scanning microscopy is crucial to understand fundamental three-dimensional (3D) dynamic processes in life sciences. However, current 3D confocal microscopy is based on an inherently slow stepwise process that consists of acquiring multiple 2D sections at different focal planes by mechanical or optical z-focus translation. Here, we show that by using an acoustically-driven optofluidic lens integrated in a commercial confocal system we can capture an entire 3D image in a single step. Our method is based on continuous axial scanning at speeds as high as 140 kHz combined with fast readout. In this way, one or more focus sweeps are produced on a pixel by pixel basis and the detected photons can be assigned to their corresponding focal plane enabling simultaneous multiplane imaging. We exemplify this method by imaging calibration and biological fluorescence samples. These results open the door to exploring new fundamental processes in science with an unprecedented time resolution.

  16. Automated identification of epidermal keratinocytes in reflectance confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gareau, Dan

    2011-03-01

    Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6+/-2.8 μm and reflectance gradient b = 3.6+/-2.1 μm at the nuclear/cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

  17. Automated spherical aberration correction in scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

    Mismatch between the refractive indexes of immersion media and glass coverslips introduces spherical aberrations in microscopes especially for high numerical aperture objectives. This contribution demonstrates an automated adjustment of the coverslip correction collar in scanning confocal microscopy to compensate for spherical aberrations due to coverslip thickness mismatch. With a motorized coverslip correction collar, the adjustment procedure consists of xz image scans, image processing, correction quality evaluation, the mismatch estimation, and eventually the optimal adjustment of the correction collar. For fast correction with less photodamage, coarse-fine Gaussian fitting algorithms are proposed and evaluated with various specimen for their estimation accuracy. The benefits of the proposed automated correction are demonstrated for various coverslips with biological specimens, showing the optimized resolution of the confocal microscope.

  18. Multimodal confocal hyperspectral imaging microscopy with wavelength sweeping source

    NASA Astrophysics Data System (ADS)

    Kim, Young-Duk; Do, Dukho; Yoo, Hongki; Gweon, DaeGab

    2015-02-01

    There exist microscopes that are able to obtain the chemical properties of a sample, because there are some cases in which it is difficult to find out causality of a phenomenon by using only the structural information of a sample. Obtaining the chemical properties of a sample is important in biomedical imaging, because most biological phenomena include changes in the chemical properties of the sample. Hyperspectral imaging (HSI) is one of the popular imaging methods for characterizing materials and biological samples by measuring the reflectance or emission spectrum of the sample. Because all materials have a unique reflectance spectrum, it is possible to analyze material properties and detect changes in the chemical properties of a sample by measuring the spectral changes with respect to the original spectrum. Because of its ability to measure the spectrum of a sample, HSI is widely used in materials identification applications such as aerial reconnaissance and is the subject of various studies in microscopy. Although there are many advantages to using the method, conventional HSI has some limitations because of its complex configuration and slow speed. In this research we propose a new type of multimodal confocal hyperspectral imaging microscopy with fast image acquisition and a simple configuration that is capable of both confocal and HSI microscopies.

  19. Imaging intracellular protein dynamics by spinning disk confocal microscopy

    PubMed Central

    Stehbens, Samantha; Pemble, Hayley; Murrow, Lindsay; Wittmann, Torsten

    2012-01-01

    The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction. PMID:22264541

  20. 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.

  1. Atherosclerotic plaque detection by confocal Brillouin and Raman microscopies

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

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

    PubMed

    Jallad, Karim N; Hedderich, Hartmut G

    2005-09-30

    A new method of analyzing kohl, a cosmetic eyeliner, using confocal Raman microscopy is reported. This technique offers an important alternative to conventional spectroscopic techniques that provide elemental/atomic composition. Raman spectra of three kohl samples have been measured between 150 and 3000 cm(-1) at room temperature. The main component of two kohl samples was found to be lead(II) sulfide (PbS). Kohl is used as a traditional cosmetic and remedy in the Middle East, Far East, and Northern Africa. Since kohl products contain very high concentrations of lead, they constitute a risk for public health, particularly for children.

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

    PubMed

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

    2016-09-01

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

  4. Quantification of transendothelial migration using three-dimensional confocal microscopy.

    PubMed

    Cain, Robert J; d'Água, Bárbara Borda; Ridley, Anne J

    2011-01-01

    Migration of cells across endothelial barriers, termed transendothelial migration (TEM), is an important cellular process that underpins the pathology of many disease states including chronic inflammation and cancer metastasis. While this process can be modeled in vitro using cultured cells, many model systems are unable to provide detailed visual information of cell morphologies and distribution of proteins such as junctional markers, as well as quantitative data on the rate of TEM. Improvements in imaging techniques have made microscopy-based assays an invaluable tool for studying this type of detailed cell movement in physiological processes. In this chapter, we describe a confocal microscopy-based method that can be used to assess TEM of both leukocytes and cancer cells across endothelial barriers in response to a chemotactic gradient, as well as providing information on their migration into a subendothelial extracellular matrix, designed to mimic that found in vivo.

  5. 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

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

    PubMed

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

    2016-05-01

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

  7. Combined FLIM and reflectance confocal microscopy for epithelial imaging

    NASA Astrophysics Data System (ADS)

    Jabbour, Joey M.; Cheng, Shuna; Shrestha, Sebina; Malik, Bilal; Jo, Javier A.; Applegate, Brian; Maitland, Kristen C.

    2012-03-01

    Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12 mm2 field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm2 with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can potentially serve as a guide in early oral cancer detection..

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

    PubMed

    Franzen, Lutz; Selzer, Dominik; Fluhr, Joachim W; Schaefer, Ulrich F; Windbergs, Maike

    2013-06-01

    Understanding the penetration behaviour of drugs into human skin is a prerequisite for the rational development and evaluation of effective dermal drug delivery. The general procedure for the acquisition of quantitative drug penetration profiles in human skin is performed by sequential segmentation and extraction. Unfortunately, this technique is destructive, laborious and lacks spatial resolution. Confocal Raman microscopy bares the potential of a chemically selective, label free and nondestructive analysis. However, the acquisition of quantitative drug depth profiles within skin by Raman microscopy is impeded by imponderable signal attenuation inside the tissue. In this study, we present a chemical semi-solid matrix system simulating the optical properties of human skin. This system serves as a skin surrogate for investigation of Raman signal attenuation under controlled conditions. Caffeine was homogeneously incorporated within the skin surrogate, and Raman intensity depth profiles were acquired. A mathematical algorithm describing the Raman signal attenuation within the surrogate was derived from these profiles. Human skin samples were incubated with caffeine, and Raman intensity depth profiles were similarly acquired. The surrogate algorithm was successfully applied to correct the drug profiles in human skin for signal attenuation. For the first time, a mathematical algorithm was established, which allows correction of Raman signal attenuation in human skin, thus facilitating reliable drug quantification in human skin by confocal Raman spectroscopy.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  10. Segmentation of skin strata in reflectance confocal microscopy depth stacks

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    Reflectance confocal microscopy is an emerging tool for imaging human skin, but currently requires expert human assessment. To overcome the need for human experts it is necessary to develop automated tools for automatically assessing reflectance confocal microscopy imagery. This work presents a novel approach to this task, using a bag of visual words approach to represent and classify en-face optical sections from four distinct strata of the skin. A dictionary of representative features is learned from whitened and normalised patches using hierarchical spherical k-means. Each image is then represented by extracting a dense array of patches and encoding each with the most similar element in the dictionary. Linear discriminant analysis is used as a simple linear classifier. The proposed framework was tested on 308 depth stacks from 54 volunteers. Parameters are tuned using 10 fold cross validation on a training sub-set of the data, and final evaluation was performed on a held out test set. The proposed method generated physically plausible profiles of the distinct strata of human skin, and correctly classified 81.4% of sections in the test set.

  11. Precise colloids with tunable interactions for confocal microscopy

    PubMed Central

    Kodger, Thomas E.; Guerra, Rodrigo E.; Sprakel, Joris

    2015-01-01

    Model colloidal systems studied with confocal microscopy have led to numerous insights into the physics of condensed matter. Though confocal microscopy is an extremely powerful tool, it requires a careful choice and preparation of the colloid. Uncontrolled or unknown variations in the size, density, and composition of the individual particles and interactions between particles, often influenced by the synthetic route taken to form them, lead to difficulties in interpreting the behavior of the dispersion. Here we describe the straightforward synthesis of copolymer particles which can be refractive index- and density-matched simultaneously to a non-plasticizing mixture of high dielectric solvents. The interactions between particles are accurately tuned by surface grafting of polymer brushes using Atom Transfer Radical Polymerization (ATRP), from hard-sphere-like to long-ranged electrostatic repulsion or mixed charge attraction. We also modify the buoyant density of the particles by altering the copolymer ratio while maintaining their refractive index match to the suspending solution resulting in well controlled sedimentation. The tunability of the inter-particle interactions, the low volatility of the solvents, and the capacity to simultaneously match both the refractive index and density of the particles to the fluid opens up new possibilities for exploring the physics of colloidal systems. PMID:26420044

  12. Precise colloids with tunable interactions for confocal microscopy

    NASA Astrophysics Data System (ADS)

    Kodger, Thomas E.; Guerra, Rodrigo E.; Sprakel, Joris

    2015-09-01

    Model colloidal systems studied with confocal microscopy have led to numerous insights into the physics of condensed matter. Though confocal microscopy is an extremely powerful tool, it requires a careful choice and preparation of the colloid. Uncontrolled or unknown variations in the size, density, and composition of the individual particles and interactions between particles, often influenced by the synthetic route taken to form them, lead to difficulties in interpreting the behavior of the dispersion. Here we describe the straightforward synthesis of copolymer particles which can be refractive index- and density-matched simultaneously to a non-plasticizing mixture of high dielectric solvents. The interactions between particles are accurately tuned by surface grafting of polymer brushes using Atom Transfer Radical Polymerization (ATRP), from hard-sphere-like to long-ranged electrostatic repulsion or mixed charge attraction. We also modify the buoyant density of the particles by altering the copolymer ratio while maintaining their refractive index match to the suspending solution resulting in well controlled sedimentation. The tunability of the inter-particle interactions, the low volatility of the solvents, and the capacity to simultaneously match both the refractive index and density of the particles to the fluid opens up new possibilities for exploring the physics of colloidal systems.

  13. CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography)

    PubMed Central

    Siegel, Nisan; Storrie, Brian; Bruce, Marc

    2016-01-01

    FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called “CINCH”. An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution. PMID:26839443

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

    PubMed

    Meller, Karl; Theiss, Carsten

    2006-03-01

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

  15. Adaptive optics in digital micromirror based confocal microscopy

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  17. Confocal Brillouin microscopy for three-dimensional mechanical imaging

    NASA Astrophysics Data System (ADS)

    Scarcelli, Giuliano; Yun, Seok Hyun

    2008-01-01

    Acoustically induced inelastic light scattering, first reported in 1922 by Brillouin, allows non-contact, direct readout of the viscoelastic properties of a material and has widely been investigated for material characterization, structural monitoring and environmental sensing. Extending the Brillouin technique from point sampling spectroscopy to imaging modality would open up new possibilities for mechanical imaging, but has been challenging because rapid spectrum acquisition is required. Here, we demonstrate a confocal Brillouin microscope based on a fully parallel spectrometer-a virtually imaged phased array-that improves the detection efficiency by nearly 100-fold over previous approaches. Using the system, we show the first cross-sectional Brillouin imaging based on elastic properties as the contrast mechanism and monitor fast dynamic changes in elastic modulus during polymer crosslinking. Furthermore, we report the first in situ biomechanical measurement of the crystalline lens in a mouse eye. These results suggest multiple applications of Brillouin microscopy in biomedical and biomaterial science.

  18. Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

    PubMed

    Lork, Marie; Delvaeye, Mieke; Gonçalves, Amanda; Van Hamme, Evelien; Beyaert, Rudi

    2016-01-01

    Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy. PMID:27613040

  19. Confocal laser scanning microscopy with spatiotemporal structured illumination.

    PubMed

    Gao, Peng; Nienhaus, G Ulrich

    2016-03-15

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

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

    PubMed Central

    Bhutani, Jaikrit; Chakinala, Raja Chandra; Bhutani, Sukriti; Sachdeva, Shruti

    2015-01-01

    Diabetes is a systemic disease associated with many complications. These can be prevented and managed effectively if detected promptly. Confocal microscopy (CFM) is a diagnostic tool which has the potential to help in early detection of disease and timely management. CFM has the potential to serve as an excellent noninvasive modality for in vivo imaging and morphological analysis, which can aid us in assessing and monitoring various infectious and pathological diseases at the cellular level. Besides ophthalmological indications, CFM has shown good sensitivity and specificity for identifying those at risk of neuropathy and foot ulceration, monitoring evolution and therapeutic response in a wide range of neuropathies apart from diabetic neuropathy. Through this communication, we aim to sensitize the endocrinologists towards cerebral cavernous malformation as a biomarker to evaluate potential outcomes and therapies in human diabetic neuropathy. PMID:25593848

  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. A generalized Potts model for confocal microscopy images

    NASA Astrophysics Data System (ADS)

    Máté, Gabriell; Heermann, Dieter W.

    2015-01-01

    Much as being among the least invasive mainstream imaging technologies in life sciences, the resolution of confocal microscopy is limited. Imaged structures, e.g., chromatin-fiber loops, have diameters around or beyond the diffraction limit, and microscopy images show seemingly random spatial density distributions only. While such images are important because the organization of the chromosomes influences different cell mechanisms, many interesting questions can also be related to the observed patterns. These concern their spatial aspects, the role of randomness, the possibility of modeling these images with a random generative process, the interaction between the densities of adjacent loci, the length-scales of these influences, etc. We answer these questions by implementing a generalization of the Potts model. We show how to estimate the model parameters, test the performance of the estimation process and numerically prove that the obtained values converge to the ground truth. Finally, we generate images with a trained model and show that they compare well to real cell images.

  3. Variational attenuation correction in two-view confocal microscopy

    PubMed Central

    2013-01-01

    Background Absorption and refraction induced signal attenuation can seriously hinder the extraction of quantitative information from confocal microscopic data. This signal attenuation can be estimated and corrected by algorithms that use physical image formation models. Especially in thick heterogeneous samples, current single view based models are unable to solve the underdetermined problem of estimating the attenuation-free intensities. Results We present a variational approach to estimate both, the real intensities and the spatially variant attenuation from two views of the same sample from opposite sides. Assuming noise-free measurements throughout the whole volume and pure absorption, this would in theory allow a perfect reconstruction without further assumptions. To cope with real world data, our approach respects photon noise, estimates apparent bleaching between the two recordings, and constrains the attenuation field to be smooth and sparse to avoid spurious attenuation estimates in regions lacking valid measurements. Conclusions We quantify the reconstruction quality on simulated data and compare it to the state-of-the art two-view approach and commonly used one-factor-per-slice approaches like the exponential decay model. Additionally we show its real-world applicability on model organisms from zoology (zebrafish) and botany (Arabidopsis). The results from these experiments show that the proposed approach improves the quantification of confocal microscopic data of thick specimen. PMID:24350574

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

    PubMed

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

    2010-12-01

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

  5. 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

  6. Rapid observation of unfixed, unstained human skin biopsy specimens with confocal microscopy and visualization

    NASA Astrophysics Data System (ADS)

    Masters, Barry R.; Aziz, David J.; Gmitro, Arthur F.; Kerr, James H.; O'Grady, Terence C.; Goldman, Leon

    1997-10-01

    The use of reflected light confocal microscopy is proposed to rapidly observe unfixed, unstained biopsy specimens of human skin. Reflected light laser scanning confocal microscopy was used to compare a freshly excised, unfixed, unstained biopsy specimen, and in vivo human skin. Optical sections from the ex vivo biopsy specimen of human skin and in vivo human skin were converted to red-green anaglyphs for 3D visualization. Contrast was derived from intrinsic differences in the scattering properties of the organelles and cells within the tissue. Individual cellular layers were observed in both tissues from the surface to the papillary dermis. Confocal microscopy of an unfixed, unstained biopsy specimen showed cells and cell nuclei of the stratum spinosum. Confocal microscopy of in vivo human skin demonstrated optical sectioning through a hair shaft on the upper hand. The combination of reflected light confocal microscopy and 3D visualization with red-green anaglyphs provides a rapid technique for observing fresh biopsies of human skin.

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

    PubMed

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

    2016-05-16

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

  8. LED uniform illumination system for DMD-based confocal microscopy

    NASA Astrophysics Data System (ADS)

    Xiao, Kaimin; Hou, Wenmei; Xu, Qixin; Peng, Bofang

    2013-10-01

    Due to the coherence of laser light source it could produce coherent noise in parallel confocal microscopy based on Digital Micromirror Device (DMD) and thus affect the resolution. LED light source instead of the laser light source can give full play because of its incoherence characterization. In this paper, free-form surface lens is used for LED secondary optics design. According to the LED characteristics and the law of refraction, we have established differential equations of free-form surface. We solved equations with the method of Runge-Kutta by Matlab and the model was built in Tracepro for optical simulation. The results show that the uniformity on the DMD is better than 90% and the lighting efficiency is higher than before. The measured data show us a much more uniform illumination on DMD and LED uniform illumination system successfully avoided the gray error which was caused by the uneven illumination. The LED driver circuit using DC power supply provides us a more stable light source. The axial optical tomography is more accurate and the reconstruction of three-dimensional image is more clearer.

  9. Confocal microscopy for intracellular co-localization of proteins.

    PubMed

    Miyashita, Toshiyuki

    2015-01-01

    Confocal laser scanning microscopy is the best method to visualize intracellular co-localization of proteins in intact cells. Because of the point scan/pinhole detection system, light contribution from the neighborhood of the scanning spot in the specimen can be eliminated, allowing high Z-axis resolution. Fluorescence detection by sensitive photomultiplier tubes allows the usage of filters with a narrow bandpath, resulting in minimal cross-talk (overlap) between two spectra. This is particularly important in demonstrating co-localization of proteins with multicolor labeling. Here, the methods outlining the detection of transiently expressed tagged proteins and the detection of endogenous proteins are described. Ideally, the intracellular co-localization of two endogenous proteins should be demonstrated. However, when antibodies raised against the protein of interest are unavailable for immunofluorescence or the available cell lines do not express the protein of interest sufficiently enough for immunofluorescence, an alternative method is to transfect cells with expression plasmids that encode tagged proteins and stain the cells with anti-tag antibodies. However, it should be noted that the tagging of proteins of interest or their overexpression could potentially alter the intracellular localization or the function of the target protein. PMID:25859973

  10. Rhinosporidium seeberi Nuclear Cycle Activities Using Confocal Microscopy.

    PubMed

    Delfino, Darly; Mendoza, Leonel; Vilela, Raquel

    2016-02-01

    Rhinosporidium seeberi is an uncultivated Ichthyosporean infecting animals, including humans. Recent studies suggested R. seeberi undergoes synchronized nuclear division without cytokinesis. We used confocal microscopy to investigate R. seeberi nuclear division cycles in formalin-fixed tissues stained with DAPI and phalloidin. We report that R. seeberi nuclei in juvenile and intermediary sporangia synchronously divided without cytokinesis. Intermediary sporangia display numerous 3-4 μm nuclei at different mitotic stages as well as a thick inner layer with strong affinity for phalloidin. Mature sporangia showed numerous 5-12 μm cell-walled endospores, each containing a 2-4 μm in diameter nucleus. Phalloidin did not bind to the inner layers of mature sporangia or endospores. The development of a "germinative zone" in the inner layer of mature sporangia containing hundreds of nuclei was also confirmed. This study establishes that during the R. seeberi life cycle synchronous nuclear divisions without cytokinesis takes place, resulting in the formation of thousands of nuclei. Cytokinesis, on the other hand, is a 1-time event and occurs in the latest stages of intermediate sporangia, after the formation of thousands of nuclei and just before mature sporangia development. PMID:26461427

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

    PubMed

    Patel, Dipika V; McGhee, Charles N

    2013-01-01

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

  12. Improving transverse resolution of confocal microscopy through spatiotemporal modulation

    NASA Astrophysics Data System (ADS)

    Wang, Baokai; Zou, Limin; Zhang, Su; Tan, Jiubin

    2015-11-01

    A new method is proposed in this paper to improve transverse resolution of a confocal microscope. By setting up the model of a confocal microscope system through spatiotemporal modulation with moving gratings or acousto-optical modulation without defocus distance under coherent light illumination and deducing two-dimensional coherent image formula and transfer function, simulation tests are run with or without spatiotemporal modulation to prove the effectiveness of the proposed method. Simulation results indicate the proposed method can be used to improve the transverse resolution of a confocal microscope system.

  13. Reflectance confocal microscopy of cutaneous melanoma. Correlation with dermoscopy and histopathology*

    PubMed Central

    Rstom, Silvia Arroyo; Libório, Lorena Silva; Paschoal, Francisco Macedo

    2015-01-01

    In vivo Confocal Microscopy is a method for non-invasive, real-time visualization of microscopic structures and cellular details of the epidermis and dermis, which has a degree of resolution similar to that obtained with histology. We present a case of cutaneous melanoma in which diagnosis was aided by confocal microscopy examination. We also correlate the observed features with the dermoscopic and histopathological findings. Confocal microscopy proved to be an useful adjunct to dermoscopy, playing an important role as a method 'between clinical evaluation and histopathology'. PMID:26131877

  14. 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.

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

    PubMed Central

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

    2016-01-01

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

  16. Resolution doubling using confocal microscopy via analogy with structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Hayashi, Shinichi

    2016-08-01

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

  17. 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

  18. Confocal Microscopy of Jammed Matter: From Elasticity to Granular Thermodynamics

    NASA Astrophysics Data System (ADS)

    Jorjadze, Ivane

    Packings of particles are ubiquitous in nature and are of interest not only to the scientific community but also to the food, pharmaceutical, and oil industries. In this thesis we use confocal microscopy to investigate packing geometry and stress transmission in 3D jammed particulate systems. By introducing weak depletion attraction we probe the accessible phase-space and demonstrate that a microscopic approach to jammed matter gives validity to statistical mechanics framework, which is intriguing because our particles are not thermally activated. We show that the fluctuations of the local packing parameters can be successfully captured by the recently proposed 'granocentric' model, which generates packing statistics according to simple stochastic processes. This model enables us to calculate packing entropy and granular temperature, the so-called 'compactivity', therefore, providing a basis for a statistical mechanics of granular matter. At a jamming transition point at which there are formed just enough number of contacts to guarantee the mechanical stability, theoretical arguments suggest a singularity which gives rise to the surprising scaling behavior of the elastic moduli and the microstructure, as observed in numerical simulations. Since the contact network in 3D is typically hidden from view, experimental test of the scaling law between the coordination number and the applied pressure is lacking in the literature. Our data show corrections to the linear scaling of the pressure with density which takes into account the creation of contacts. Numerical studies of vibrational spectra, in turn, reveal sudden features such as excess of low frequency modes, dependence of mode localization and structure on the pressure. Chapter four describes the first calculation of vibrational density of states from the experimental 3D data and is in qualitative agreement with the analogous computer simulations. We study the configurational role of the pressure and demonstrate

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

    PubMed Central

    Lin, Tong; Gong, Lan

    2016-01-01

    Abstract The aim of the study was to evaluate the morphological changes of meibomian glands (MGs) in primary blepharospasm (PBS) by in vivo laser scanning confocal microscopy (LSCM) and to investigate the correlations between clinical data of PBS and LSCM parameters of MGs. This prospective and case–control study recruited 30 consecutive PBS patients and 30 age- and gender-matched healthy controls. After questionnaire assessments of ocular surface disease index (OSDI), Jankovic rating scale, and blepharospasm disability index, all subjects underwent blink rate evaluation, tear film break-up time (TBUT), corneal fluorescein staining (CFS), Schirmer test, MG expressibility, meibum quality, MG dropout, and LSCM examination of the MGs. The main LSCM outcomes included the mean MG acinar area and density, orifice diameter, meibum secretion reflectivity, acinar irregularity, and inhomogeneity of interstice and acinar wall. The PBS patients had significantly higher blink rate, higher OSDI and CFS scores, lower TBUT and Schirmer test value, and worse MG expressibility than the controls (All P < 0.05), whereas meibum quality showed no difference (P > 0.05). The PBS patients showed lower values of MG acinar area, orifice diameter and meibum secretion reflectivity, and higher scores of acinar irregularity and inhomogeneity of interstices than the controls (All P < 0.05). For the PBS patients, the severity of blepharospasm evaluated by JCR scale was strong correlated with MG acinar area (P < 0.001), orifice diameter (P = 0.002), meibum secretion reflectivity (P = 0.002), and MG acinar irregularity (P = 0.013). The MG expressibility was significantly correlated to MG acinar area (P = 0.039), orifice diameter (P < 0.001), and MG acinar irregularity (P = 0.014). The OSDI score was moderate correlated with MG acinar irregularity (P = 0.016), whereas the TBUT value was positively correlated with MG acinar area (P = 0.045) and negatively correlated to MG acinar

  20. High-resolution confocal Raman microscopy using pixel reassignment.

    PubMed

    Roider, Clemens; Ritsch-Marte, Monika; Jesacher, Alexander

    2016-08-15

    We present a practical modification of fiber-coupled confocal Raman scanning microscopes that is able to provide high confocal resolution in conjunction with high light collection efficiency. For this purpose, the single detection fiber is replaced by a hexagonal lenslet array in combination with a hexagonally packed round-to-linear multimode fiber bundle. A multiline detector is used to collect individual Raman spectra for each fiber. Data post-processing based on pixel reassignment allows one to improve the lateral resolution by up to 41% compared to a single fiber of equal light collection efficiency. We present results from an experimental implementation featuring seven collection fibers, yielding a resolution improvement of about 30%. We believe that our implementation represents an attractive upgrade for existing confocal Raman microscopes that employ multi-line detectors. PMID:27519099

  1. Reflectance confocal microscopy of red blood cells: simulation and experiment

    PubMed Central

    Zeidan, Adel; Yelin, Dvir

    2015-01-01

    Measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient’s health. In this work, we have simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the morphological parameters and the resulting characteristic interference patterns of the cell. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry that imaged the cells in a linear flow without artificial staining. By matching the simulated patterns to confocal images of the cells, this method could be used for measuring cell morphology in three dimensions and for studying their physiology. PMID:26600999

  2. WHOLE INSECT AND MAMMALIAN EMBRYO IMAGING WITH CONFOCAL MICROSCOPY: MORPHOLOGY AND APOPTOSIS

    EPA Science Inventory

    Background: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates...

  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. Clinical usefulness of reflectance confocal microscopy in the management of facial lentigo maligna melanoma.

    PubMed

    Alarcón, I; Carrera, C; Puig, S; Malvehy, J

    2014-04-01

    Facial lentigo maligna melanoma can be a diagnostic challenge in daily clinical practice as it has similar clinical and morphological features to other lesions such as solar lentigines and pigmented actinic keratoses. Confocal microscopy is a noninvasive technique that provides real-time images of the epidermis and superficial dermis with cellular-level resolution. We describe 3 cases of suspected facial lentigo maligna that were assessed using dermoscopy and confocal microscopy before histopathology study. In the first case, diagnosed as lentigo maligna melanoma, presurgical mapping by confocal microscopy was performed to define the margins more accurately. In the second and third cases, with a clinical and dermoscopic suspicion of lentigo maligna melanoma, confocal microscopy was used to identify the optimal site for biopsy. PMID:24657027

  5. Sugarcane cell wall structure and lignin distribution investigated by confocal and electron microscopy.

    PubMed

    Sant'Anna, Celso; Costa, Lilian T; Abud, Yuri; Biancatto, Lucas; Miguens, Flávio Costa; de Souza, Wanderley

    2013-08-01

    Lignocellulosic plant cell wall is considered a potential source for second generation biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a prerequisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron microscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and surrounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lignin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose-rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO4 cytochemistry revealed that lignin was predominantly distributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant-based biofuel production. PMID:23733560

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Paddock, Stephen W; Eliceiri, Kevin W

    2014-01-01

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

  9. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY AND FOUNDATIONS FOR QUANTITATION

    EPA Science Inventory

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The reliability of the CLSM to obtain specific measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. For man...

  10. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR CALIBRATION, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

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

  11. 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

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

    NASA Astrophysics Data System (ADS)

    Gareau, Daniel

    2014-03-01

    The reflectance confocal microscope has made translational progress in dermatology. 0.5 micrometer lateral resolution, 0.75mm field-of-view and excellent temporal resolution at ~15 frames/second serve the VivaScope well in the clinic, but it may be overlooked in basic research. This work reviews high spatiotemporal confocal microscopy and presents images acquired of various samples: zebra fish embryo where melanocytes with excellent contrast overly the spinal column, chicken embryo, where myocardium is seen moving at 15 frames/ second, calcium spikes in dendrites (fluorescence mode) just beyond the temporal resolution, and human skin where blood cells race through the artereovenous microvasculature. For an introduction to confocal microscopy, see: http://dangareau.net.s69818.gridserver.com/science/confocal-microscopy

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

    PubMed

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

    2014-08-01

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

  14. Confocal microscopy for astrocyte in vivo imaging: Recycle and reuse in microscopy

    PubMed Central

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

    2013-01-01

    In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo. PMID:23658537

  15. Image segmentation for integrated multiphoton microscopy and reflectance confocal microscopy imaging of human skin in vivo

    PubMed Central

    Chen, Guannan; Lui, Harvey

    2015-01-01

    Background Non-invasive cellular imaging of the skin in vivo can be achieved in reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) modalities to yield complementary images of the skin based on different optical properties. One of the challenges of in vivo microscopy is the delineation (i.e., segmentation) of cellular and subcellular architectural features. Methods In this work we present a method for combining watershed and level-set models for segmentation of multimodality images obtained by an integrated MPM and RCM imaging system from human skin in vivo. Results Firstly, a segmentation model based on watershed is introduced for obtaining the accurate structure of cell borders from the RCM image. Secondly,, a global region based energy level-set model is constructed for extracting the nucleus of each cell from the MPM image. Thirdly, a local region-based Lagrange Continuous level-set approach is used for segmenting cytoplasm from the MPM image. Conclusions Experimental results demonstrated that cell borders from RCM image and boundaries of cytoplasm and nucleus from MPM image can be obtained by our segmentation method with better accuracy and effectiveness. We are planning to use this method to perform quantitative analysis of MPM and RCM images of in vivo human skin to study the variations of cellular parameters such as cell size, nucleus size and other mophormetric features with skin pathologies. PMID:25694949

  16. Imaging of small particles using wide-field confocal microscopy

    NASA Astrophysics Data System (ADS)

    Morgan, Stephen P.; Sawyer, N. B. E.; Somekh, Michael G.; See, Chung Wah; Shekunov, B. Y.; Astrakharchik, E.

    2003-10-01

    Particle measurement is important in many applications such as the manufacture of drugs and paints, and aerosols. In bioimaging there is interest understanding the imaging of nanoparticles and subcellular scatterers. We present in this paper a wide field, phase measuring confocal microscope that can be used for such measurements. The wide field confocal response is obtained by illuminating both sample and reference arms of an interferometric microscope with nominally identical speckle patterns. When the speckle patterns are highly correlated the interference is significant. Contributions from out of focus planes result in uncorrelated speckle patterns and no interference. This provides a wide field confocal response. High speed measurements are enabled by parallel phase stepping using polarization optics. We have also developed a vector diffraction microscope model, using Mie theory as a scattering function, to validate the images of small particles. Correctly scaling the amplitudes of the unscattered and scattered electric fields enables co-polar transmission imaging to be modeled. Finally it is demonstrated that the phase is a more sensitive measurement of particle size than the amplitude.

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

    PubMed

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

    2016-10-01

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

  18. Quantification of whey in fluid milk using confocal Raman microscopy and artificial neural network.

    PubMed

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

    2015-06-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. In-vivo multi-spectral confocal microscopy

    NASA Astrophysics Data System (ADS)

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

    2005-03-01

    A multi-spectral confocal microendoscope (MCME) for in-vivo imaging has been developed. The MCME employs a flexible fiber-optic catheter coupled to a slit-scan confocal microscope with an imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The focus mechanism allows for imaging to a maximum tissue depth of 200 microns. The 3mm diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope. The confocal nature of the system provides optical sectioning with 3 micron lateral resolution and 30 micron axial resolution. The system incorporates two laser sources and is therefore capable of simultaneous acquisition of spectra from multiple dyes using dual excitation. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 8nm to 16nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersion characteristics of the prism. The MCME is designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. The primary applications for the system include diagnosis of disease in the gastro-intestinal tract and female reproductive system. In-vitro, and ex-vivo multi-spectral results are presented.

  4. A coherent model for turbid imaging with confocal microscopy

    PubMed Central

    Glazowski, Christopher E.; Zavislan, James

    2013-01-01

    We present an engineering model of coherent imaging within a turbid volume, such as human tissues, with a confocal microscope. The model is built to analyze the statistical effect of aberrations and multiply scattered light on the resulting image. Numerical modeling of theory is compared with experimental results. We describe the construction of a stable phantom that represents the statistical effect of object turbidity on the image recorded. The model and phantom can serve as basis for system optimization in turbid imaging. PMID:23577285

  5. Adaptive optics confocal microscopy using direct wavefront sensing.

    PubMed

    Tao, Xiaodong; Fernandez, Bautista; Azucena, Oscar; Fu, Min; Garcia, Denise; Zuo, Yi; Chen, Diana C; Kubby, Joel

    2011-04-01

    Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images in deep-tissue imaging. We introduce a confocal fluorescence microscope with adaptive optics, which can correct aberrations based on direct wavefront measurements using a Shack-Hartmann wavefront sensor with a fluorescent bead used as a point source reference beacon. The results show a 4.3× improvement in the Strehl ratio and a 240% improvement in the signal intensity for fixed mouse tissues at depths of up to 100 μm.

  6. Confocal microscopy through a multimode fiber using optical correlation

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed

    Babes, Liane; Kubes, Paul

    2016-01-01

    Intravital microscopy has evolved into an invaluable technique to study the complexity of tumors by visualizing individual cells in live organisms. Here, we describe a method for employing intravital spinning disk confocal microscopy to picture high-resolution tumor-stroma interactions in real time. We depict in detail the surgical procedures to image various tumor microenvironments and different cellular components in the liver.

  8. Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

    PubMed

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

    2015-03-01

    High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas.

  9. Characterization of lased enamel organic matrix using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Hsu, Chin-Ying S.; Girija, Veerappan

    2001-10-01

    In the past decade several studies have demonstrated the increased acid resistance in enamel demineralization after laser irradiation. However, the exact mechanism of action to this effect still remains a speculation. Recently, the role of organic matrix was revealed to be significant in the laser-induced inhibition of enamel demineralization. The aim of the present study was to characterize the lipid component of organic matrix in mature lazed enamel and unlazed enamel histochemically using a hydrophobic fluorescent probe with Confocal Laser Scanning Microscope (CLSM). Partial decalcification of thin enamel sections was carried out using 0.5 M of EDTA in a stainless steel grid for 5 hours, following fixation with 3.5% paraformaldehyde. Thereafter the sections were stained with Nile red coupled with CLSM. The intensity of the light reflection was analyzed under the same conditions for all specimens, ruling out the autofluorescence in the control sections. Confocal imaging revealed a diffuse and increased fluorescence of the lipid stain in the lazed areas suggesting that the swelling and coating of organic matrix on the surface of enamel crystals in the peri and interprismatic spaces is rendering the increased acid resistance. These findings will substantiate the proposed organic blocking theory in partially explaining the laser-induced prevention of enamel demineralization.

  10. Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector.

    PubMed

    Sánchez-Ortiga, Emilio; Sheppard, Colin J R; Saavedra, Genaro; Martínez-Corral, Manuel; Doblas, Ana; Calatayud, Arnau

    2012-04-01

    We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning.

  11. Group refractive index reconstruction with broadband interferometric confocal microscopy

    PubMed Central

    Marks, Daniel L.; Schlachter, Simon C.; Zysk, Adam M.; Boppart, Stephen A.

    2010-01-01

    We propose a novel method of measuring the group refractive index of biological tissues at the micrometer scale. The technique utilizes a broadband confocal microscope embedded into a Mach–Zehnder interferometer, with which spectral interferograms are measured as the sample is translated through the focus of the beam. The method does not require phase unwrapping and is insensitive to vibrations in the sample and reference arms. High measurement stability is achieved because a single spectral interferogram contains all the information necessary to compute the optical path delay of the beam transmitted through the sample. Included are a physical framework defining the forward problem, linear solutions to the inverse problem, and simulated images of biologically relevant phantoms. PMID:18451922

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery. PMID:25309455

  14. Super-resolution spinning-disk confocal microscopy using optical photon reassignment.

    PubMed

    Azuma, Takuya; Kei, Takayuki

    2015-06-01

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

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

    NASA Astrophysics Data System (ADS)

    Yang, Yong-fa; Li, Qi

    2014-12-01

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

  16. Corneal confocal microscopy: Recent progress in the evaluation of diabetic neuropathy

    PubMed Central

    Papanas, Nikolaos; Ziegler, Dan

    2015-01-01

    The present brief review discusses recent progress with corneal confocal microscopy for the evaluation of diabetic sensorimotor polyneuropathy. Corneal confocal microscopy is a new, non-invasive and reproducible diagnostic modality, and it can also be easily applied for patient follow up. It enables new perspectives of studying the natural history of diabetic sensorimotor polyneuropathy, severity of nerve fiber pathology and documenting early nerve fiber regeneration after therapeutic intervention. It shows moderate to high sensitivity and specificity for the timely diagnosis of diabetic sensorimotor polyneuropathy. Currently, corneal confocal microscopy is mainly used in specialized centers, but deserves more widespread application for the assessment of diabetic sensorimotor polyneuropathy. Finally, further progress is required in terms of technical improvements for automated nerve fiber quantification and for analysis of larger images. PMID:26221515

  17. Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy

    PubMed Central

    Yokogawa, Hideaki; Kobayashi, Akira; Mori, Natsuko; Sugiyama, Kazuhisa

    2015-01-01

    Purpose To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Methods Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Results Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Conclusion Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite. PMID:26445524

  18. 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.

  19. Impaired Intracellular Ca2+ Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Plank, David M.; Sussman, Mark A.

    2005-06-01

    Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2015-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    NASA Astrophysics Data System (ADS)

    Lunter, Dominique; Daniels, Rolf

    2016-03-01

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

  4. Case Report: melanoma and melanocytic nevus differentiation with reflectance confocal microscopy.

    PubMed Central

    Łudzik, Joanna; Witkowski, Alexander M; Pellacani, Giovanni

    2015-01-01

    Historically, melanoma has been typically diagnosed by naked-eye examination and confirmed with invasive biopsy. However, recently the use of reflectance confocal microscopy enables non-invasive bedside diagnosis of clinically equivocal lesions. We present a case in which reflectance confocal microscopy was used to evaluate two skin lesions in the same patient confirming the diagnosis of a melanoma and potentially avoiding invasive biopsy in the second benign melanocytic lesion.  Clinicians should be aware of the availability of new non-invasive technologies that can aid in early diagnosis of malignant skin tumors and potentially reduce the number of benign lesion excisions. PMID:26236471

  5. Imaging anisotropy using differential polarization laser scanning confocal microscopy.

    PubMed

    Steinbach, Gábor; Pomozi, István; Zsiros, Ottó; Menczel, László; Garab, Gyozo

    2009-01-01

    We have constructed differential polarization (DP) attachments to a laser scanning microscope (LSM) for imaging the main DP quantities of anisotropic microscopic objects. The DP-LSM operates with high-frequency modulation and subsequent demodulation and displays the main DP quantities pixel by pixel. These, for linearly polarized light, include: (i) linear birefringence (LB), which is exhibited by structurally and/or optically anisotropic material; (ii) linear dichroism (LD), which carries information on the anisotropic distribution of the molecules, i.e. of their absorbance transition dipole vectors, in the sample; (iii) fluorescence-detected LD (FDLD), which carries the same information for fluorescent dyes upon excitations with two orthogonally polarized light beams; (iv) anisotropy of the fluorescence emission (r), excited with non-polarized light, which is determined by the distribution of the emission transition dipole vectors in the sample and is analogous with LD and (v) the degree of polarization of the fluorescence emission (P), excited with polarized light, which depends on the depolarization of the emission e.g. due to the rotation of molecules during their excitation lifetimes. In fluorescence regimes, the DP images can be recorded in the confocal regime of the microscope, which thus warrants good spatial resolution and the possibility of mapping the anisotropy in three dimensions. In this paper, we outline the design and technical realization of our DP-LSM and give a few examples on DP imaging of different biological samples.

  6. Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

    PubMed Central

    Shimozawa, Togo; Yamagata, Kazuo; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M.; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

    2013-01-01

    A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to “pinhole cross-talk,” which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging. PMID:23401517

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-09-15

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

  9. Ex vivo confocal microscopy imaging to identify tumor tissue on freshly removed brain sample.

    PubMed

    Forest, Fabien; Cinotti, Elisa; Yvorel, Violaine; Habougit, Cyril; Vassal, François; Nuti, Christophe; Perrot, Jean-Luc; Labeille, Bruno; Péoc'h, Michel

    2015-09-01

    Confocal microscopy is a technique able to realize "optic sections" of a tissue with increasing applications. We wondered if we could apply an ex vivo confocal microscope designed for dermatological purpose in a routine use for the most frequent brain tumors. The aim of this work was to identify tumor tissue and its histopathological hallmarks, and to assess grading criteria used in neuropathological practice without tissue loss on freshly removed brain tissue. Seven infiltrating gliomas, nine meningiomas and three metastases of carcinomas were included. We compared imaging results obtained with the confocal microscope to frozen sections, smears and tissue sections of formalin-fixed tissue. Our results show that ex vivo confocal microscopy imaging can be applied to brain tumors in order to quickly identify tumor tissue without tissue loss. It can differentiate tumors and can assess most of grading criteria. Confocal microscopy could represent a new tool to identify tumor tissue on freshly removed sample and could help in selecting areas for biobanking of tumor tissue.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-07-01

    Clinical management of alopecia represents one of the major issues in dermatology. Scalp biopsies are not easily accepted because of the high bleeding and sensitive anatomical area. Trichoscopy is routinely used for diagnosis of alopecia, but in several cases lack to provide sufficient information on the status of the disease. Recently, reflectance confocal microscopy demonstrated its usefulness for the evaluation of several inflammatory skin condition and preliminary reports about alopecia have been proposed in the literature. The aim was to identify the confocal features characterizing scarring and non-scarring alopecia. Reflectance confocal microscopy from 86 patients affected by scarring (28 lichen planopilaris and 9 lupus erythematosus) and non-scarring alopecia (30 androgenic alopecia and 19 alopecia areata), were retrospectively, blinded evaluated. Good concordance between different readers on the confocal criteria has been assessed. Statistical significant features, specific for scarring alopecia and non-scarring alopecia have been identified. In this study, data on reflectance confocal microscopy features useful for the differential diagnosis between scarring and non-scarring alopecia have been identified. Further studies focusing on the use of this non-invasive technique in the therapeutic follow-up and distinction of sub-entities of alopecia are still required.

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

    PubMed Central

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

    2016-01-01

    A large percentage of breast cancer patients treated with breast conserving surgery need to undergo multiple surgeries due to positive margins found during post-operative margin assessment. Carcinomas could be removed completely during the initial surgery and additional surgery avoided if positive margins can be determined intra-operatively. Spectrally-encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to rapidly image the entire surgical margin at sub-cellular resolution and accurately determine margin status intra-operatively. In this paper, in order to test feasibility of using SECM for intra-operative margin assessment, we have evaluated the diagnostic accuracy of SECM for detecting various types of breast cancers. Forty-six surgically-removed breast specimens were imaged with a SECM system. Side-by-side comparison between SECM and histologic images showed that SECM images can visualize key histomorphologic patterns of normal/benign and malignant breast tissues. Small (500 µm × 500 µm) spatially-registered SECM and histologic images (n=124 for each) were diagnosed independently by three pathologists with expertise in breast pathology. Diagnostic accuracy of SECM for determining malignant tissues was high, average sensitivity of 0.91, specificity of 0.93, positive predictive value of 0.95, and negative predictive value of 0.87. Intra-observer agreement and inter-observer agreement for SECM were also high, 0.87 and 0.84, respectively. Results from this study suggest that SECM may be developed into an intra-operative margin assessment tool for guiding breast cancer excisions. PMID:26779830

  13. Dermoscopy, confocal laser microscopy, and hi-tech evaluation of vascular skin lesions: diagnostic and therapeutic perspectives.

    PubMed

    Grazzini, Marta; Stanganelli, Ignazio; Rossari, Susanna; Gori, Alessia; Oranges, Teresa; Longo, Anna Sara; Lotti, Torello; Bencini, Pier Luca; De Giorgi, Vincenzo

    2012-01-01

    Vascular skin lesions comprise a wide and heterogeneous group of malformations and tumors that can be correctly diagnosed based on natural history and physical examination. However, considering the high incidence of such lesions, a great number of them can be misdiagnosed. In addition, it is not so rare that an aggressive amelanotic melanoma can be misdiagnosed as a vascular lesion. In this regard, dermoscopy and confocal laser microscopy examination can play a central role in increasing the specificity of the diagnosis of such lesions. In fact, the superiority of these tools over clinical examination has encouraged dermatologists to adopt these devices for routine clinical practice, with a progressive spread of their use. In this review, we will go through the dermoscopic and the confocal laser microscopy of diagnosis of most frequent vascular lesions (i.e., hemangiomas angiokeratoma, pyogenic granuloma, angiosarcoma) taking into particular consideration the differential diagnosis with amelanotic melanoma. PMID:22950556

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

    PubMed

    Zapata, Félix; García-Ruiz, Carmen

    2016-07-01

    Explosives are increasingly being used for terrorist attacks to cause devastating explosions. The detection of their postblast residues after an explosion is a high challenge, which has been barely investigated, particularly using spectroscopic techniques. In this research, a novel methodology using confocal Raman microscopy has been developed for the analysis of postblast residues from 10 open-air explosions caused by 10 different explosives (TNT, RDX, PETN, TATP, HMTD, dynamite, black powder, ANFO, chloratite, and ammonal) commonly used in improvised explosive devices. The methodology for the determination of postblast particles from explosives consisted of examining the samples surfaces with both the naked eye, first, and microscopically (10× and 50×), immediately afterward; and finally, analyzing the selected residues by confocal Raman spectroscopy in order to identify the postblast particles from explosives. Interestingly, confocal Raman microscopy has demonstrated to be highly suitable to rapidly, selectively, and noninvasively analyze postblast microscopic particles from explosives up to the nanogram range. PMID:27281604

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

    PubMed

    Zapata, Félix; García-Ruiz, Carmen

    2016-07-01

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

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

    PubMed Central

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

    2012-01-01

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

  17. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells.

    PubMed

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

    2016-04-01

    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  1. MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES, EMBRYOS AND FETAL LIMBS USING CONFOCAL MICROSCOPY

    EPA Science Inventory

    The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional section...

  2. Confocal microscopy studies of morphology and apoptosis: ovaries, limbs, embryos and insects

    EPA Science Inventory

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

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

    PubMed Central

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

    2016-01-01

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

  4. Optical coherence tomography combined with confocal microscopy for investigation of interfaces in class V cavities

    NASA Astrophysics Data System (ADS)

    Rominu, Mihai; Sinescu, Cosmin; Petrescu, Emanuela; Haiduc, Claudiu; Rominu, Roxana; Enescu, Marius; Hughes, Michael; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

    2009-07-01

    Standardized class V cavities, prepared in human extracted teeth, were filled with Premise (Kerr) composite. The specimens were thermo cycled. The interfaces were examined using a system employing two simultaneous imaging channels, an en-face Optical Coherence Tomography channel and a confocal microscopy channel.

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

    PubMed Central

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

    2016-01-01

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

  6. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR QUANTIFYING CYTOMETRIC APPLICATIONS WITH SPECTROSCOPIC INSTRUMENTS

    EPA Science Inventory

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-07-01

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

  9. 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...

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

    PubMed

    Babes, Liane; Kubes, Paul

    2016-01-01

    Intravital microscopy has evolved into an invaluable technique to study the complexity of tumors by visualizing individual cells in live organisms. Here, we describe a method for employing intravital spinning disk confocal microscopy to picture high-resolution tumor-stroma interactions in real time. We depict in detail the surgical procedures to image various tumor microenvironments and different cellular components in the liver. PMID:27581024

  11. Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin

    NASA Astrophysics Data System (ADS)

    Song, Eunjoo; Ahn, YoonJoon; Ahn, Jinhyo; Ahn, Soyeon; Kim, Changhwan; Choi, Sanghoon; Boutilier, Richard Martin; Lee, Yongjoong; Kim, Pilhan; Lee, Ho

    2015-10-01

    We examined the optical clearing assisted confocal microscopy of the transgenic mouse skin. The pinna and dorsal skin were imaged with a confocal microscope after the application of glycerol and FocusClear. In case of the glycerol-treated pinna, the clearing was minimal due to the inefficient permeability. However, the imaging depth was improved when the pinna was treated with FocusClear. In case of dorsal skin, we were able to image deeply to the subcutaneous connective tissue with both agents. Various skin structures such as the vessel, epithelium cells, cartilage, dermal cells, and hair follicles were clearly imaged.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2015-02-15

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

  15. Rapid diagnosis of tinea incognito using handheld reflectance confocal microscopy: a paradigm shift in dermatology?

    PubMed

    Navarrete-Dechent, Cristián; Bajaj, Shirin; Marghoob, Ashfaq A; Marchetti, Michael A

    2015-06-01

    Dermatophytoses are common skin infections. Traditional diagnostic tests such as skin scrapings for light microscopy examination, fungal cultures and biopsies remain imperfect due to false-negative test results, cost, time required to perform the procedure, time delays in test results and/or a requirement for an invasive procedure. Herein, we present a case of an 80-year-old female whose tinea incognito was non-invasively diagnosed within seconds using handheld reflectance confocal microscopy (RCM). As non-invasive skin imaging continues to improve, we expect light-based office microscopy to be replaced with technologies such as RCM, which has multiple and continually expanding diagnostic applications.

  16. In-vivo immunofluorescence confocal microscopy of herpes simplex virus type 1 keratitis

    NASA Astrophysics Data System (ADS)

    Kaufman, Stephen C.; Laird, Jeffery A.; Beuerman, Roger W.

    1996-05-01

    The white-light confocal microscope offers an in vivo, cellular-level resolution view of the cornea. This instrument has proven to be a valuable research and diagnostic tool for the study of infectious keratitis. In this study, we investigate the direct visualization of herpes simplex virus type 1 (HSV-1)-infected corneal epithelium, with in vivo confocal microscopy, using HSV-1 immunofluorescent antibodies. New Zealand white rabbits were infected with McKrae strain of HSV-1 in one eye; the other eye of each rabbit was used as an uninfected control. Four days later, the rabbits were anesthetized and a cellulose sponge was applied to each cornea, and a drop of direct HSV fluorescein-tagged antibody was placed on each sponge every 3 to 5 minutes for 1 hour. Fluorescence confocal microscopy was then performed. The HSV-infected corneas showed broad regions of hyperfluorescent epithelial cells. The uninfected corneas revealed no background fluorescence. Thus, using the confocal microscope with a fluorescent cube, we were able to visualize HSV-infected corneal epithelial cells tagged with a direct fluorescent antibody. This process may prove to be a useful clinical tool for the in vivo diagnosis of HSV keratitis.

  17. 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.

  18. Shrinking of ultrathin polyelectrolyte multilayer capsules upon annealing: A confocal laser scanning microscopy and scanning force microscopy study

    NASA Astrophysics Data System (ADS)

    Leporatti, S.; Gao, C.; Voigt, A.; Donath, E.; Möhwald, H.

    Heating-induced morphological changes of micrometer size capsules prepared by step-wise deposition of oppositely charged polyelectrolytes onto melamine formaldehyde (MF) latex particles and biological cells with subsequent dissolution of the core have been investigated by confocal laser scanning microscopy (CLSM) and scanning force microscopy (SFM). For poly(styrenesulfonate-Na salt)/poly(allylamine hydrochloride) polyelectrolyte capsules a remarkable heating-induced shrinking is observed. An increase of the wall thickness corresponding to the capsule diameter decrease is found. The morphology of these microcapsules after temperature treatment is characterized. The thickening of the polyelectrolyte multilayer is interpreted in terms of a configurational entropy increase via polyanion-polycation bond rearrangement.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  20. Three-dimensional resolution and contrast-enhanced confocal microscopy with array detection.

    PubMed

    Ge, Baoliang; Wang, Yifan; Huang, Yujia; Kuang, Cuifang; Fang, Yue; Xiu, Peng; Rong, Zihao; Liu, Xu

    2016-05-01

    What we believe is a novel method for improving confocal microscopy's resolution and contrast in 3D space is proposed. Based on a conventional confocal microscopy setup, we use an array detector composed of 32 photomultiplier tubes (PMTs) to replace one point-detector, where the location offset of each PMT caused a different effective point spread function (PSF). By applying array detection and the fluorescence emission difference method of an image with a solid PSF and another with a donut-shaped PSF, we can enhance lateral resolution about 27% in real time with only one scan, and improve the axial resolving ability by about 22% simultaneously. Experimental results of both fluorescent beads and living cells are presented to verify the applicability and effectiveness of our method. PMID:27128062

  1. Confocal Raman microscopy to monitor extracellular matrix during dental pulp stem cells differentiation

    NASA Astrophysics Data System (ADS)

    Salehi, Hamideh; Collart-Dutilleul, Pierre-Yves; Gergely, Csilla; Cuisinier, Frédéric J. G.

    2015-07-01

    Regenerative medicine brings promising applications for mesenchymal stem cells, such as dental pulp stem cells (DPSCs). Confocal Raman microscopy, a noninvasive technique, is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800 to 3000 cm-1 region (C-H stretching) and the 960 cm-1 peak (ν1 PO43-) were collected (to image cells and phosphate, respectively), and the ratio of two peaks 1660 over 1690 cm-1 (amide I bands) to measure the collagen cross-linking has been calculated. Raman spectra of DPSCs after 21 days differentiation reveal several phosphate peaks: ν1 (first stretching mode) at 960 cm-1, ν2 at 430 cm-1, and ν4 at 585 cm-1 and collagen cross-linking can also be calculated. Confocal Raman microscopy enables monitoring osteogenic differentiation in vitro and can be a credible tool for clinical stem cell based research.

  2. Near-IR fluorescence and reflectance confocal microscopy for imaging of quantum dots in mammalian skin

    PubMed Central

    Mortensen, Luke J.; Glazowski, Christopher E.; Zavislan, James M.; DeLouise, Lisa A.

    2011-01-01

    Understanding the skin penetration of nanoparticles (NPs) is an important concern due to the increasing presence of NPs in consumer products, including cosmetics. Technical challenges have slowed progress in evaluating skin barrier and NP factors that contribute to skin penetration risk. To limit sampling error and other problems associated with histological processing, many researchers are implementing whole tissue confocal or multiphoton microscopies. This work introduces a fluorescence and reflectance confocal microscopy system that utilizes near-IR excitation and emission to detect near-IR lead sulfide quantum dots (QDs) through ex vivo human epidermis. We provide a detailed prediction and experimental analysis of QD detection sensitivity and demonstrate detection of QD skin penetration in a barrier disrupted model. The unique properties of near-IR lead-based QDs will enable future studies that examine the impact of further barrier-disrupting agents on skin penetration of QDs and elucidate mechanistic insight into QD tissue interactions at the cellular level. PMID:21698023

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

    PubMed

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

    2009-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  5. Improvements of axial resolution in confocal microscopy with fan-shaped apertures.

    PubMed

    Ma, Ye; Kuang, Cuifang; Gong, Wei; Xue, Li; Zheng, Yao; Wang, Yifan; Si, Ke; Liu, Xu

    2015-02-20

    Based on diffraction theory, this paper theoretically demonstrates improvements in axial resolution in confocal microscopy with fan-shaped apertures. It provides the optimal geometric parameters of the fan-shaped pupils to give the maximum axial resolution for a given pinhole size. To fully understand the overall performance, the signal-to-background ratio and the signal level with regard to the geometric parameters are discussed.

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

    PubMed

    Ulrich, Martina; Lange-Asschenfeldt, Susanne

    2013-06-01

    Confocal laser scanning microscopy (CLSM) represents an emerging technique for the noninvasive histomorphological analysis of skin in vivo and has shown its applicability for dermatological research as well as its value as an adjunct tool in the clinical management of skin cancer patients. Herein, we aim to give an overview on the current clinical indications for CLSM in dermatology and also highlight the diverse applications of CLSM in dermatological research. PMID:23338938

  7. Template confined synthesis of amorphous carbon nanotubes and its confocal Raman microscopy

    SciTech Connect

    Maity, Supratim; Roychowdhury, Tuhin; Chattopadhyay, Kalyan Kumar

    2014-04-24

    Amorphous carbon nanotubes (aCNTs) were synthesized by AAO (anodic aluminum oxide) template at a temperature 500 °C in nitrogen atmosphere using the citric acid as a carbon source without the help of any catalyst particles. Morphological analysis of the as prepared samples was carried out by field emission scanning electron microscopy (FESEM). Confocal Raman imaging has been studied and an attempt has been made to find out the graphitic (sp{sup 2}) and disordered phase of the CNTs.

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

    NASA Astrophysics Data System (ADS)

    Ulrich, Martina; Lange-Asschenfeldt, Susanne

    2013-06-01

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

  9. Combining confocal Raman microscopy and freeze-drying for quantification of substance penetration into human skin.

    PubMed

    Franzen, Lutz; Anderski, Juliane; Planz, Viktoria; Kostka, Karl-Heinz; Windbergs, Maike

    2014-12-01

    In the area of dermatological research, the knowledge of rate and extent of substance penetration into the human skin is essential not only for evaluation of therapeutics, but also for risk assessment of chemicals and cosmetic ingredients. Recently, confocal Raman microscopy emerged as a novel analytical technique for analysis of substance skin penetration. In contrast to destructive drug extraction and quantification, the technique is non-destructive and provides high spatial resolution in three dimensions. However, the generation of time-resolved concentration depth profiles is restrained by ongoing diffusion of the penetrating substance during analysis. To prevent that, substance diffusion in excised human skin can instantly be stopped at defined time points by freeze-drying the sample. Thus, combining sample preparation by freeze-drying with drug quantification by confocal Raman microscopy yields a novel analytical platform for non-invasive and quantitative in vitro analysis of substance skin penetration. This work presents the first proof-of-concept study for non-invasive quantitative substance depth profiling in freeze-dried excised human stratum corneum by confocal Raman microscopy. PMID:25219950

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

    PubMed Central

    Kiratli, Hayyam; Mocan, Mehmet C.; İrkeç, Murat

    2016-01-01

    This report aims to describe the facilitating role of in vivo confocal microscopy in differentiating inflammatory cells from a metastatic process in a patient with uveal melanoma and multiple systemic metastases who developed anterior uveitis while under ipilimumab treatment. A 43-year-old woman developed systemic metastases 11 months after treatment of amelanotic choroidal melanoma in her right eye with 30 Gy fractionated stereotactic radiotherapy. She first received temozolomide and then 4 cycles of ipilimumab 3 mg/kg/day. After the third cycle, severe anterior uveitis with coarse pigment clumps on the lens was seen in the left eye. Her left visual acuity declined from 20/20 to 20/80. Confocal microscopy revealed globular keratic precipitates with hyperreflective inclusions and endothelial blebs all suggestive of granulomatous uveitis. The uveitic reaction subsided after a 3-week course of topical corticosteroids, and her visual acuity was 20/20 again. Although uveal melanoma metastatic to the intraocular structures of the fellow eye is exceedingly rare and metastasis masquerading uveitis without any identifiable uveal lesion is even more unusual, it was still mandatory to rule out this distant possibility in our particular patient who already had widespread systemic metastases. Confocal microscopy was a useful complementary tool by identifying the inflammatory features of the keratic precipitates. PMID:27790127

  11. Multi-beam confocal microscopy based on a custom image sensor with focal-plane pinhole array effect.

    PubMed

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

    2013-01-28

    Multi-beam confocal microscopy without any physical pinhole was demonstrated. As a key device, a custom CMOS image sensor realizing a focal-plane pinhole array effect by special pixel addressing and discarding of the unwanted photocarriers was developed. The axial resolution in the confocal mode measured by FWHM for a planar mirror was 8.9 μm, which showed that the confocality has been achieved with the proposed CMOS image sensor.

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

    PubMed

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

    2016-03-01

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

  13. Tracking the Dephosphorylation of Resveratrol Triphosphate in Skin by Confocal Raman Microscopy

    PubMed Central

    Zhang, Guojin; Flach, Carol R.; Mendelsohn, Richard

    2007-01-01

    Polyphenolic resveratrol has been identified as a potent antioxidant acting as both a free radical scavenger and an inhibitor of enzyme oxidative activity. However, the reactive propensity of resveratrol also limits its use in topical formulations. A transient derivative of resveratrol, resveratrol triphosphate, has been designed to provide a means for the delayed delivery of the active compound in skin tissue where endogenous enzymes capable of dephosphorylation reside. Confocal Raman microscopy studies of intact pigskin biopsies treated with modified resveratrol provided information about the spatial distribution and time-dependence of permeation and conversion to the native active form. Conversion to the active form was not observed when skin samples were exposed to steam, a procedure that likely inactivates endogenous skin enzymes. In addition, treatment with the triphosphate compared to the parent compound revealed a more homogeneous distribution of resveratrol throughout the stratum corneum and viable epidermis when the former was applied. Thus, the bioavailability of resveratrol in the epidermis appears to be enhanced upon application of the pro-molecule compared to resveratrol. PMID:17826862

  14. Progress in reflectance confocal microscopy for imaging oral tissues in vivo

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  15. Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

    PubMed Central

    Leigh, Steven Y.; Chen, Ye; Liu, Jonathan T.C.

    2014-01-01

    A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively ‘labels’ in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues. PMID:24940534

  16. Crystallization kinetics of calcium oxalate hydrates studied by scanning confocal interference microscopy

    NASA Astrophysics Data System (ADS)

    Grohe, Bernd; Rogers, Kem A.; Goldberg, Harvey A.; Hunter, Graeme K.

    2006-10-01

    Scanning confocal interference microscopy (SCIM) is an optical technique that allows the visualization of structures below the limits of classical optical microscopy (≪250 nm). This study represents the first use of SCIM to analyze the formation of calcium oxalate crystals, the major constituent of kidney stones. Crystals were nucleated and grown on the glass bottom of Petri dishes in the presence and absence of the polyelectrolyte inhibitor poly- L-aspartic acid (poly-asp). In the absence of poly-asp, monoclinic calcium oxalate monohydrate (COM) nucleated from {1 0 0} or {0 1 0} faces. The first observed particles were 70-120 nm in diameter and grew by a step-like progression in the [0 0 1] and [0 1 0] directions. Addition of poly-asp had several effects on calcium oxalate formation. First, the number of particles was increased, but their sizes were decreased. Second, the rate of COM growth in the [0 0 1] direction was decreased to a greater extent than the rate along [0 1 0]. Third, the formation of tetragonal calcium oxalate dihydrate (COD) crystals was favored. Fourth, the rates of COD growth along <1 1 0> and allied directions were decreased, whereas that parallel to <0 0 1> is increased. Sequences of highly resolved growth fronts show step displacement for COM and moving crystal edges for COD. Analysis of image sequences suggested that growth is strongly affected by competing and alternating processes, in which diffusion processes are rate-limiting and induce nonlinear growth. This study shows that SCIM is a powerful technique for the quantitative analysis of crystallization processes and for determining the mode of action of inhibitors.

  17. High-speed line-scanning confocal holographic microscopy for quantitative phase imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Liu, Changgeng; Knitter, Sebastian; Cong, Zhilong; Sencan, Ikbal; Cao, Hui; Choma, Michael A.

    2016-03-01

    We present a high speed, phase-sensitive, line-scanning reflectance confocal interference microscope. We achieved rapid confocal imaging using a fast line-scan camera and quantitative phase imaging using off-axis digital holography on a 1D, line-by-line basis. In our prototype system, a He-Ne laser (~1.2 mW) was used to demonstrate the principle of operation. Using a 20 kHz line scan rate (1024 pixels per line scan), we achieved a video-rate frame rate of 20 Hz for 1024x500 pixel en-face confocal images (20 MHz total pixel rate). By using an objective lens of a NA 0.65, we achieved an axial and lateral resolution of ~3.5 micrometers and ~0.8 micrometers, respectively. By z-stack imaging of a custom silicon target with a stepped structure, we confirmed that the axial sectioning of the interference microscope is similar to that of a traditional line-scan confocal microscope (our microscope with the reference arm blocked). The utility of phase-sensitive holographic detection in line-scan confocal was demonstrated in two ways. First, using a custom axial height phantom fabricated using chrome deposition, we demonstrated variations in phase corresponding to heights in the 100 nm range with a contrast-to-noise ratio of ~31 dB. Second, we demonstrate digital refocusing of an out-of-focus holographic image. The mechanism of confocality in our line-scan system is 1D physical pinholing. Our ongoing work aims to add an additional mechanism of confocality by using low spatial coherence sources to impose interferometric pinholing.

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

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

  19. Dual-slit confocal light sheet microscopy for in vivo whole-brain imaging of zebrafish

    PubMed Central

    Yang, Zhe; Mei, Li; Xia, Fei; Luo, Qingming; Fu, Ling; Gong, Hui

    2015-01-01

    In vivo functional imaging at single-neuron resolution is an important approach to visualize biological processes in neuroscience. Light sheet microscopy (LSM) is a cutting edge in vivo imaging technique that provides micron-scale spatial resolution at high frame rate. Due to the scattering and absorption of tissue, however, conventional LSM is inadequate to resolve cells because of the attenuated signal to noise ratio (SNR). Using dual-beam illumination and confocal dual-slit detection, here a dual-slit confocal LSM is demonstrated to obtain the SNR enhanced images with frame rate twice as high as line confocal LSM method. Through theoretical calculations and experiments, the correlation between the slit’s width and SNR was determined to optimize the image quality. In vivo whole brain structural imaging stacks and the functional imaging sequences of single slice were obtained for analysis of calcium activities at single-cell resolution. A two-fold increase in imaging speed of conventional confocal LSM makes it possible to capture the sequence of the neurons’ activities and help reveal the potential functional connections in the whole zebrafish’s brain. PMID:26137381

  20. Taylor series expansion based multidimensional image reconstruction for confocal and 4pi microscopy

    NASA Astrophysics Data System (ADS)

    Dilipkumar, Shilpa; Pratim Mondal, Partha

    2013-08-01

    We propose and experimentally demonstrate a three-dimensional (3D) image reconstruction methodology based on Taylor series approximation (TSA) in a Bayesian image reconstruction formulation. TSA incorporates the requirement of analyticity in the image domain, and acts as a finite impulse response filter. This technique is validated on images obtained from widefield, confocal laser scanning fluorescence microscopy and two-photon excited 4pi (2PE-4pi) fluorescence microscopy. Studies on simulated 3D objects, mitochondria-tagged yeast cells (labeled with Mitotracker Orange) and mitochondrial networks (tagged with Green fluorescent protein) show a signal-to-background improvement of 40% and resolution enhancement from 360 to 240 nm. This technique can easily be extended to other imaging modalities (single plane illumination microscopy (SPIM), individual molecule localization SPIM, stimulated emission depletion microscopy and its variants).

  1. Towards real-time image deconvolution: application to confocal and STED microscopy

    PubMed Central

    Zanella, R.; Zanghirati, G.; Cavicchioli, R.; Zanni, L.; Boccacci, P.; Bertero, M.; Vicidomini, G.

    2013-01-01

    Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). We test the proposed algorithms both on synthetic and real data of confocal and STED microscopy. Combining the SGP method with the GPU implementation we achieve a speed-up factor from about a factor 25 to 690 (with respect the conventional algorithm). The excellent results obtained on STED microscopy images demonstrate the synergy between super-resolution techniques and image-deconvolution. Further, the real-time processing allows conserving one of the most important property of STED microscopy, i.e the ability to provide fast sub-diffraction resolution recordings. PMID:23982127

  2. Fibre optic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo.

    PubMed Central

    Delaney, P M; King, R G; Lambert, J R; Harris, M R

    1994-01-01

    Fibre optic confocal imaging (FOCI) is a new type of microscopy which has been recently developed (Delaney et al. 1993). In contrast to conventional light microscopy, FOCI and other confocal techniques allow clear imaging of subsurface structures within translucent objects. However, unlike conventional confocal microscopes which are bulky (because of a need for accurate alignment of large components) FOCI allows the imaging end to be miniaturised and relatively mobile. FOCI is thus particularly suited for clear subsurface imaging of structures within living animals or subjects. The aim of the present study was to assess the suitability of using FOCI for imaging of subsurface structures within the colon, both in vitro (human and rat biopsies) and in vivo (in rats). Images were obtained in fluorescence mode (excitation 488 nm, detection above 515 nm) following topical application of fluorescein. By this technique the glandular structure of the colon was imaged. FOCI is thus suitable for subsurface imaging of the colon in vivo. Images Fig. 2 Fig. 3 PMID:8157487

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  4. Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

    Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12 mm2 of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called "strip mosaicing," which was demonstrated on a 10-×-10 mm2 of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10 mm2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5 cm2 piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

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

    PubMed Central

    Demirkilinc Biler, Elif; Guven Yilmaz, Suzan; Palamar, Melis; Hamrah, Pedram

    2015-01-01

    Purpose. To report clinical and in vivo confocal microscopy (IVCM) findings of two patients with ocular ochronosis secondary due to alkaptonuria. Materials and Methods. Complete ophthalmologic examinations, including IVCM (HRT II/Rostock Cornea Module, Heidelberg, Germany), anterior segment optical coherence tomography (AS-OCT) (Topcon 3D spectral-domain OCT 2000, Topcon Medical Systems, Paramus, NJ, USA), corneal topography (Pentacam, OCULUS Optikgeräte GmbH, Wetzlar, Germany), and anterior segment photography, were performed. Results. Biomicroscopic examination showed bilateral darkly pigmented lesions of the nasal and temporal conjunctiva and episclera in both patients. In vivo confocal microscopy of the lesions revealed prominent degenerative changes, including vacuoles and fragmentation of collagen fibers in the affected conjunctival lamina propria and episclera. Hyperreflective pigment granules in different shapes were demonstrated in the substantia propria beneath the basement membrane. AS-OCT of Case 1 demonstrated hyporeflective areas. Fundus examination was within normal limits in both patients, except tilted optic discs with peripapillary atrophy in one of the patients. Corneal topography, thickness, and macular OCT were normal bilaterally in both cases. Conclusion. The degenerative and anatomic changes due to ochronotic pigment deposition in alkaptonuria can be demonstrated in detail with IVCM and AS-OCT. Confocal microscopic analysis in ocular ochronosis may serve as a useful adjunct in diagnosis and monitoring of the disease progression. PMID:26788390

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

    NASA Astrophysics Data System (ADS)

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

    2011-05-01

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

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

    PubMed Central

    Wielowieyska-Szybińska, Dorota; Białek-Galas, Kamila; Podolec, Katarzyna

    2014-01-01

    Reflectance confocal microscopy (RCM) is a modern, non-invasive diagnostic method that enables real-time imaging of epidermis and upper layers of the dermis with a nearly histological precision and high contrast. The application of this technology in skin imaging in the last few years has resulted in the progress of dermatological diagnosis, providing virtual access to the living skin erasing the need for conventional histopathology. The RCM has a potential of wide application in the dermatological diagnostic process with a particular reference to benign and malignant skin tumors. This article provides a summary of the latest reports and previous achievements in the field of RCM application in the diagnostic process of skin neoplasms. A range of dermatological indications and general characteristics of confocal images in various types of tumors are presented. PMID:25610353

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

    PubMed Central

    Reddy, D Shyam Prasad; Sherlin, Herald J; Ramani, Pratibha; Prakash, P Ajay

    2012-01-01

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

  9. Simultaneous multiplane imaging for 3D confocal microscopy using high-speed z-scanning multiplexing

    NASA Astrophysics Data System (ADS)

    Duocastella, Marti; Vicidomini, Giuseppe; Diaspro, Alberto

    2015-03-01

    One of the key frontiers in optical imaging is to maximize the spatial information retrieved from a sample while minimizing acquisition time. Confocal laser scanning microscopy is a powerful imaging modality that allows real-time and high-resolution acquisition of two-dimensional (2D) sections. However, in order to obtain information from threedimensional (3D) volumes it is currently limited by a stepwise process that consists of acquiring multiple 2D sections from different focal planes by slow z-focus translation. Here, we present a novel method that enables the capture of an entire 3D sample in a single step. Our approach is based on an acoustically-driven varifocal lens integrated in a commercial confocal system that enables axial focus scanning at speeds of 140 kHz or above. Such high-speed allows for one or multiple focus sweeps on a pixel by pixel basis. By using a fast acquisition card, we can assign the photons detected at each pixel to their corresponding focal plane allowing simultaneous multiplane imaging. We exemplify this novel 3D confocal microscopy technique by imaging different biological fluorescent samples and comparing them with those obtained using traditional z-scanners. Based on these results, we find that image quality in this novel approach is similar to that obtained with traditional confocal methods, while speed is only limited by signal-to-noise-ratio. As the sensitivity of photodetectors increases and more efficient fluorescent labeling is developed, this novel 3D method can result in significant reduction in acquisition time allowing the study of new fundamental processes in science.

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

    PubMed

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

    2016-01-01

    Optical interrogation of suspicious skin lesions is standard care in the management of skin cancer worldwide. Morphological and functional markers of malignancy are often combined to improve expert human diagnostic power. We propose the evaluation of the combination of two independent optical biomarkers of skin tumours concurrently. The morphological modality of reflectance confocal microscopy (RCM) is combined with the functional modality of laser Doppler flowmetry, which is capable of quantifying tissue perfusion. To realize the idea, we propose laser feedback interferometry as an implementation of RCM, which is able to detect the Doppler signal in addition to the confocal reflectance signal. Based on the proposed technique, we study numerical models of skin tissue incorporating two optical biomarkers of malignancy: (i) abnormal red blood cell velocities and concentrations and (ii) anomalous optical properties manifested through tissue confocal reflectance, using Monte Carlo simulation. We also conduct a laboratory experiment on a microfluidic channel containing a dynamic turbid medium, to validate the efficacy of the technique. We quantify the performance of the technique by examining a signal to background ratio (SBR) in both the numerical and experimental models, and it is shown that both simulated and experimental SBRs improve consistently using this technique. This work indicates the feasibility of an optical instrument, which may have a role in enhanced imaging of skin malignancies. PMID:27598157

  11. 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

  12. 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.

  13. Extraction of Individual Filaments from 2D Confocal Microscopy Images of Flat Cells.

    PubMed

    Basu, Saurav; Chi Liu; Rohde, Gustavo Kunde

    2015-01-01

    A crucial step in understanding the architecture of cells and tissues from microscopy images, and consequently explain important biological events such as wound healing and cancer metastases, is the complete extraction and enumeration of individual filaments from the cellular cytoskeletal network. Current efforts at quantitative estimation of filament length distribution, architecture and orientation from microscopy images are predominantly limited to visual estimation and indirect experimental inference. Here we demonstrate the application of a new algorithm to reliably estimate centerlines of biological filament bundles and extract individual filaments from the centerlines by systematically disambiguating filament intersections. We utilize a filament enhancement step followed by reverse diffusion based filament localization and an integer programming based set combination to systematically extract accurate filaments automatically from microscopy images. Experiments on simulated and real confocal microscope images of flat cells (2D images) show efficacy of the new method.

  14. Physicochemical composition of osteoporotic bone in the trichothiodystrophy premature aging mouse determined by confocal Raman microscopy.

    PubMed

    van Apeldoorn, Aart A; de Boer, Jan; van Steeg, Harry; Hoeijmakers, Jan H J; Otto, Cees; van Blitterswijk, Clemens A

    2007-01-01

    Although it has been established that premature aging trichothiodystrophy (TTD) mice display typical signs of osteoporosis, exact changes in physicochemical properties of these mice have not been elucidated. We used confocal Raman microscopy and histology to study femora of TTD mice. We measured femora isolated from xeroderma pigmentosum group A (XPA)/TTD double mutant mice to establish that Raman microscopy can be applied to measure differences in bone composition. Raman data from XPA/TTD mice showed remarkable changes in bone mineral composition. Moreover, we observed a severe form of osteoporosis, with strongly reduced cortical bone thickness. We used Raman microscopy to analyze bone composition in eight wild-type and eight TTD animals, and observed decreased levels of phosphate and carbonate in the cortex of femora isolated from TTD mice. In contrast, the bands representing the bone protein matrix were not affected in these mice.

  15. Plastic casts and confocal laser scanning microscopy applied to the observation of enamel tubules in the red Kangaroo (Macropus rufus).

    PubMed

    Suzuki, Masatsugu; Ushijima, Natsumi; Kohno, Ayako; Sawa, Yoshihiko; Yoshida, Shigemitsu; Sekikawa, Mitsuo; Ohtaishi, Noriyuki

    2003-03-01

    Scanning electron microscopy for plastic casts and confocal laser scanning microscopy for Villanueva bone-stained ground sections were used together to observe enamel tubules in red kangaroo molars. Although the tubular structures such as terminals, bends, expansions, splits, divergences and rejoinings in this species were within the variations of marsupial species, their morphological characteristics were demonstrated with extremely clear and persuasive images. Thus, the combined observations of plastic casts by scanning electron microscopy and Villanueva bone-stain sections by confocal laser scanning microscopy were found to be of value for the investigation of enamel tubules and tubular structures in other hard tissues.

  16. Confocal and electron microscopy to characterize sialoglycoconjugates in mouse sublingual gland acinar cells.

    PubMed

    Menghi, G; Bondi, A M; Marchetti, L; Ballarini, P; Materazzi, G

    1998-08-01

    Double lectin labeling for confocal microscopy and lectin-protein A-gold binding for electron microscopy were applied to the mouse sublingual gland in order to study surface and cytoplasmic sialoglycoconjugates. For this purpose, serially cut sections were submitted to sialidase followed by incubation with lectins recognizing usually acceptor sugars for terminal sialic acids. At the electron microscope level, the residues subtended to sialic acid were individually identified on adjacent sections by an indirect technique of labeling, whereas with confocal microscopy the above sugars were simultaneously visualized on the same section by a double staining method using fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectins. Acinar cells were found to contain the terminal sequence sialic acid-beta-galactose in abundance while the sequence sialic acid-alpha-N-acetylgalactosamine appeared to be present in modest amounts. Both sialoglycoconjugates were homogeneously codistributed inside acinar cells. The combination with a saponification method also allowed the occurrence of C4 acetylated sialic acids linked to beta-galactose to be discovered, at the electron microscope level, on acinar cell secretory products.

  17. 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.

  18. 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

  19. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  1. Waterproofing in Arabidopsis: Following Phenolics and Lipids In situ by Confocal Raman Microscopy.

    PubMed

    Prats Mateu, Batirtze; Hauser, Marie Theres; Heredia, Antonio; Gierlinger, Notburga

    2016-01-01

    Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes, and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm) at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299, 1600, and 3400 cm(-1) band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobizing changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum) as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol) and phenolic acids were acquired. PMID:26973831

  2. Waterproofing in Arabidopsis: Following phenolics and lipids in situ by Confocal Raman Microscopy

    NASA Astrophysics Data System (ADS)

    Prats Mateu, Batirtze; Hauser, Marie-Theres; Heredia, Antonio; Gierlinger, Notburga

    2016-02-01

    Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm) at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299 cm-1, 1600 cm-1 and 3400 cm-1 band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobising changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum) as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol) and phenolic acids were acquired. Keywords: Arabidopsis thaliana, lignin, cutin, wax, Raman, cuticle, waterproofing

  3. Waterproofing in Arabidopsis: Following Phenolics and Lipids In situ by Confocal Raman Microscopy

    PubMed Central

    Prats Mateu, Batirtze; Hauser, Marie Theres; Heredia, Antonio; Gierlinger, Notburga

    2016-01-01

    Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes, and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm) at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299, 1600, and 3400 cm−1 band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobizing changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum) as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol) and phenolic acids were acquired. PMID:26973831

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

    NASA Astrophysics Data System (ADS)

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

    2000-12-01

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

  5. Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

    PubMed

    Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

    2014-05-01

    The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  9. Amiodarone-Induced Multiorgan Toxicity with Ocular Findings on Confocal Microscopy

    PubMed Central

    Turk, Ugur; Turk, Bengu Gerceker; Yılmaz, Suzan Guven; Tuncer, Esref; Alioğlu, Emin; Dereli, Tugrul

    2015-01-01

    Amiodarone is an antiarrhythmic medication that can adversely effect various organs including lungs, thyroid gland, liver, eyes, skin, and nerves. The risk of adverse effects increases with high doses and prolonged use. We report a 54-year-old female who presented with multiorgan toxicity after 8 months of low dose (200 mg/day) amiodarone treatment. The findings of confocal microscopy due to amiodarone-induced keratopathy are described. Amiodarone may cause multiorgan toxicity even at lower doses and for shorter treatment periods. PMID:25949090

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

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

    PubMed Central

    Tilli, Maddalena T; Parrish, Angela R; Cotarla, Ion; Jones, Laundette P; Johnson, Michael D; Furth, Priscilla A

    2008-01-01

    Background Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. Methods We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. Results In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. Conclusion In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary. PMID:18215290

  13. Programmable Illumination and High-Speed, Multi-Wavelength, Confocal Microscopy Using a Digital Micromirror

    PubMed Central

    Martial, Franck P.; Hartell, Nicholas A.

    2012-01-01

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

  14. Filtering, reconstruction, and measurement of the geometry of nuclei from hippocampal neurons based on confocal microscopy data.

    PubMed

    Queisser, Gillian; Wittmann, Malte; Bading, Hilmar; Wittum, Gabriel

    2008-01-01

    The cell nucleus is often considered a spherical structure. However, the visualization of proteins associated with the nuclear envelope in rat hippocampal neurons indicates that the geometry of nuclei is far more complex. The shape of cell nuclei is likely to influence the nucleo-cytoplasmic exchange of macromolecules and ions, in particular calcium, a key regulator of neuronal gene expression. We developed a tool to retrieve the 3-D view of cell nuclei from laser scanning confocal microscopy data. By applying an inertia-based filter, based on a special structure detection mechanism, the signal-to-noise ratio of the image is enhanced, the signal is smoothed, gaps in the membrane are closed, while at the same time the geometric properties, such as diameters of the membrane, are preserved. After segmentation of the image data, the microscopy data are sufficiently processed to extract surface information of the membrane by creating an isosurface with a marching tetrahedra algorithm combined with a modified Dijkstra graph-search algorithm. All methods are tested on artificial data, as well as on real data, which are recorded with a laser scanning confocal microscope. Significant advantages of the inertia-based filter can be observed when comparing it to other state of the art nonlinear diffusion filters. An additional program is written to calculate surface and volume of cell nuclei. These results represent the first step toward establishing a geometry-based model of the-dynamics of cytoplasmic and nuclear calcium. PMID:18315367

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

    NASA Astrophysics Data System (ADS)

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

    2013-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  17. Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

    2012-12-01

    Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.

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

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

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

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

    PubMed

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-09-15

    In vivo corneal confocal microscopy (IVCCM) is a novel, reproducible, easy and noninvasive technique that allows the study of the different layers of the cornea at a cellular level. As cornea is the most innervated organ of human body, several studies investigated the use of corneal confocal microscopy to detect diabetic neuropathies, which are invalidating and deadly complications of diabetes mellitus. Corneal nerve innervation has been shown impaired in subjects with diabetes and a close association between damages of peripheral nerves due to the diabetes and alterations in corneal sub-basal nerve plexus detected by IVCCM has been widely demonstrated. Interestingly, these alterations seem to precede the clinical onset of diabetic neuropathies, paving the path for prevention studies. However, some concerns still prevent the full implementation of this technique in clinical practice. In this review we summarize the most recent and relevant evidences about the use of IVCCM for the diagnosis of peripheral sensorimotor polyneuropathy and of autonomic neuropathy in diabetes. New perspectives and current limitations are also discussed. PMID:27660697

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

    PubMed Central

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-01-01

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

  3. Adaptive and Background-Aware GAL4 Expression Enhancement of Co-registered Confocal Microscopy Images.

    PubMed

    Trapp, Martin; Schulze, Florian; Novikov, Alexey A; Tirian, Laszlo; J Dickson, Barry; Bühler, Katja

    2016-04-01

    GAL4 gene expression imaging using confocal microscopy is a common and powerful technique used to study the nervous system of a model organism such as Drosophila melanogaster. Recent research projects focused on high throughput screenings of thousands of different driver lines, resulting in large image databases. The amount of data generated makes manual assessment tedious or even impossible. The first and most important step in any automatic image processing and data extraction pipeline is to enhance areas with relevant signal. However, data acquired via high throughput imaging tends to be less then ideal for this task, often showing high amounts of background signal. Furthermore, neuronal structures and in particular thin and elongated projections with a weak staining signal are easily lost. In this paper we present a method for enhancing the relevant signal by utilizing a Hessian-based filter to augment thin and weak tube-like structures in the image. To get optimal results, we present a novel adaptive background-aware enhancement filter parametrized with the local background intensity, which is estimated based on a common background model. We also integrate recent research on adaptive image enhancement into our approach, allowing us to propose an effective solution for known problems present in confocal microscopy images. We provide an evaluation based on annotated image data and compare our results against current state-of-the-art algorithms. The results show that our algorithm clearly outperforms the existing solutions. PMID:26743993

  4. Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source.

    PubMed

    Träutlein, D; Adler, F; Moutzouris, K; Jeromin, A; Leitenstorfer, A; Ferrando-May, E

    2008-03-01

    The performance of a confocal microscopy setup based on a single femtosecond fiber system is explored over a broad range of pump wavelengths for both linear and nonlinear imaging techniques. First, the benefits of a laser source in linear fluorescence excitation that is continuously tunable over most of the visible spectrum are demonstrated. The influences of subpicosecond pulse durations on the bleaching behavior of typical fluorophores are discussed. We then utilize the tunable near-infrared output of the femtosecond system in connection with a specially designed prism compressor for dispersion control. Pulses as short as 33 fs are measured in the confocal region. As a consequence, 2 mW of average power are sufficient for two-photon microscopy in an organotypic sample from the mouse brain. This result shows great prospect for deep-tissue imaging in the optimum transparency window around 1100 nm. In a third experiment, we prove that our compact setup is powerful enough to exploit even higher-order nonlinearities such as three-photon absorption that we use to induce spatially localized photodamage in DNA.

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

    PubMed Central

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-01-01

    In vivo corneal confocal microscopy (IVCCM) is a novel, reproducible, easy and noninvasive technique that allows the study of the different layers of the cornea at a cellular level. As cornea is the most innervated organ of human body, several studies investigated the use of corneal confocal microscopy to detect diabetic neuropathies, which are invalidating and deadly complications of diabetes mellitus. Corneal nerve innervation has been shown impaired in subjects with diabetes and a close association between damages of peripheral nerves due to the diabetes and alterations in corneal sub-basal nerve plexus detected by IVCCM has been widely demonstrated. Interestingly, these alterations seem to precede the clinical onset of diabetic neuropathies, paving the path for prevention studies. However, some concerns still prevent the full implementation of this technique in clinical practice. In this review we summarize the most recent and relevant evidences about the use of IVCCM for the diagnosis of peripheral sensorimotor polyneuropathy and of autonomic neuropathy in diabetes. New perspectives and current limitations are also discussed. PMID:27660697

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

    PubMed

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-09-15

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

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

    PubMed Central

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

    2016-01-01

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

  8. Investigation of the jamming transition using confocal microscopy of dense colloidal suspensions and complex biomaterials

    NASA Astrophysics Data System (ADS)

    Cianci, Gianguido C.

    Jamming is the process by which a liquid stops flowing and assumes some properties commonly associated with solids. Everyday examples include the setting of a bowl of Jell-O(TM) or the vitrification of a molten mixture of silica, lime and soda at the end of a glassblower's blowpipe. In these examples, the system resists flow and can even support some pressure just like a crystalline solid. At the same time its structure is not crystalline and is in fact hardly distinguishable from that of a liquid. Here we focus our attention on two separate but related questions presented by the jamming (or glass) transition. The dynamical properties of a glass (e.g. viscosity) depend on the time elapsed since vitrification. This slow evolution is called aging and is currently poorly understood. For example a static, or structural, measure of the age of a glass has not yet been identified and aging can only be detected by following the system's dynamics. We consider the aging of the microscopic structure in out-of-equilibrium glasses by measuring local tetrahedral geometry in two systems: a monodisperse colloidal suspension observed by confocal microscopy and a binary Lennard-Jones glass simulated by molecular dynamics. While these models differ in some details they are both widely used in the study of glasses. Tetrahedral structure is found to be a poor indicator of age in the experiments though it weakly correlates with the slowing dynamics. In contrast, the structure of the simulated glass shows clear signs of aging. In both cases, tetrahedral structure samples geometry in a non-trivial way. Similarly, a static measure of a liquid's proximity to the glass transition has remained elusive. We present preliminary results from a magnetic tweezers experiment aimed at dynamically exciting a dormant length scale in a binary "supercooled" colloidal liquid. We observe the sample's local response to a point disturbance from a magnetic probe. We find that the disturbance decays

  9. Confocal microscopy of a newly identified protein associated with heart development in the Mexican axolotl.

    PubMed

    Erginel-Unaltuna, N; Dube, D K; Salsbury, K G; Lemanski, L F

    1995-01-01

    confocal microscopy. In addition, mutant embryos at stage 37-38 were studied for the presence and distribution of the N1-protein on cross-sections through their heart regions. The N1-protein staining was significantly reduced in mutant hearts when compared to normal. PMID:8581063

  10. Confocal mosaicing microscopy of basal-cell carcinomas ex vivo: progress in digital staining to simulate histology-like appearance

    NASA Astrophysics Data System (ADS)

    Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; DiMarzio, Charles; Rajadhyaksha, Milind

    2011-03-01

    Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Using acridine orange (1 milliMolar, 20 seconds) to stain nuclei, basal cell carcinomas were detected in fluorescence confocal mosaics of Mohs surgical excisions with sensitivity of 96.6% and specificity of 89.2%. A possible barrier toward clinical acceptance is that confocal mosaics are based on a single mode of contrast and appear in grayscale, whereas histology is based on two (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple-and-pink. Toward addressing this barrier, we report progress in developing a multispectral analytical model for digital staining: fluorescence confocal mosaics, which show only nuclei, are digitally stained purple and overlaid on reflectance confocal mosaics, which show only cellular cytoplasm and dermis, and digitally stained pink, to mimic the appearance of histology. Comparison of digitally stained confocal mosaics by our Mohs surgeon to the corresponding Mohs histology shows good correlation for normal and tumor detail. Digitally stained confocal mosaicing microscopy may allow direct examination of freshly excised tissue and serve as an adjunct for rapid pathology at-the-bedside.

  11. Prototype study on a miniaturized dual-modality imaging system for photoacoustic microscopy and confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Sung-Liang; Xie, Zhixing; Guo, L. Jay; Wang, Xueding

    2014-03-01

    It is beneficial to study tumor angiogenesis and microenvironments by imaging the microvasculature and cells at the same time. Photoacoustic microscopy (PAM) is capable of sensitive three-dimensional mapping of microvasculature, while fluorescence microscopy may be applied to assessment of tissue pathology. In this work, a fiber-optic based PAM and confocal fluorescence microscopy (CFM) dual-modality imaging system was designed and built, serving as a prototype of a miniaturized dual-modality imaging probe for endoscopic applications. As for the design, we employed miniature components, including a microelectromechanical systems (MEMS) scanner, a miniature objective lens, and a small size optical microring resonator as an acoustic detector. The system resolutions were calibrated as 8.8 μm in the lateral directions for both PAM and CFM, and 19 μm and 53 μm in the axial direction for PAM and CFM, respectively. Images of the animal bladders ex vivo were demonstrated to show the ability of the system in imaging not only microvasculature but also cellular structure.

  12. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo.

    PubMed

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-02-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  13. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo

    PubMed Central

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J.; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-01-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

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

    PubMed

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

    2013-03-01

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

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

    PubMed

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

    2015-03-01

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

  16. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  17. Fault localization and analysis in semiconductor devices with optical-feedback infrared confocal microscopy

    SciTech Connect

    Sarmiento, Raymund; Cemine, Vernon Julius; Tagaca, Imee Rose; Salvador, Arnel; Mar Blanca, Carlo; Saloma, Caesar

    2007-11-01

    We report on a cost-effective optical setup for characterizing light-emitting semiconductor devices with optical-feedback confocal infrared microscopy and optical beam-induced resistance change.We utilize the focused beam from an infrared laser diode to induce local thermal resistance changes across the surface of a biased integrated circuit (IC) sample. Variations in the multiple current paths are mapped by scanning the IC across the focused beam. The high-contrast current maps allow accurate differentiation of the functional and defective sites, or the isolation of the surface-emittingp-i-n devices in the IC. Optical beam-induced current (OBIC) is not generated since the incident beam energy is lower than the bandgap energy of the p-i-n device. Inhomogeneous current distributions in the IC become apparent without the strong OBIC background. They are located at a diffraction-limited resolution by referencing the current maps against the confocal reflectance image that is simultaneously acquired via optical-feedback detection. Our technique permits the accurate identification of metal and semiconductor sites as well as the classification of different metallic structures according to thickness, composition, or spatial inhomogeneity.

  18. Lithographically-fabricated channel arrays for confocal x-ray fluorescence microscopy and XAFS

    NASA Astrophysics Data System (ADS)

    Woll, Arthur R.; Agyeman-Budu, David; Choudhury, Sanjukta; Coulthard, Ian; Finnefrock, Adam C.; Gordon, Robert; Hallin, Emil; Mass, Jennifer

    2014-03-01

    Confocal X-ray Fluorescence Microscopy (CXRF) employs overlapping focal regions of two x-ray optics—a condenser and collector—to directly probe a 3D volume. The minimum-achievable size of this probe volume is limited by the collector, for which polycapillaries are generally the optic of choice. Recently, we demonstrated an alternative collection optic for CXRF, consisting of an array of micron-scale collimating channels, etched in silicon, and arranged like spokes of a wheel directed towards a single source position. The optic, while successful, had a working distance of only 0.2 mm and exhibited relatively low total collection efficiency, limiting its practical application. Here, we describe a new design in which the collimating channels are formed by a staggered array of pillars whose side-walls taper away from the channel axis. This approach improves both collection efficiency and working distance, while maintaining excellent spatial resolution. We illustrate these improvements with confocal XRF data obtained at the Cornell High Energy Synchrotron Source (CHESS) and the Advanced Photon Source (APS) beamline 20-ID-B.

  19. 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.

  20. Structure and chemical composition of the dentin-enamel junction analyzed by Confocal Raman Microscopy

    NASA Astrophysics Data System (ADS)

    Desoutter, A.; Salehi, H.; Slimani, A.; Marquet, P.; Jacquot, B.; Tassery, H.; Cuisinier, F. J. G.

    2014-02-01

    The structure and chemical composition of the human dentin-enamel junction (DEJ) was studied using confocal Raman microscopy - a chemical imaging technique. Slices of non-fixed, sound teeth were prepared with an Isomet diamond saw and scanned with Witec Alpha300R system. The combination of different characteristics peaks of phosphate, carbonate and organic matrix (respectively 960, 1072 and 1545 cm-1), generates images representing the chemical composition of the DEJ area. Images are also calculated using peak ratios enabling precise determination of the chemical composition across the DEJ. Then, with two characterized peaks, different pictures are calculated to show the ratio of two components. The images of the spatial distribution of mineral phosphate (960cm-1) to organic matrix (1545 cm-1) ratios, mineral carbonates (1072cm-1) to mineral phosphate ratios; and mineral carbonates to organic matrix ratios were reconstructed. Cross sectional and calculated graphic profile show the variations of the different chemical component ratios through the enamel and the dentin. Phosphate to organic ratio shows an accumulation of organic material under the enamel surface. The cross sectional profile of these pictures shows a high phosphate content compared to enamel in the vicinity of the DEJ. The Confocal Raman imaging technique can be used to further provide full chemical imaging of tooth, particularly of the whole DEJ and to study enamel and dentin decay.

  1. 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.

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

    PubMed

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

    2014-06-01

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

  3. The use of reflectance confocal microscopy for monitoring response to therapy of skin malignancies

    PubMed Central

    Ulrich, Martina; Lange-Asschenfeldt, Susanne; Gonzalez, Salvador

    2012-01-01

    Summary Reflectance confocal microscopy (RCM) is a new non-invasive imaging technique that enables visualizing cells and structures in living skin in real-time with resolution close to that of histological analysis. RCM has been successfully implemented in the assessment of benign and malignant lesions. Most importantly, it also enables monitoring dynamic changes in the skin over time and in response to different therapies, e.g., imiquimod, photodynamic therapy, and others. Given the often traumatic nature of skin cancer that affects both the physiology and the psychology of the patients, it is crucial to have methods that enable monitoring the response to treatment but that minimize the distress and discomfort associated with such process. This article provides a very brief overview of the fundamentals of RCM and then focuses on its recent employment as a monitoring tool in skin cancer and other pathologies that may require frequent follow-up. PMID:23785598

  4. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1991-05-01

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

  6. Consistency and distribution of reflectance confocal microscopy features for diagnosis of cutaneous T cell lymphoma

    NASA Astrophysics Data System (ADS)

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

    2012-01-01

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

  7. 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.

  8. 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.

  9. Use of Confocal Microscopy in the Search for Optical Counterparts to Gamma-Ray Bursts

    NASA Astrophysics Data System (ADS)

    Sharma, A.; Jacobson, R. E.

    1995-09-01

    A number of star-like images have been found on archival photographic plates, however there is debate as to whether these are optical emissions relating to Gamma-Ray bursts, or simply plate defects. Results from photographic science are summarized so that the characteristic shape of different types of images on photographic plates can be theoretically predicted. Explanation for the formation of the relief image and reasons for its continued study are described. New methods in the use of laser scanning confocal microscopy are presented to study the relief image and the depthwise disposition of silver so that a test procedure can be established to end the speculation surrounding the astrophysical truth of stellar-like images on photographic plates.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Detection of apoptosis caused by anticancer drug paclitaxel in MCF-7 cells by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Salehi, H.; Middendorp, E.; Végh, A.-G.; Ramakrishnan, S.-K.; Gergely, C.; Cuisinier, F. J. G.

    2013-02-01

    Confocal Raman Microscopy, a non-invasive, label free imaging technique is used to study apoptosis in living MCF-7 cells. The images are based on Raman spectra of cells components. K-mean clustering was used to determine mitochondria position in cells and cytochrome c distribution inside the cells was based on correlation analysis. Cell apoptosis is defined as cytochrome c diffusion in cytoplasm. Co-localization of cytochrome c is found within mitochondria after three hours of incubation with 10 μM paclitaxel. Our results demonstrate that the presence of paclitaxel at this concentration in the culture media for 3 hours does not induce apoptosis of MCF7 cells via a caspase independent pathway.

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

    PubMed Central

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

    2016-01-01

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

  14. Three-dimensional visualization of termite (Apicotermitinae) enteric valve using confocal laser scanning microscopy.

    PubMed

    Host, B; Twyffels, L; Roisin, Y; Vanderwinden, J-M

    2014-08-01

    Humivorous termites are dominant members of tropical rainforest soil communities. In the soil-feeding subfamily Apicotermitinae (Termitidae), the enteric valve connecting the first section of the hindgut to the paunch often displays a complex sclerotized armature everted towards the lumen of the paunch. This structure is central in termite taxonomy but its function remains hypothetical. Here, we evaluate the potential of confocal laser scanning microscopy to provide detailed imaging of the valve of Anoplotermes parvus, by comparison with bright-field microscopy and scanning electron microscopy. We detected a strong far-red emission of the enteric valve armature that sharply contrasted with the surrounding tissues, providing a convenient method to highlight minute structural elements of the valve and its three-dimensional structure. The method is easy to use and is applicable to standard archival material as demonstrated by images of enteric valves of four other Apicotermitinae species. It may represent a valuable asset for the study of termite enteric valves, for the purpose of taxonomy or functional morphology. PMID:24947115

  15. 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.

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

    PubMed

    Steinbach, Gábor; Kaňa, Radek

    2016-04-01

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

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

    PubMed

    Steinbach, Gábor; Kaňa, Radek

    2016-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  19. 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

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

    PubMed Central

    Schlachter, Simon C.; Kang, DongKyun; Gora, Michalina J.; Vacas-Jacques, Paulino; Wu, Tao; Carruth, Robert W.; Wilsterman, Eric J.; Bouma, Brett E.; Woods, Kevin; Tearney, Guillermo J.

    2013-01-01

    Spectrally encoded confocal microscopy (SECM) is a reflectance confocal microscopy technology that uses a diffraction grating to illuminate different locations on the sample with distinct wavelengths. SECM can obtain line images without any beam scanning devices, which opens up the possibility of high-speed imaging with relatively simple probe optics. This feature makes SECM a promising technology for rapid endoscopic imaging of internal organs, such as the esophagus, at microscopic resolution. SECM imaging of the esophagus has been previously demonstrated at relatively low line rates (5 kHz). In this paper, we demonstrate SECM imaging of large regions of esophageal tissues at a high line imaging rate of 100 kHz. The SECM system comprises a wavelength-swept source with a fast sweep rate (100 kHz), high output power (80 mW), and a detector unit with a large bandwidth (100 MHz). The sensitivity of the 100-kHz SECM system was measured to be 60 dB and the transverse resolution was 1.6 µm. Excised swine and human esophageal tissues were imaged with the 100-kHz SECM system at a rate of 6.6 mm2/sec. Architectural and cellular features of esophageal tissues could be clearly visualized in the SECM images, including papillae, glands, and nuclei. These results demonstrate that large-area SECM imaging of esophageal tissues can be successfully conducted at a high line imaging rate of 100 kHz, which will enable whole-organ SECM imaging in vivo. PMID:24049684

  1. In Vivo Confocal Microscopy of the Ocular Surface: From Bench to Bedside

    PubMed Central

    Villani, Edoardo; Baudouin, Christophe; Efron, Nathan; Hamrah, Pedram; Kojima, Takashi; Patel, Sanjay V.; Pflugfelder, Stephen C.; Zhivov, Andrey; Dogru, Murat

    2014-01-01

    In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move “from bench to bedside”. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis. PMID

  2. In Vivo Confocal Microscopy Demonstrates Bilateral Loss of Endothelial Cells in Unilateral Herpes Simplex Keratitis

    PubMed Central

    Müller, Rodrigo T.; Pourmirzaie, Roxanna; Pavan-Langston, Deborah; Cavalcanti, Bernardo M.; Aggarwal, Shruti; Colón, Clara; Jamali, Arsia; Cruzat, Andrea; Hamrah, Pedram

    2015-01-01

    Purpose To report bilateral corneal endothelial cell density (ECD), as well as its correlation with subbasal nerve changes, in patients with unilateral herpes simplex keratitis (HSK). Methods Thirty-six eyes of 36 patients with corneal scarring caused by HSK, as well as their respective contralateral clinically unaffected eyes, were prospectively studied and compared with 26 eyes of 26 healthy volunteers. In vivo confocal microscopy and corneal sensation of the central cornea were performed bilaterally in all patients and in one random eye of controls. The ECD and subbasal corneal nerve density, including the lengths of total nerves, main trunks, and branches were evaluated and correlated to central corneal sensation. Results The ECD was significantly lower in eyes affected with HSK than in controls (2304 ± 578 vs. 2940 ± 370 cells/mm2, P < 0.0001). Surprisingly, lower ECD was also detected in contralateral clinically unaffected eyes (2548 ± 423), compared to controls (P = 0.02). Both affected and contralateral eyes showed decrease in total nerve length, compared to controls (10.0 ± 6.3 vs. 17.6 ± 6.3 vs. 21.9 ± 4.3 mm/mm2, respectively; P < 0.05 for all). The ECD correlated positively with total nerve length (r = 0.39, P = 0.0009) and with corneal sensation (r = 0.31, P = 0.009). Conclusions In vivo confocal microscopy findings demonstrated alterations in corneal ECD in both affected and clinically unaffected contralateral eyes of patients with unilateral HSK. Moreover, the positive significant correlation between the ECD and the subbasal nerve density may suggest a potential link between corneal innervation and corneal endothelial cell homeostasis. PMID:26225629

  3. Effects of acids used in the microabrasion technique: Microhardness and confocal microscopy analysis

    PubMed Central

    Pini, Núbia-Inocencya-Pavesi; Ambrosano, Gláucia-Maria-Bovi; da Silva, Wander-José; Aguiar, Flávio-Henrique-Baggio; Lovadino, José-Roberto

    2015-01-01

    Background This study evaluated the effects of the acids used in the microabrasion on enamel. Material and Methods Seventy enamel/dentine blocks (25 mm2) of bovine incisors were divided into 7 groups (n=10). Experimental groups were treated by active/passive application of 35% H3PO4 (E1/E2) or 6.6% HCl (E3/E4). Control groups were treated by microabrasion with H3PO4+pumice (C5), HCl+silica (C6), or no treatment (C7). The superficial (SMH) and cross-sectional (CSMH; depths of 10, 25, 50, and 75 µm) microhardness of enamel were analyzed. Morphology was evaluated by confocal laser-scanning microscopy (CLSM). Data were analyzed by analysis of variance (Proc Mixed), Tukey, and Dunnet tests (α=5%). Results Active application (E1 and E3) resulted in higher microhardness than passive application (E2 and E4), with no difference between acids. For most groups, the CSMH decreased as the depth increased. All experimental groups and negative controls (C5 and C6) showed significantly reduced CSMH values compared to the control. A significantly higher mean CSMH result was obtained with the active application of H3PO4 (E1) compared to HCl (E3). Passive application did not result in CSMH differences between acids. CLSM revealed the conditioning pattern for each group. Conclusions Although the acids displayed an erosive action, use of microabrasive mixture led to less damage to the enamel layers. Key words:Enamel microabrasion, enamel microhardness, confocal laser scanning microscopy. PMID:26535098

  4. 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

  5. The application of laser scanning confocal microscopy to the examination of hairs and textile fibers: an initial investigation.

    PubMed

    Kirkbride, K Paul; Tridico, Silvana R

    2010-02-25

    An initial investigation of the application of laser scanning confocal microscopy to the examination of hairs and fibers has been conducted. This technique allows the production of virtual transverse and longitudinal cross-sectional images of a wide range of hairs and fibers. Special mounting techniques are not required; specimens that have been mounted for conventional microscopy require no further treatment. Unlike physical cross-sectioning, in which it is difficult to produce multiple cross-sections from a single hair or fiber and the process is destructive, confocal microscopy allows the examiner to image the cross-section at any point in the field of view along the hair or fiber and it is non-destructive. Confocal microscopy is a fluorescence-based technique. The images described in this article were collected using only the autofluorescence exhibited by the specimen (i.e. fluorescence staining was not necessary). Colorless fibers generally and hairs required excitation at 405 nm in order to stimulate useful autofluorescence; longer wavelength excitation was suitable for dyed fibers. Although confocal microscopy was found to be generally applicable to the generation virtual transverse cross-sections from a wide range of hairs and fibers, on some occasions the autofluorescence signal was attenuated by heavy pigmentation or the presence of an opaque medulla in hairs, and by heavy delustering or the presence of air-filled voids in the case of fibers. In these situations only partial cross-sections were obtained.

  6. ERG AND CHD1 HETEROGENEITY IN PROSTATE CANCER: USE OF CONFOCAL MICROSCOPY IN ASSESSMENT OF MICROSCOPIC FOCI

    PubMed Central

    Tereshchenko; Zhong, H; Santanam, U; Petrosky, W; Stein, MN; Dvorzhinski, D; Ganesan, S; Singer, E; Moore, D; Tischfield, J; DiPaola, RS

    2014-01-01

    Background Biomarkers predicting tumor response are important to emerging targeted therapeutics. Complimentary methods to assess and understand genetic changes and heterogeneity within only few cancer cells in tissue will be a valuable addition for assessment of tumors such as prostate cancer that often have insufficient tumor for next generation sequencing in a single biopsy core. Methods Using confocal microscopy to identify cell-to-cell relationships in situ, we studied the most common gene rearrangement in prostate cancer (TMPRSS2 and ERG) and the tumor suppressor CHD1 in 56 patients who underwent radical prostatectomy. Contingency tables and the chi-square test were used to determine associations between mutation status of the genes and tumor phenotype. Results Wild type ERG was found in 22 of 56 patients; ERG copy number was increased in 10/56, and ERG rearrangements confirmed in 24/56 patients. In 24 patients with ERG rearrangements, the mechanisms of rearrangement were heterogeneous, with deletion in 14/24, a split event in 7/24, and both deletions and split events in the same tumor focus in 3/24 patients. Overall, 14/45 (31.1%) of patients had CHD1 deletion, with the majority of patients with CHD1 deletions (13/14) correlating with ERG-rearrangement negative status (p< 0.01). Conclusions These results demonstrate the ability of confocal microscopy and FISH to identify the cell-to-cell differences in common gene fusions such as TMPRSS2–ERG that may arise independently within the same tumor focus. These data support the need to study complimentary approaches to assess genetic changes that may stratify therapy based on predicted sensitivities. PMID:25175909

  7. Three-dimensional reconstructions from optical sections of thick mouse inner ears using confocal microscopy.

    PubMed

    Kopecky, B J; Duncan, J S; Elliott, K L; Fritzsch, B

    2012-12-01

    Three-dimensional (3D) reconstructions of the vertebrate inner ear have provided novel insights into the development of this complex organ. 3D reconstructions enable superior analysis of phenotypic differences between wild type and mutant ears but can result in laborious work when reconstructed from physically sectioned material. Although nondestructive optical sectioning light sheet microscopy may ultimately prove the ideal solution, these technologies are not yet commercially available, or in many instances are not monetarily feasible. Here we introduce a simple technique to image a fluorescently labelled ear at different stages throughout development at high resolution enabling 3D reconstruction of any component of the inner ear using confocal microscopy. We provide a step-by-step manual from tissue preparation to imaging to 3D reconstruction and analysis including a rationale and troubleshooting guide at each step for researchers with different equipment, protocols, and access to resources to successfully incorporate the principles of this method and customize them to their laboratory settings.

  8. 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.

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

    PubMed

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

    2016-04-01

    The accuracy of electron backscatter diffraction (EBSD) and confocal Raman microscopy (CRM) for small-scale strain mapping are assessed using the multi-axial strain field surrounding a wedge indentation in Si as a test vehicle. The strain field is modeled using finite element analysis (FEA) that is adapted to the near-indentation surface profile measured by atomic force microscopy (AFM). The assessment consists of (1) direct experimental comparisons of strain and deformation and (2) comparisons in which the modeled strain field is used as an intermediate step. Direct experimental methods (1) consist of comparisons of surface elevation and gradient measured by AFM and EBSD and of Raman shifts measured and predicted by CRM and EBSD, respectively. Comparisons that utilize the combined FEA-AFM model (2) consist of predictions of distortion, strain, and rotation for comparison with EBSD measurements and predictions of Raman shift for comparison with CRM measurements. For both EBSD and CRM, convolution of measurements in depth-varying strain fields is considered. The interconnected comparisons suggest that EBSD was able to provide an accurate assessment of the wedge indentation deformation field to within the precision of the measurements, approximately 2×10(-4) in strain. CRM was similarly precise, but was limited in accuracy to several times this value. PMID:26939030

  10. Electrochemical preparation of silver and gold nanoparticles: Characterization by confocal and surface enhanced Raman microscopy

    NASA Astrophysics Data System (ADS)

    Plieth, W.; Dietz, H.; Anders, A.; Sandmann, G.; Meixner, A.; Weber, M.; Kneppe, H.

    2005-12-01

    Localized silver and gold nanoparticles, electrochemically prepared by means of the double-pulse technique, were investigated with respect to their optical and spectroscopic properties by scanning confocal microscopy combined with surface enhanced Raman spectroscopy (SERS) and subsequent comparison with the local image of scanning electron microscopy (SEM). Analogous to the silver cluster preparation technique, controlled electrodeposition of gold nanoparticles was demonstrated, varying size from 10 to 500 nm and particle density. The maximum SERS enhancement factors found in the measurements were: (i) 10 10 for silver particles and (ii) 10 8 for gold particles. The optical and spectroscopic data of the local nanoparticle structures investigated showed that SERS is a local phenomenon, because (i) only few particles are Raman active particles, (ii) strongest enhancements in SERS are obtained from particle agglomerates, (iii) typically the Raman radiation is emitted from irregular structures like the necks between two or more particles agglomerated. In the investigated range from 10 to 500 nm no significant influence of the particle size was observed.

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

    PubMed

    Cianconi, Luigi; Conte, Gabriele; Mancini, Manuele

    2011-01-01

    This study evaluated the shear bond strength, failure modes, and confocal microscopy of two different amalgam alloy restorations lined with five adhesive systems. Two regular-set high-copper dental amalgam alloys, Amalcap Plus and Valiant Ph.D, and five commercially available adhesive systems were selected. One hundred and twenty freshly-extracted human third molars were used for the study. The results were statistically evaluated using two-factor analysis of variance (ANOVA). The shear bond strength (SBS) of amalgam to dentin was significantly affected by both the adhesive (p<0.0001) and amalgam alloy (p<0.0002). Regarding mode of failure (MF), among samples restored with Valiant Ph.D, 31 of 50 exhibited adhesive failure, and 19 displayed mixed failure. Laser optical microscopy (OM) of the bonded interface revealed the presence of a good hybrid layer was evident in all experimental groups. Higher bond strengths were measured for four of the five adhesives when used in combination with the spherical alloy. PMID:21383518

  12. Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

    PubMed

    Mas, Abraham; Amenós, Montse; Lois, L Maria

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected. PMID:27424751

  13. In toto imaging of embryogenesis with confocal time-lapse microscopy.

    PubMed

    Megason, Sean G

    2009-01-01

    Microscopy has been one of the most direct and powerful tools since the beginning of biological research. Continued advances such as confocal and two-photon fluorescence microscopy and fluorescent proteins now make imaging useful at a variety of spatial scales (molecules, circuits, cells, tissues, and even whole embryos) and temporal scales (

  14. Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

    PubMed Central

    Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin

    2016-01-01

    Objectives We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. Conclusions The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis. PMID:27525165

  15. Three-dimensional chromatin distribution in neuroblastoma nuclei shown by confocal scanning laser microscopy

    NASA Astrophysics Data System (ADS)

    Brakenhoff, G. J.; van der Voort, H. T. M.; van Spronsen, E. A.; Linnemans, W. A. M.; Nanninga, N.

    1985-10-01

    The relationship between cell shape and function has long been of interest1-9. However, although the behaviour of the cytoskeleton during the cell cycle has been studied extensively10-12 variations in the shape and three-dimensional substructure of the nucleus are less well documented. The spatial distribution of chromatin has previously been studied by a mathematical analysis of the optical densities of stained nuclei13-15, allowing an indirect derivation of the three-dimensional distribution of chromatin. More direct information on chromatin organization can be obtained from electron-microscopic serial sections, although this is very laborious. Using an iterative deconvolution algorithm, Agard and Sedat16 achieved a degree of optical sectioning in conventional fluorescence microscopy and reconstructed the three-dimensional arrangement of polytene chromosomes. We report here on the three-dimensional structure of cultured mammalian cells as visualized by confocal scanning laser microscopy (CSLM). The exceptionally short depth of field of this imaging technique provides direct optical sectioning which, together with its higher resolution, makes CSLM extremely useful for studying the three-dimensional morphology of biological structures17-19.

  16. Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy.

    PubMed

    Talamás-Lara, Daniel; Talamás-Rohana, Patricia; Fragoso-Soriano, Rogelio Jaime; Espinosa-Cantellano, Martha; Chávez-Munguía, Bibiana; González-Robles, Arturo; Martínez-Palomo, Adolfo

    2015-10-01

    Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica.

  17. Lipid and protein distribution in epithelial cells assessed with confocal microscopy

    NASA Astrophysics Data System (ADS)

    Peterson, Kajsa H.; Randen, Michael; Hays, Richard M.; Magnusson, Karl-Eric

    1992-06-01

    Confocal laser scanning microscopy, image processing, and volume visualization were used to characterize the 3-D distribution of lectin receptors, lipid probes, and actin cytoskeleton in epithelial cells. Small intestine-like cells were grown on glass or filter supports and apically labelled with different fluorescent lipid and lectin probes. The restriction of the probes by the tight junctions was studied in living cells. Series of confocal x-y sections were transferred to an image processing system for analysis. The fluorescence intensity within a specified area of all x-y sections was plotted as a function of the vertical position of the sections. The curve inclination was used to describe the degree of restriction to the probes. It was found that lectins were more confined to the apical part than the lipids, which showed varying degree of redistribution to the basolateral membrane. Volume rendering, and specifically animated sequences with varying viewpoint and opacity mapping, were used to visualize the structure of actin cytoskeleton and distribution of lipid and lectin probes. In toad bladder epithelial cells, actin was labelled before and after treatment with the antidiuretic hormone vasopressin. The hormone-induced redistribution of actin in the apical and lateral portion of the cells was measured on x-z scanned images. Ratios of apical-to-lateral intensity were calculated. It was found that the decrease in the ratios after vasopressin treatment was around 30%. The decrease was due to loss of actin apically. This is supposed to facilitate apical fusion of vesicles containing the water-channel forming proteins, being important in water homeostasis.

  18. Improving axial resolution in confocal microscopy with new high refractive index mounting media.

    PubMed

    Fouquet, Coralie; Gilles, Jean-François; Heck, Nicolas; Dos Santos, Marc; Schwartzmann, Richard; Cannaya, Vidjeacoumary; Morel, Marie-Pierre; Davidson, Robert Stephen; Trembleau, Alain; Bolte, Susanne

    2015-01-01

    Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.

  19. Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

    PubMed Central

    Fouquet, Coralie; Gilles, Jean-François; Heck, Nicolas; Dos Santos, Marc; Schwartzmann, Richard; Cannaya, Vidjeacoumary; Morel, Marie-Pierre; Davidson, Robert Stephen; Trembleau, Alain; Bolte, Susanne

    2015-01-01

    Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required. PMID:25822785

  20. Automated acquisition and analysis of airway surface liquid height by confocal microscopy

    PubMed Central

    Choi, Hyun-Chul; Kim, Christine Seul Ki

    2015-01-01

    The airway surface liquid (ASL) is a thin-liquid layer that lines the luminal side of airway epithelia. ASL contains many molecules that are involved in primary innate defense in the lung. Measurement of ASL height on primary airway cultures by confocal microscopy is a powerful tool that has enabled researchers to study ASL physiology and pharmacology. Previously, ASL image acquisition and analysis were performed manually. However, this process is time and labor intensive. To increase the throughput, we have developed an automatic ASL measurement technique that combines a fully automated confocal microscope with novel automatic image analysis software that was written with image processing techniques derived from the computer science field. We were able to acquire XZ ASL images at the rate of ∼1 image/s in a reproducible fashion. Our automatic analysis software was able to analyze images at the rate of ∼32 ms/image. As proofs of concept, we generated a time course for ASL absorption and a dose response in the presence of SPLUNC1, a known epithelial sodium channel inhibitor, on human bronchial epithelial cultures. Using this approach, we determined the IC50 for SPLUNC1 to be 6.53 μM. Furthermore, our technique successfully detected a difference in ASL height between normal and cystic fibrosis (CF) human bronchial epithelial cultures and detected changes in ATP-stimulated Cl−/ASL secretion. We conclude that our automatic ASL measurement technique can be applied for repeated ASL height measurements with high accuracy and consistency and increased throughput. PMID:26001773

  1. Development of a viability standard curve for microencapsulated probiotic bacteria using confocal microscopy and image analysis software.

    PubMed

    Moore, Sarah; Kailasapathy, Kasipathy; Phillips, Michael; Jones, Mark R

    2015-07-01

    Microencapsulation is proposed to protect probiotic strains from food processing procedures and to maintain probiotic viability. Little research has described the in situ viability of microencapsulated probiotics. This study successfully developed a real-time viability standard curve for microencapsulated bacteria using confocal microscopy, fluorescent dyes and image analysis software.

  2. Any Way You Slice It-A Comparison of Confocal Microscopy Techniques.

    PubMed

    Jonkman, James; Brown, Claire M

    2015-07-01

    The confocal fluorescence microscope has become a popular tool for life sciences researchers, primarily because of its ability to remove blur from outside of the focal plane of the image. Several different kinds of confocal microscopes have been developed, each with advantages and disadvantages. This article will cover the grid confocal, classic confocal laser-scanning microscope (CLSM), the resonant scanning-CLSM, and the spinning-disk confocal microscope. The way each microscope technique works, the best applications the technique is suited for, the limitations of the technique, and new developments for each technology will be presented. Researchers who have access to a range of different confocal microscopes (e.g., through a local core facility) should find this paper helpful for choosing the best confocal technology for specific imaging applications. Others with funding to purchase an instrument should find the article helpful in deciding which technology is ideal for their area of research.

  3. Any Way You Slice It—A Comparison of Confocal Microscopy Techniques

    PubMed Central

    Jonkman, James

    2015-01-01

    The confocal fluorescence microscope has become a popular tool for life sciences researchers, primarily because of its ability to remove blur from outside of the focal plane of the image. Several different kinds of confocal microscopes have been developed, each with advantages and disadvantages. This article will cover the grid confocal, classic confocal laser-scanning microscope (CLSM), the resonant scanning-CLSM, and the spinning-disk confocal microscope. The way each microscope technique works, the best applications the technique is suited for, the limitations of the technique, and new developments for each technology will be presented. Researchers who have access to a range of different confocal microscopes (e.g., through a local core facility) should find this paper helpful for choosing the best confocal technology for specific imaging applications. Others with funding to purchase an instrument should find the article helpful in deciding which technology is ideal for their area of research. PMID:25802490

  4. 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.

  5. Confocal Raman Microscopy of Hybrid-Supported Phospholipid Bilayers within Individual C18-Functionalized Chromatographic Particles.

    PubMed

    Kitt, Jay P; Harris, Joel M

    2016-09-01

    Measuring lipid-membrane partitioning of small molecules is critical to predicting bioavailability and investigating molecule-membrane interactions. A stable model membrane for such studies has been developed through assembly of a phospholipid monolayer on n-alkane-modified surfaces. These hybrid bilayers have recently been generated within n-alkyl-chain (C18)-modified porous silica and used in chromatographic retention studies of small molecules. Despite their successful application, determining the structure of hybrid bilayers within chromatographic silica is challenging because they reside at buried interfaces within the porous structure. In this work, we employ confocal Raman microscopy to investigate the formation and temperature-dependent structure of hybrid-phospholipid bilayers in C18-modified, porous-silica chromatographic particles. Porous silica provides sufficient surface area within a confocal probe volume centered in an individual particle to readily measure, with Raman microscopy, the formation of an ordered hybrid bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the surface C18 chains. The DMPC surface density was quantified from the relative Raman scattering intensities of C18 and phospholipid acyl chains and found to be ∼40% of a DMPC vesicle membrane. By monitoring Raman spectra acquired versus temperature, the bilayer main phase transition was observed to be broadened and shifted to higher temperature compared to a DMPC vesicle, in agreement with differential scanning calorimetry (DSC) results. Raman scattering of deuterated phospholipid was resolved from protonated C18 chain scattering, showing that the lipid acyl and C18 chains melt simultaneously in a single phase transition. The surface density of lipid in the hybrid bilayer, the ordering of both C18 and lipid acyl chains upon bilayer formation, and decoupling of C18 methylene C-H vibrations by deuterated lipid acyl chains all suggest an interdigitated acyl chain

  6. Simultaneous pH measurement in endocytic and cytosolic compartments in living cells using confocal microscopy.

    PubMed

    Lucien, Fabrice; Harper, Kelly; Pelletier, Pierre-Paul; Volkov, Leonid; Dubois, Claire M

    2014-01-01

    Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported(1). Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases(2). Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells(3). This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis(3,4). Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can

  7. 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

  8. Confocal Raman Microscopy of Hybrid-Supported Phospholipid Bilayers within Individual C18-Functionalized Chromatographic Particles.

    PubMed

    Kitt, Jay P; Harris, Joel M

    2016-09-01

    Measuring lipid-membrane partitioning of small molecules is critical to predicting bioavailability and investigating molecule-membrane interactions. A stable model membrane for such studies has been developed through assembly of a phospholipid monolayer on n-alkane-modified surfaces. These hybrid bilayers have recently been generated within n-alkyl-chain (C18)-modified porous silica and used in chromatographic retention studies of small molecules. Despite their successful application, determining the structure of hybrid bilayers within chromatographic silica is challenging because they reside at buried interfaces within the porous structure. In this work, we employ confocal Raman microscopy to investigate the formation and temperature-dependent structure of hybrid-phospholipid bilayers in C18-modified, porous-silica chromatographic particles. Porous silica provides sufficient surface area within a confocal probe volume centered in an individual particle to readily measure, with Raman microscopy, the formation of an ordered hybrid bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the surface C18 chains. The DMPC surface density was quantified from the relative Raman scattering intensities of C18 and phospholipid acyl chains and found to be ∼40% of a DMPC vesicle membrane. By monitoring Raman spectra acquired versus temperature, the bilayer main phase transition was observed to be broadened and shifted to higher temperature compared to a DMPC vesicle, in agreement with differential scanning calorimetry (DSC) results. Raman scattering of deuterated phospholipid was resolved from protonated C18 chain scattering, showing that the lipid acyl and C18 chains melt simultaneously in a single phase transition. The surface density of lipid in the hybrid bilayer, the ordering of both C18 and lipid acyl chains upon bilayer formation, and decoupling of C18 methylene C-H vibrations by deuterated lipid acyl chains all suggest an interdigitated acyl chain

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

    PubMed Central

    2012-01-01

    Background Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) allows selective replacement of the endothelium. Post-operative haze and particles can affect the interface quality and, ultimately, visual outcome. In this study, we evaluated DSAEK interface with in vivo laser confocal microscopy (LCM) in order to: (i) correlate interface status with best corrected visual acuity, and (ii) with time from surgery; (iii) correlate interface particle number with best corrected visual acuity. Host-donor interface was imaged and graded using a published reflectivity scale. Particles at the interface were counted. Methods 18 eyes of 16 patients (6 males and 10 females); mean age: 74 ± 8.3 years which underwent DSAEK were examined by means of in vivo laser confocal microscopy between 1 and 24 months after surgery. Host-donor interface was imaged and graded using a published reflectivity scale. Particles present at the interface were counted. Results Interface reflectivity was 2.17 ± 1.2 and significantly correlated with visual acuity (Spearman correlation coefficient −0.83; P < 0.001), and with time after surgery (Spearman correlation coefficient −0.87; P < 0.001). Visual acuity was 0.67 ± 0.27. The number of particles was 205 ± 117.8; no correlation was found between this number and visual acuity (Spearman correlation coefficient −0.41; P = 0.15). Conclusion DSAEK interface imaged with LCM is helpful in diagnosing poor host-donor interface quality in DSAEK surgery. A good quality interface is related to a better visual acuity. Moreover, the quality of the interface appears to improve as time passes from the surgery. Interface quality is related with visual acuity and improves with time from surgery. LCM should be considered as an added tool in post-DSAEK follow-up of patients. Finally, our study shows that the presence of particles does not influence visual outcome. PMID:22853313

  10. Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

    PubMed Central

    Lucien, Fabrice; Harper, Kelly; Pelletier, Pierre-Paul; Volkov, Leonid; Dubois, Claire M.

    2014-01-01

    Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used

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

    NASA Astrophysics Data System (ADS)

    Ballard, Stephen G.

    1990-08-01

    For 350 years, the optical microscope has had a powerful symbiotic relationship with biology. Until this century, optical microscopy was the only means of examining cellular structure; in return, biologists have contributed greatly to the evolution of microscope design and technique. Recent advances in the detection and processing of optical images, together with methods for labelling specific biological molecules, have brought about a resurgence in the application of optical microscopy to the biological sciences. One of the areas in which optical microscopy is breaking new ground is in elucidating the large scale organization of chromatin in chromosomes and cell nuclei. Nevertheless, imaging the contents of the cell nucleus is a difficult challenge for light microscopy, for two principal reasons. First, the dimensions of all but the largest nuclear structures (nucleoli, vacuoles) are close to or below the resolving power of far field optics. Second, the native optical contrast properties of many important chromatin structures (eg. chromosome domains, centromere regions) are very weak, or essentially zero. As an extreme example, individual genes probably have nothing to distinguish them other than their sequence of DNA bases, which cannot be directly visualized with any current form of microscopy. Similarly, the interphase nucleus shows no direct visible evidence of focal chromatin domains. Thus, imaging of such entities depends heavily on contrast enhancement methods. The most promising of these is labelling DNA in situ using sequence-specific probes that may be visualized using fluorescent dyes. We have applied this method to detecting individual genes in metaphase chromosomes and interphase nuclei, and to imaging a number of DNA-containing structures including chromosome domains, metaphase chromosomes and centromere regions. We have also demonstrated the applicability of in situ fluorescent labelling to detecting numerical and structural abnormalities both in

  12. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

    PubMed

    Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

    2013-12-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space.

  13. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

    PubMed

    Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

    2013-12-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. PMID:23242824

  14. Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms

    NASA Astrophysics Data System (ADS)

    Bouchard, Matthew B.; Voleti, Venkatakaushik; Mendes, César S.; Lacefield, Clay; Grueber, Wesley B.; Mann, Richard S.; Bruno, Randy M.; Hillman, Elizabeth M. C.

    2015-02-01

    We report a three-dimensional microscopy technique—swept, confocally-aligned planar excitation (SCAPE) microscopy—that allows volumetric imaging of living samples at ultrahigh speeds. Although confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image three-dimensional volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light sheet in a single-objective, en face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless three-dimensional imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.

  15. 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.

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

    PubMed

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

    2009-01-01

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

  17. Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy.

    PubMed

    Donaldson, L A; Kroese, H W; Hill, S J; Franich, R A

    2015-09-01

    A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl-substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4-nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4-nitrophenyl β-D-glucopyrano-side (monosaccharide), 4-nitrophenyl β-D-lactopyranoside (disaccharide), 2-chloro-4-nitrophenyl β-D-maltotrioside (trisaccharide), and 4-nitrophenyl α-D-maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4-nitrophenyl α-D-maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface.

  18. 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.

  19. Inflammation in dry eye associated with rheumatoid arthritis: cytokine and in vivo confocal microscopy study.

    PubMed

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

    2013-01-01

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

  20. Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

    PubMed Central

    Bohórquez, Diego; Haque, Fariha; Medicetty, Satish; Liddle, Rodger A.

    2015-01-01

    Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue. PMID:26273796

  1. Corneal Confocal Microscopy to Assess Diabetic Neuropathy: An Eye on the Foot

    PubMed Central

    Tavakoli, Mitra; Petropoulos, Ioannis N.; Malik, Rayaz A.

    2013-01-01

    Accurate detection and quantification of human diabetic peripheral neuropathy are important to define at-risk patients, anticipate deterioration, and assess new therapies. Easily performed clinical techniques such as neurological examination, assessment of vibration perception or insensitivity to the 10 g monofilament only assess advanced neuropathy, i.e., the at-risk foot. Techniques that assess early neuropathy include neurophysiology (which assesses only large fibers) and quantitative sensory testing (which assesses small fibers), but they can be highly subjective while more objective techniques, such as skin biopsy for intra-epidermal nerve fiber density quantification, are invasive and not widely available. The emerging ophthalmic technique of corneal confocal microscopy allows quantification of corneal nerve morphology and enables clinicians to diagnose peripheral neuropathy in diabetes patients, quantify its severity, and potentially assess therapeutic benefit. The present review provides a detailed critique of the rationale, a practical approach to capture images, and a basis for analyzing and interpreting the images. We also critically evaluate the diagnostic ability of this new noninvasive ophthalmic test to diagnose diabetic and other peripheral neuropathies. PMID:24124944

  2. In Vivo Confocal Microscopy in Dry Eye Disease and Related Conditions

    PubMed Central

    Alhatem, Albert; Cavalcanti, Bernardo; Hamrah, Pedram

    2015-01-01

    A new era of ocular imaging has recently begun with the advent of in vivo confocal microscopy (IVCM), shedding more light on the pathophysiology, diagnosis, and potential treatment strategies for dry eye disease. IVCM is a noninvasive and powerful tool that allows detection of changes in ocular surface epithelium, immune and inflammatory cells, corneal nerves, keratocytes, and meibomian gland structures on a cellular level. Ocular surface structures in dry eye-related conditions have been assessed and alterations have been quantified using IVCM. IVCM may aid in the assessment of dry eye disease prognosis and treatment, as well as lead to improved understanding of the pathophysiological mechanisms in this complex disease. Further, due to visualization of subclinical findings, IVCM may allow detection of disease at much earlier stages and allow stratification of patients for clinical trials. Finally, by providing an objective methodology to monitor treatment efficacy, image-guided therapy may allow the possibility of tailoring treatment based on cellular changes, rather than on clinical changes alone. PMID:23163268

  3. A confocal microscopy-based atlas of tissue architecture in the tapeworm Hymenolepis diminuta.

    PubMed

    Rozario, Tania; Newmark, Phillip A

    2015-11-01

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

  4. Functional imaging of living Paramecium by means of confocal and two-photon excitation fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto; Fronte, Paola; Raimondo, Marco; Fato, Marco; DeLeo, Gianluca; Beltrame, Francesco; Cannone, Fabio; Chirico, Giberto; Ramoino, Paola

    2002-05-01

    Confocal and Two-photon excitation laser scanning microscopy allow gathering three-dimensional and temporal information from biological systems exploiting fluorescence labeling and autofluorescence properties. In this work we study biological events linked to functionality in Paramecium primaurelia. The internalization of material in ciliated one-celled organisms (protozoa) occurs via different mechanisms, even if most of nutrients, particulate or not, is taken up by food vacuoles formed at the bottom of the oral cavity. The endocytosis of small-sized molecules occurs at the parasomal sacs, located next the ciliar basal bodies. Vital fluorescent dyes (BSA-FITC, WGA-FITC, dextran-Texas Red, cholesteryl-Bodipy) and autofluorescence were used to study formation, movement, and fusion of vesicles during endocytosis and phagocytosis of Paramecium primaurelia. By immobilizing living cells pulsed with food vacuole and endosome markers at successive times after chasing in unlabeled medium, the intracellular movement and fusion of food vacuoles and of endosomes were visualized. A temporal analysis of fluorescence images and the false-color technique were used. Starting from time series or 3D data sets composite images were generated by associating with each originally acquired image a different color corresponding to each sampling point in time and along the z-axis. Second Harmonic Generation Imaging attempts are also outlined.

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

    PubMed

    Calapez, Alexandre; Rosa, Agostinho

    2010-09-01

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

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

    PubMed

    Biolchini, Carla de Lamare; Neves, Renata Heisler; Hulstijn, Maarten; Gomes, Delir Corrêa; Machado-Silva, José Roberto

    2006-09-01

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

  7. In vivo reflectance confocal microscopy detects pigmentary changes in melasma at a cellular level resolution.

    PubMed

    Kang, Hee Young; Bahadoran, Philippe; Suzuki, Itaru; Zugaj, Didier; Khemis, Abdallah; Passeron, Thierry; Andres, Philippe; Ortonne, Jean-Paul

    2010-08-01

    Melasma is a frequent pigmentary disorder caused by abnormal melanin deposits in the skin. In vivo reflectance confocal microscopy (RCM) is a repetitive imaging tool that provides real-time images of the skin at nearly histological resolution. As melanin is the strongest endogenous contrast in human skin, pigmentary disorders are the most suitable candidates for RCM examination but RCM features of melasma have never been reported. This study investigates the pilot use of RCM in melasma to provide a set of well-described morphological criteria with histological correlations. RCM images were acquired from melasma skin and compared to adjacent control skin in 26 patients. Skin biopsies were obtained from eight patients. In the epidermis, RCM showed in all patients a significant increase in hyperrefractile cobblestoning cells. These cells corresponded to hyperpigmented basal keratinocytes in histology. In six patients, dendritic cells corresponding to activated melanocytes were also found in the epidermis. In the dermis, RCM identified in nine patients plump bright cells corresponding to melanophages. Interestingly, for a given patient, the topographic distribution of melanophages in melasma lesions was very heterogeneous. RCM also showed a significant increase in solar elastosis and blood vessels in the dermis. RCM is a non-invasive technique that detects pigmentary changes in melasma at a cellular level resolution. Therefore, RCM provides an innovative way to classify melasma by pigment changes.

  8. 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.

  9. 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.

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

    PubMed

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

    2016-01-01

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

  11. Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions.

    PubMed

    Seidel, Thomas; Edelmann, J-C; Sachse, Frank B

    2016-05-01

    Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 µm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale. PMID:26399990

  12. Implementation of fluorescence confocal mosaicing microscopy by "early adopter" Mohs surgeons: a review of recent progress

    NASA Astrophysics Data System (ADS)

    Jain, Manu; Rajadhyaksha, Milind; Nehal, Kishwer

    2016-03-01

    Confocal mosaicing microscopy (CMM) enables rapid imaging of large areas of fresh tissue ex vivo without the processing that is necessary for conventional histology. When performed with fluorescence mode using acridine orange (nuclear specific dye) it enhances nuclei-to-dermis contrast that enables detection of all types of BCCs including thin strands of infiltrative basal cell carcinomas (BCCs). Thus far, this technique has been mostly validated in research setting for the analysis of BCC tumor margins. Recently, CMM has been adopted and implemented in real clinical settings by some surgeons as an alternative tool to frozen section (FS) during Mohs surgery. In this review article we summarize the development of CMM guided imaging of ex vivo tissues from bench to bedside. We also present its current state of application in routine clinical workflow not only for the assessment of BCC margin but also for other skin cancers such as melanoma, SCC, and some infectious diseases where FS is not routinely performed. Lastly, we also discuss the potential limitations of this technology as well as future developments. As this technology advances further, it may serve as an adjunct to standard histology and enable rapid surgical pathology of skin cancers at the bedside.

  13. Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

    PubMed

    Mravec, Filip; Obruca, Stanislav; Krzyzanek, Vladislav; Sedlacek, Petr; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Nebesarova, Jana

    2016-05-01

    Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation. PMID:27190240

  14. 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.

  15. 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.

  16. Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy.

    PubMed

    Donaldson, L A; Kroese, H W; Hill, S J; Franich, R A

    2015-09-01

    A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl-substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4-nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4-nitrophenyl β-D-glucopyrano-side (monosaccharide), 4-nitrophenyl β-D-lactopyranoside (disaccharide), 2-chloro-4-nitrophenyl β-D-maltotrioside (trisaccharide), and 4-nitrophenyl α-D-maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4-nitrophenyl α-D-maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface. PMID:25925133

  17. Three-dimensional assessment of bone turnover using computed microtomography and laser-scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

    Prevrhal, Sven; Jiang, Yebin; Zhao, Jenny; Genant, Harry K.

    2000-06-01

    Objective: Metabolic activity in trabecular bone is an important indicator in the therapy of bone diseases like osteoporosis. It is reflected by the amount of osteoid (young, not yet mineralized bone) and young calcified tissue (YCT). Our aim was to replace standard 2D histomorphometry with a 3D approach for osteoid and YCT measurement. Measurement Methods: Excised lumbar vertebrae of 5 ovariectomized (OVX) and 5 control rats were 3D-scanned with computed micro-tomography ((mu) CT, isotropic spatial resolution 20 micrometer3) and laser scanning confocal microscopy (LSCM, 20X magnification, 1X1X2 micrometer3 spatial resolution). (mu) CT shows trabecular bone structure; LSCM shows osteoid and YCT by fluorescent light. Image Processing Methods: The fraction of bone to tissue volume (BV/TV) and the number of trabeculae (Tb.N) were calculated from globally thresholded (mu) CT images. LSCM images were enhanced using top-hat transform, globally thresholded and morphologically closed. Separate regions were labeled by volume growing. We measured feature volume to background volume ratio and number of features per unit volume. Results and Conclusions: In the specimens obtained from the OVX rats, a significant increase in the volume fractions of osteoid and YCT could be seen. The (mu) CT-LSCM approach presents a significant improvement over time-consuming, standard histomorphometry. The image processing for both modalities could be achieved automatically.

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

    PubMed

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

    2016-01-01

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

  19. Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

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

  20. Feasibility of intraoperative imaging during Mohs surgery with reflectance confocal microscopy

    NASA Astrophysics Data System (ADS)

    Flores, Eileen S.; Cordova, Miguel; Kose, Kivanc; Phillips, William; Nehal, Kishwer; Rajadhyaksha, Milind

    2014-03-01

    Mohs surgery for the removal of non-melanoma skin cancers (NMSCs) is performed in stages, while being guided by the examination for residual tumor with frozen pathology. However, preparation of frozen pathology at each stage is timeconsuming and labor-intensive. Real-time intraoperative reflectance confocal microscopy (RCM) may enable rapid detection of residual tumor directly in surgical wounds on patients. We report initial feasibility on twenty-one patients, using 35% AlCl3 for nuclear contrast. Imaging was performed in quadrants in the wound, to simulate the Mohs surgeon's examination of pathology. Images and videos of the epidermal and dermal margins were found to be of clinically acceptable quality. Bright nuclear morphology was identified at the epidermal margin. The presence of residual BCC/SCC tumor and normal skin features could be detected in the peripheral and deep dermal margins. Nuclear morphology was detectable in residual BCC/SCC tumors. Intraoperative RCM imaging may enable detection of residual tumor, directly on Mohs patients, and may serve as an adjunct for frozen pathology. However, a stronger source of contrast will be necessary, and also a smaller device with an automated approach for imaging in the entire wound in a rapid and controlled manner for clinical utility.

  1. Confocal Raman microscopy of self-assembled GeSi/Si(001) Islands

    SciTech Connect

    Mashin, A. I.; Nezhdanov, A. V.; Filatov, D. O. Isakov, M. A.; Shengurov, V. G.; Chalkov, V. Yu.; Denisov, S. A.

    2010-11-15

    The method of confocal Raman microscopy is used for the first time to study the spatial distribution of elemental composition and elastic strains in self-assembled GexSi{sub 1-x}/Si(001) islands grown by the method of sublimation molecular-beam epitaxy in the GeH{sub 4} ambient. The lines related to vibrational modes Si-Si, Ge-Ge, and Si-Ge are identified in the Raman spectra measured in the regions with dimensions <100 nm on the surface of the samples. The spatial distribution of the Ge atomic fraction x in the Ge{sub x}Si{sub 1-x} alloy and of the elastic strain {epsilon} (averaged in depth over the island layer) have been calculated from the maps of the Raman shifts of the corresponding lines over the sample surface. The dependences of x and {epsilon} on the islands' growth temperature and on the nominal thickness of the deposited Ge layer have been studied.

  2. Morphological confocal microscopy in arthropods and the enhancement of autofluorescence after proteinase K extraction.

    PubMed

    Valdecasas, Antonio G; Abad, Angela

    2011-02-01

    Procedures to study the molecular and morphological characteristics of microscopic organisms are often incompatible with each other. Therein, the realization of alternatives that make the characterization of these features compatible and simultaneously permit the deposition of the original material as a voucher sample into a reference collection is one of the foremost goals of biodiversity studies. In this study, we show that genomic extraction does not necessarily compromise the detailed study of the external morphology of microscopic organisms, and to do so, we used a group of aquatic mites (Acari, Hydrachnidia) as a test group. Hydrachnidia morphology is difficult to study when specimens have been stored in pure ethanol; however, proteinase K extraction leaves them flexible and easy to dissect, while, at the same time, maintaining all of their diagnostic features intact. Furthermore, autofluorescence is significantly enhanced after proteinase extraction. Our study was conducted with aquatic mites that were stored in absolute ethanol in the field and processed for DNA extraction using a Qiagen QIAamp minikit. Before and after molecular extraction, a laser scanning confocal microscopy morphological examination was carried out.

  3. Jamming of a soft granular system of hollow elastic shells in 3D using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Jose, Jissy; van Blaaderen, Alfons; Imhof, Arnout

    2014-03-01

    We introduce a new system for jammed matter research consisting of monodisperse, fluorescent, hollow deformable shells, dispersed in an index matched solvent. The interesting fact about these elastic shells is that they undergo buckling: in each contact one of the shells receives an indentation from its neighbor under compressive stress. This kind of deformation is different from the soft granular systems experimentally studied so far like photo elastic disks, emulsions and foams, where the particles are flattened in the region of contact and conserve their volume. Using confocal microscopy and image analysis routines (ImageJ software) we identified the 3D position of the particles with sub pixel resolution. The force law to find the contact forces between pairs of particle is derived from the theory of elasticity of thin shells, where force is proportional to the square root of indentation depth. The distribution of normalized contact forces showed a similar trend like other jammed systems with a peak around the mean and a tail that decayed faster than exponential away from jamming threshold. Further, we also investigated the structure of the jammed packings and contact number distribution with distance to jamming.

  4. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study.

    PubMed

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

    2016-01-01

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

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

    PubMed

    Calapez, Alexandre; Rosa, Agostinho

    2010-09-01

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

  6. 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.

  7. A confocal microscopy-based atlas of tissue architecture in the tapeworm Hymenolepis diminuta.

    PubMed

    Rozario, Tania; Newmark, Phillip A

    2015-11-01

    Tapeworms are pervasive and globally distributed parasites that infect millions of humans and livestock every year, and are the causative agents of two of the 17 neglected tropical diseases prioritized by the World Health Organization. Studies of tapeworm biology and pathology are often encumbered by the complex life cycles of disease-relevant tapeworm species that infect hosts such as foxes, dogs, cattle, pigs, and humans. Thus, studies of laboratory models can help overcome the practical, ethical, and cost-related difficulties faced by tapeworm parasitologists. The rat intestinal tapeworm Hymenolepis diminuta is easily reared in the laboratory and has the potential to enable modern molecular-based experiments that will greatly contribute to our understanding of multiple aspects of tapeworm biology, such as growth and reproduction. As part of our efforts to develop molecular tools for experiments on H. diminuta, we have characterized a battery of lectins, antibodies, and common stains that label different tapeworm tissues and organ structures. Using confocal microscopy, we have assembled an "atlas" of H. diminuta organ architecture that will be a useful resource for helminthologists. The methodologies we describe will facilitate characterization of loss-of-function perturbations using H. diminuta. This toolkit will enable a greater understanding of fundamental tapeworm biology that may elucidate new therapeutic targets toward the eradication of these parasites. PMID:26049090

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

    PubMed

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

    2016-01-01

    Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20-30 and 50-70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin. PMID:27088865

  9. In vivo confocal microscopy of basal cell carcinoma: a systematic review of diagnostic accuracy.

    PubMed

    Kadouch, D J; Schram, M E; Leeflang, M M; Limpens, J; Spuls, P I; de Rie, M A

    2015-10-01

    Basal cell carcinoma (BCC) is the most prevalent type of skin cancer. Histologic analysis of punch biopsy or direct excision specimen is used to confirm clinical diagnosis. In vivo reflectance confocal microscopy (RCM) is a non-invasive imaging modality that could facilitate early diagnosis and minimize unnecessary invasive procedures. We systematically reviewed diagnostic accuracy (sensitivity and specificity) of RCM in diagnosing primary BCCs to judge its usefulness. Eligible studies were reviewed for methodological quality using the QUADAS-2 tool. We used the bivariate random-effects model to calculate summary estimates of sensitivity and specificity. Six studies met the selection criteria and were included for analysis. The meta-analysis showed a summary estimate of sensitivity 0.97 (95% CI, 0.90-0.99) and specificity 0.93 (95% CI, 0.88-0.96). All but one of the QUADAS-2 items showed a high or unclear risk of bias with regards to patient selection. RCM may be a promising diagnostic tool, but the limited number of available studies and potential risk of bias of included studies do not allow us to draw firm conclusions. Future accuracy studies should take these limitations into account. PMID:26290493

  10. Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions.

    PubMed

    Seidel, Thomas; Edelmann, J-C; Sachse, Frank B

    2016-05-01

    Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 µm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale.

  11. In vivo reflectance confocal microscopy evaluation of cheilitis glandularis: a report of 5 cases.

    PubMed

    Lourenço, Silvia V; Kos, Eliana; Borguezan Nunes, Thais; Bologna, Sheyla B; Sangueza, Martin; Nico, Marcello M S

    2015-03-01

    Cheilitis glandularis (CG) is an uncommon condition of unknown origin; it is clinically characterized by variable degrees of macrocheilia associated with red dilated ostia of minor salivary glands on the vermilion area, which secrete viscous saliva. Histopathological characteristics of CG are comprised of chronic sialadenitis with engorged acinar lobules and dilated ducts; CG also features chronic sun damage (actinic cheilitis and squamous cell carcinoma). These changes may be localized, and a punch biopsy specimen might fail to reveal enough criteria to support the diagnosis of CG. Reflectance confocal microscopy (RCM) is a noninvasive imaging technique that enables an in vivo en face visualization of tissues with a resolution close to conventional histopathology. Its use allows analysis of the entire lip, without excision. We reported the evaluation of 5 cases of CG based on clinical RCM and histopathological correlation. RCM examination of the lip vermilion mainly revealed a bright aspect of the superficial epithelial layers, which corresponded to labial keratosis. Alteration of the classical epithelial honeycomb pattern was observed in RCM, which corresponded to epithelial changes in actinic cheilitis at histopathology. Round, dark empty spaces intermingling the epithelium, corresponded to the ectopic excretory salivary gland ducts that open their ostia within the lip vermilion. In the lamina propria, the most striking feature was superficial salivary gland lobules, seen as dark gray lobular structures. Our study, demonstrated the use of RCM in the evaluation of CG, showing that a correlation between the clinical, digital RCM images and histopathology improved the diagnostic skills in CG evaluation.

  12. Intraoperative imaging during Mohs surgery with reflectance confocal microscopy: initial clinical experience

    PubMed Central

    Flores, Eileen S.; Cordova, Miguel; Kose, Kivanc; Phillips, William; Rossi, Anthony; Nehal, Kishwer; Rajadhyaksha, Milind

    2015-01-01

    Abstract. Mohs surgery for the removal of nonmelanoma skin cancers (NMSCs) is performed in stages, while being guided by the examination for residual tumor with frozen pathology. However, preparation of frozen pathology at each stage is time consuming and labor intensive. Real-time intraoperative reflectance confocal microscopy (RCM), combined with video mosaicking, may enable rapid detection of residual tumor directly in the surgical wounds on patients. We report our initial experience on 25 patients, using aluminum chloride for nuclear contrast. Imaging was performed in quadrants in the wound to simulate the Mohs surgeon’s examination of pathology. Images and videos of the epidermal and dermal margins were found to be of clinically acceptable quality. Bright nuclear morphology was identified at the epidermal margin and detectable in residual NMSC tumors. The presence of residual tumor and normal skin features could be detected in the peripheral and deep dermal margins. Intraoperative RCM imaging may enable detection of residual tumor directly on patients during Mohs surgery, and may serve as an adjunct for frozen pathology. Ultimately, for routine clinical utility, a stronger tumor-to-dermis contrast may be necessary, and also a smaller microscope with an automated approach for imaging in the entire wound in a rapid and controlled manner. PMID:25706821

  13. Automated Delineation of Dermal-Epidermal Junction In Reflectance Confocal Microscopy Image Stacks Of Human Skin

    PubMed Central

    Park, Brian

    2014-01-01

    Reflectance confocal microscopy (RCM) images skin non-invasively, with optical sectioning and nuclear-level resolution comparable to that of pathology. Based on assessment of the dermal-epidermal junction (DEJ) and morphologic features in its vicinity, skin cancer can be diagnosed in vivo with high sensitivity and specificity. However, the current visual, qualitative approach for reading images leads to subjective variability in diagnosis. We hypothesize that machine learning-based algorithms may enable a more quantitative, objective approach. Testing and validation was performed with two algorithms that can automatically delineate the DEJ in RCM stacks of normal human skin. The test set was composed of 15 fair and 15 dark skin stacks (30 subjects) with expert labellings. In dark skin, in which the contrast is high due to melanin, the algorithm produced an average error of 7.9±6.4μm. In fair skin, the algorithm delineated the DEJ as a transition zone, with average error of 8.3±5.8μm for the epidermis-to-transition zone boundary and 7.6±5.6μm for the transition zone-to-dermis. Our results suggest that automated algorithms may quantitatively guide the delineation of the DEJ, to assist in objective reading of RCM images. Further development of such algorithms may guide assessment of abnormal morphological features at the DEJ. PMID:25184959

  14. Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Wei, Qiaoling; Le, Qihua; Hong, Jiaxu; Xiang, Jun; Wei, Anji; Xu, Jianjiang

    2015-01-01

    Purpose: To investigate the correlations between conjunctival inflammatory status and meibomian gland (MG) morphology in vernal keratoconjunctivitis (VKC) patients by using in vivo confocal microscopy (CM). Materials and Methods: Nineteen VKC patients (7 limbal, 7 tarsal, and 5 mixed forms) and 16 normal volunteers (controls) were enrolled. All subjects underwent CM scanning to obtain the images of upper palpebral conjunctiva and MGs. Inflammatory cell (IC) density in palpebral conjunctival epithelial and stromal layers, Langerhans cell (LC) density at lid margins and the stroma adjacent to the MG, and MG acinar unit density (MGAUD) were recorded. The longest and shortest diameters of MG acinar were measured. The Kruskal-Wallis test was used to compare the parameter differences whereas the Spearman's rank correlation analysis was applied to determine their correlations. Results: Among all groups, no significant statistical differences were found in epithelial and stromal IC densities, mean values of MG acinar unit densities, or longest and shortest diameters. Both LC parameters in the tarsal-mixed groups were significantly higher than those in the limbal and control groups. All LC densities of VKC patients showed a positive correlation with MGAUD and shortest diameter. Conclusions: In VKC patients, the conjunctival inflammatory status could be associated with the MG status. In vivo CM is a noninvasive, efficient tool in the assessment of MG status and ocular surface. PMID:26044472

  16. Demeclocycline as a contrast agent for detecting brain neoplasms using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Wirth, Dennis; Smith, Thomas W.; Moser, Richard; Yaroslavsky, Anna N.

    2015-04-01

    Complete resection of brain tumors improves life expectancy and quality. Thus, there is a strong need for high-resolution detection and microscopically controlled removal of brain neoplasms. The goal of this study was to test demeclocycline as a contrast enhancer for the intraoperative detection of brain tumors. We have imaged benign and cancerous brain tumors using multimodal confocal microscopy. The tumors investigated included pituitary adenoma, meningiomas, glioblastomas, and metastatic brain cancers. Freshly excised brain tissues were stained in 0.75 mg ml-1 aqueous solution of demeclocyline. Reflectance images were acquired at 402 nm. Fluorescence signals were excited at 402 nm and registered between 500 and 540 nm. After imaging, histological sections were processed from the imaged specimens and compared to the optical images. Fluorescence images highlighted normal and cancerous brain cells, while reflectance images emphasized the morphology of connective tissue. The optical and histological images were in accordance with each other for all types of tumors investigated. Demeclocyline shows promise as a contrast agent for intraoperative detection of brain tumors.

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

    PubMed Central

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

    2016-01-01

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

  18. The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

    PubMed Central

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

    2016-01-01

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

  19. Corneal Confocal Microscopy Detects Neuropathy in Patients with Type 1 Diabetes without Retinopathy or Microalbuminuria

    PubMed Central

    Chan, Agnes W. S.; Alam, Uazman; Fadavi, Hassan; Marshall, Andrew; Asghar, Omar; Efron, Nathan; Tavakoli, Mitra; Malik, Rayaz A.

    2015-01-01

    Objective Corneal innervation is increasingly used as a surrogate marker of human diabetic peripheral neuropathy (DPN) however its temporal relationship with the other microvascular complications of diabetes is not fully established. In this cross-sectional, observational study we aimed to assess whether neuropathy occurred in patients with type 1 diabetes, without retinopathy or microalbuminuria. Materials and Methods All participants underwent detailed assessment of peripheral neuropathy [neuropathy disability score (NDS), vibration perception threshold (VPT), peroneal motor nerve conduction velocity (PMNCV), sural sensory nerve conduction velocity (SSNCV) and in vivo corneal confocal microscopy (IVCCM)], retinopathy (digital fundus photography) and albuminuria status [albumin: creatinine ratio (ACR)]. Results 53 patients with Type 1 diabetes with (n=37) and without retinopathy (n=16) were compared to control subjects (n=27). SSNCV, corneal nerve fibre (CNFD) and branch (CNBD) density and length (CNFL) were reduced significantly (p<0.001) in diabetic patients without retinopathy compared to control subjects. Furthermore, CNFD, CNBD and CNFL were also significantly (p<0.001) reduced in diabetic patients without microalbuminuria (n=39), compared to control subjects. Greater neuropathic severity was associated with established retinopathy and microalbuminuria. Conclusions IVCCM detects early small fibre damage in the absence of retinopathy or microalbuminuria in patients with Type 1 diabetes. PMID:25853247

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

    PubMed Central

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

    2016-01-01

    Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20–30 and 50–70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 μm, 6.0 μm and 5.5 μm respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin. PMID:27088865

  2. Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

    PubMed

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

    2016-09-19

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

  3. Intraoperative imaging during Mohs surgery with reflectance confocal microscopy: initial clinical experience

    NASA Astrophysics Data System (ADS)

    Flores, Eileen S.; Cordova, Miguel; Kose, Kivanc; Phillips, William; Rossi, Anthony; Nehal, Kishwer; Rajadhyaksha, Milind

    2015-06-01

    Mohs surgery for the removal of nonmelanoma skin cancers (NMSCs) is performed in stages, while being guided by the examination for residual tumor with frozen pathology. However, preparation of frozen pathology at each stage is time consuming and labor intensive. Real-time intraoperative reflectance confocal microscopy (RCM), combined with video mosaicking, may enable rapid detection of residual tumor directly in the surgical wounds on patients. We report our initial experience on 25 patients, using aluminum chloride for nuclear contrast. Imaging was performed in quadrants in the wound to simulate the Mohs surgeon's examination of pathology. Images and videos of the epidermal and dermal margins were found to be of clinically acceptable quality. Bright nuclear morphology was identified at the epidermal margin and detectable in residual NMSC tumors. The presence of residual tumor and normal skin features could be detected in the peripheral and deep dermal margins. Intraoperative RCM imaging may enable detection of residual tumor directly on patients during Mohs surgery, and may serve as an adjunct for frozen pathology. Ultimately, for routine clinical utility, a stronger tumor-to-dermis contrast may be necessary, and also a smaller microscope with an automated approach for imaging in the entire wound in a rapid and controlled manner.

  4. Corneal Confocal Microscopy Detects Neuropathy in Subjects With Impaired Glucose Tolerance

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Vesely, P; Boyde, A

    2001-06-01

    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 microm and in time to 1/25th of a second. Mapping the cell space at 0.2 microm x 0.2 microm (XY = in instantaneous best focal plane) x 0.5 microm (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.

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

    PubMed Central

    Mokhtarzadeh, Maryam; Casey, Richard

    2011-01-01

    Purpose. The basis of fluorescein-associated superficial punctate staining in dry eyes is controversial. Prior explanations include fluorescein pooling in surface erosive defects, intercellular trapping of fluorescein, and intracellular staining in dead cells. In this study, the hypothesis that punctate erosions are individual cells with enhanced fluorescence was tested. Methods. Ten impression cytology membrane materials were compared, to optimize cellular yield in buccal mucosa and cornea. Clinicocytologic correlation of punctate fluorescent spots was performed in four dry eye patients. Individual punctate spots were localized by fiducial marks in photographs, before and after removal with impression membranes, and were traced in fluorescence microscopy and cytologic staining. Two-way contingency table analysis was used to determine the correlation of punctate spots with cells removed by the membrane. Clinicopathologic correlation of punctate spots was performed in 10 corneas removed in dry eye patients by transplantation for concurrent diseases. Punctate fluorescence was tracked in specimens by fiducial marks and epifluorescence. The distribution of fluorescent spots in specific cell layers of the cornea was determined by confocal microscopy. Results. Cellular yield was greatest with impressions from polytetrafluoroethylene (PTFE [Teflon]; BioPore; Millipore, Billerica, MA) membrane compared with its closest rival (P = 0.019). Punctate fluorescent spots, most of which disappeared after impression cytology (71%), correlated with cells on the membranes (P = 0.009). The punctate spots were more frequent in the superficial cell layers of the cornea (80%) compared with the deepest two layers (0%) (P < 0.00049). Conclusions. Punctate epithelial erosions correspond to enhanced fluorescence in epithelial cells predominantly in superficial layers of the cornea and would be more aptly named fluorescent epithelial cells (FLECs). PMID:21212176

  7. Normative Values for Corneal Nerve Morphology Assessed Using Corneal Confocal Microscopy: A Multinational Normative Data Set

    PubMed Central

    Tavakoli, Mitra; Ferdousi, Maryam; Petropoulos, Ioannis N.; Morris, Julie; Pritchard, Nicola; Zhivov, Andrey; Ziegler, Dan; Pacaud, Danièle; Romanchuk, Kenneth; Perkins, Bruce A.; Lovblom, Leif E.; Bril, Vera; Singleton, J. Robinson; Smith, Gordon; Boulton, Andrew J.M.; Efron, Nathan

    2015-01-01

    OBJECTIVE Corneal confocal microscopy is a novel diagnostic technique for the detection of nerve damage and repair in a range of peripheral neuropathies, in particular diabetic neuropathy. Normative reference values are required to enable clinical translation and wider use of this technique. We have therefore undertaken a multicenter collaboration to provide worldwide age-adjusted normative values of corneal nerve fiber parameters. RESEARCH DESIGN AND METHODS A total of 1,965 corneal nerve images from 343 healthy volunteers were pooled from six clinical academic centers. All subjects underwent examination with the Heidelberg Retina Tomograph corneal confocal microscope. Images of the central corneal subbasal nerve plexus were acquired by each center using a standard protocol and analyzed by three trained examiners using manual tracing and semiautomated software (CCMetrics). Age trends were established using simple linear regression, and normative corneal nerve fiber density (CNFD), corneal nerve fiber branch density (CNBD), corneal nerve fiber length (CNFL), and corneal nerve fiber tortuosity (CNFT) reference values were calculated using quantile regression analysis. RESULTS There was a significant linear age-dependent decrease in CNFD (−0.164 no./mm2 per year for men, P < 0.01, and −0.161 no./mm2 per year for women, P < 0.01). There was no change with age in CNBD (0.192 no./mm2 per year for men, P = 0.26, and −0.050 no./mm2 per year for women, P = 0.78). CNFL decreased in men (−0.045 mm/mm2 per year, P = 0.07) and women (−0.060 mm/mm2 per year, P = 0.02). CNFT increased with age in men (0.044 per year, P < 0.01) and women (0.046 per year, P < 0.01). Height, weight, and BMI did not influence the 5th percentile normative values for any corneal nerve parameter. CONCLUSIONS This study provides robust worldwide normative reference values for corneal nerve parameters to be used in research and clinical practice in the study of diabetic and other peripheral

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

    PubMed

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

    2011-10-20

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

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

    PubMed

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

    2011-01-01

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

  10. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques

    PubMed Central

    Jemielita, Matthew; Taormina, Michael J.; DeLaurier, April; Kimmel, Charles B.; Parthasarathy, Raghuveer

    2013-01-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. (A) Schematic: Light sheet microscopy of zebrafish embryos. Opercle-forming osteoblasts following twenty-four hours of (B) light sheet imaging, showing normal growth, and (C) spinning disk confocal imaging, showing aberrant growth. PMID:23242824

  11. Image restoration for confocal microscopy: improving the limits of deconvolution, with application to the visualization of the mammalian hearing organ.

    PubMed

    Boutet de Monvel, J; Le Calvez, S; Ulfendahl, M

    2001-05-01

    Deconvolution algorithms have proven very effective in conventional (wide-field) fluorescence microscopy. Their application to confocal microscopy is hampered, in biological experiments, by the presence of important levels of noise in the images and by the lack of a precise knowledge of the point spread function (PSF) of the system. We investigate the application of wavelet-based processing tools to deal with these problems, in particular wavelet denoising methods, which turn out to be very effective in application to three-dimensional confocal images. When used in combination with more classical deconvolution algorithms, these methods provide a robust and efficient restoration scheme allowing one to deal with difficult imaging conditions. To make our approach applicable in practical situations, we measured the PSF of a Biorad-MRC1024 confocal microscope under a large set of imaging conditions, including in situ acquisitions. As a specific biological application, we present several examples of restorations of three-dimensional confocal images acquired inside an intact preparation of the hearing organ. We also provide a quantitative assessment of the gain in quality achieved by wavelet-aided restorations over classical deconvolution schemes, based on a set of numerical experiments that we performed with test images.

  12. Confocal microscopy: A valid approach to evaluate the three-dimensional characteristics of root-end cavities

    PubMed Central

    Rodríguez-Martos, Ramón; Castellanos-Cosano, Lizett; Yáñez-Vico, Rosa; Segura-Egea, Juan J.; Gutiérrez-Pérez, José L.

    2013-01-01

    Objective: To analyze, using confocal microscope, the three-dimensional characteristics of the root-end cavity preparations completed in root apices of extracted teeth determining their area, perimeter, circularity and cavo-surface angle. Study design: Thirty-two single-rooted extracted teeth underwent endodontic treatment and apical resection. Root-end cavities were prepared according to 4 protocols, as follows: Group1, stainless steel ultrasonic tips (SST) at 33 KHz power; Group 2, SST at 30 KHz power; Group 3, diamond-coated ultrasonic tips (DCT) at 30 KHz power; and Group 4, DCT at 33 KHz power. Finally, root-end cavity was evaluated using a confocal microscope, recording its area, perimeter, circularity and cavo-surface angle. Results: The largest cavity perimeter was found in the Group 2 (4.8 ± 1.6 mm) (p & 0.05). Root-end cavities performed using SST showed larger areas than those performed with DCT (p = 0.03). The power of vibration or the tip type did not show correlation with the perimeter, circularity and cavo-surface angle of the root-end cavity (p & 0.05). Conclusions: Confocal microscopy is a useful approach to study the three-dimensional characteristics of the root-end cavity. Key words:Confocal microscopy, root-end cavity, surgical root canal treatment, ultrasonic tips. PMID:23524419

  13. Visualization of calcium and zinc ions in Saccharomyces cerevisiae cells treated with PEFs (pulse electric fields) by laser confocal microscopy.

    PubMed

    Urszula, Pankiewicz; Jerzy, Jamroz; Sujka, Monika; Kowalski, Radosław

    2015-12-01

    The aim of the present work was to visualize the areas of increased concentration of calcium and zinc ions inside Saccharomyces cerevisiae cells with the use of confocal microscopy and to make an attempt to asses semi-quantitatively their concentration within the limits of the cells. Semi-quantitative analysis revealed that fluorescence inside cells from control samples was three-times lower than that observed for cells from the sample enriched with calcium. Differences in distribution of fluorescence intensity between cells originated from the samples enriched with zinc and control samples were also observed. On the basis of the optical sections, the 3D reconstructions of ion-rich areas distribution in the cell were made. The obtained results showed that confocal microscopy is a useful technique for visualization of the areas in S. cerevisiae cells which contain higher amount of calcium and zinc and it may be also used for semi-quantitative analysis.

  14. Nanoparticle flow velocimetry with image phase correlation for confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Jun, Brian H.; Giarra, Matthew; Yang, Haisheng; Main, Russell; Vlachos, Pavlos P.

    2016-10-01

    We present a new particle image correlation technique for resolving nanoparticle flow velocity using confocal laser scanning microscopy (CLSM). The two primary issues that complicate nanoparticle scanning laser image correlation (SLIC)-based velocimetry are (1) the use of diffusion-dominated nanoparticles as flow tracers, which introduce a random decorrelating error into the velocity estimate, and (2) the effects of the scanning laser image acquisition, which introduces a bias error. To date, no study has quantified these errors or demonstrated a means to deal with them in SLIC velocimetry. In this work, we build upon the robust phase correlation (RPC) and existing methods of SLIC to quantify and mitigate these errors. First, we implement an ensemble RPC instead of using an ensemble standard cross-correlation, and develop a SLIC optimal filter that maximizes the correlation strength in order to reliably and accurately detect the correlation peak representing the most probable average displacement of the nanoparticles. Secondly, we developed an analytical model of the SLIC measurement bias error due to image scanning of diffusion-dominated tracer particles. We show that the bias error depends only on the ratio of the mean velocity of the tracer particles to that of the laser scanner and we use this model to correct the induced errors. We validated our technique using synthetic images and experimentally obtained SLIC images of nanoparticle flow through a micro-channel. Our technique reduced the error by up to a factor of ten compared to other SLIC algorithms for the images tested in this study. Moreover, our optimized RPC filter reduces the number of image pairs required for the convergence of the ensemble correlation by two orders of magnitude compared to the standard cross correlation. This feature has broader implications to ensemble correlation methods and should be further explored in depth in the future.

  15. 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. PMID:18767050

  16. Small fiber neuropathy in Parkinson's disease: A clinical, pathological and corneal confocal microscopy study

    PubMed Central

    Kass-Iliyya, Lewis; Javed, Saad; Gosal, David; Kobylecki, Christopher; Marshall, Andrew; Petropoulos, Ioannis N.; Ponirakis, Georgios; Tavakoli, Mitra; Ferdousi, Maryam; Chaudhuri, Kallol Ray; Jeziorska, Maria; Malik, Rayaz A.; Silverdale, Monty A.

    2015-01-01

    Autonomic and somatic denervation is well established in Parkinson's disease (PD). Objectives (1) To determine whether corneal confocal microscopy (CCM) can non-invasively demonstrate small nerve fiber damage in PD. (2) To identify relationships between corneal nerve parameters, intraepidermal nerve fiber density (IENFD) and clinical features of PD. Methods Twenty-six PD patients and 26 controls underwent CCM of both eyes. 24/26 PD patients and 10/26 controls underwent skin biopsies from the dorsa of both feet. PD patients underwent assessment of parasympathetic function [deep breathing heart rate variability (DB-HRV)], autonomic symptoms [scale for outcomes in Parkinson's disease – autonomic symptoms (SCOPA-AUT)], motor symptoms [UPDRS-III “ON”] and cumulative Levodopa dose. Results PD patients had significantly reduced corneal nerve fiber density (CNFD) with increased corneal nerve branch density (CNBD) and corneal nerve fiber length (CNFL) compared to controls. CNBD and CNFL but not CNFD correlated inversely with UPDRS-III and SCOPA-AUT. All CCM parameters correlated strongly with DB-HRV. There was no correlation between CCM parameters and disease duration, cumulative Levodopa dose or pain. IENFD was significantly reduced in PD compared to controls and correlated with CNFD and UPDRS-III. However, unlike CCM measures, IENFD correlated with disease duration and cumulative Levodopa dose but not with autonomic dysfunction. Conclusion CCM identifies corneal nerve fiber pathology, which correlates with autonomic symptoms, parasympathetic deficits and motor scores in patients with PD. IENFD is also reduced and correlates with CNFD and motor symptoms but not parasympathetic deficits, indicating it detects different aspects of peripheral nerve pathology in PD. PMID:26578039

  17. Compressive sensing in reflectance confocal microscopy of skin images: a preliminary comparative study

    NASA Astrophysics Data System (ADS)

    Arias, Fernando X.; Sierra, Heidy; Rajadhyaksha, Milind; Arzuaga, Emmanuel

    2016-03-01

    Compressive Sensing (CS)-based technologies have shown potential to improve the efficiency of acquisition, manipulation, analysis and storage processes on signals and imagery with slight discernible loss in data performance. The CS framework relies on the reconstruction of signals that are presumed sparse in some domain, from a significantly small data collection of linear projections of the signal of interest. As a result, a solution to the underdetermined linear system resulting from this paradigm makes it possible to estimate the original signal with high accuracy. One common approach to solve the linear system is based on methods that minimize the L1-norm. Several fast algorithms have been developed for this purpose. This paper presents a study on the use of CS in high-resolution reflectance confocal microscopy (RCM) images of the skin. RCM offers a cell resolution level similar to that used in histology to identify cellular patterns for diagnosis of skin diseases. However, imaging of large areas (required for effective clinical evaluation) at such high-resolution can turn image capturing, processing and storage processes into a time consuming procedure, which may pose a limitation for use in clinical settings. We present an analysis on the compression ratio that may allow for a simpler capturing approach while reconstructing the required cellular resolution for clinical use. We provide a comparative study in compressive sensing and estimate its effectiveness in terms of compression ratio vs. image reconstruction accuracy. Preliminary results show that by using as little as 25% of the original number of samples, cellular resolution may be reconstructed with high accuracy.

  18. Analysis of Microstructure of the Cardiac Conduction System Based on Three-Dimensional Confocal Microscopy

    PubMed Central

    Romero, Daniel; Camara, Oscar; Sachse, Frank; Sebastian, Rafael

    2016-01-01

    The specialised conducting tissues present in the ventricles are responsible for the fast distribution of the electrical impulse from the atrio-ventricular node to regions in the subendocardial myocardium. Characterisation of anatomical features of the specialised conducting tissues in the ventricles is highly challenging, in particular its most distal section, which is connected to the working myocardium via Purkinje-myocardial junctions. The goal of this work is to characterise the architecture of the distal section of the Purkinje network by differentiating Purkinje cells from surrounding tissue, performing a segmentation of Purkinje fibres at cellular scale, and mathematically describing its morphology and interconnections. Purkinje cells from rabbit hearts were visualised by confocal microscopy using wheat germ agglutinin labelling. A total of 16 3D stacks including labeled Purkinje cells were collected, and semi-automatically segmented. State-of-the-art graph metrics were applied to estimate regional and global features of the Purkinje network complexity. Two types of cell types, tubular and star-like, were characterised from 3D segmentations. The analysis of 3D imaging data confirms the previously suggested presence of two types of Purkinje-myocardium connections, a 2D interconnection sheet and a funnel one, in which the narrow side of a Purkinje fibre connect progressively to muscle fibres. The complex network analysis of interconnected Purkinje cells showed no small-world connectivity or assortativity properties. These results might help building more realistic computational PK systems at high resolution levels including different cell configurations and shapes. Better knowledge on the organisation of the network might help in understanding the effects that several treatments such as radio-frequency ablation might have when the PK system is disrupted locally. PMID:27716829

  19. A meta-analysis of reflectance confocal microscopy for the diagnosis of malignant skin tumours.

    PubMed

    Xiong, Y D; Ma, S; Li, X; Zhong, X; Duan, C; Chen, Q

    2016-08-01

    Early diagnosis is extremely important for treatment and prognosis of skin cancer. Reflectance confocal microscopy (RCM) is a recently developed technique used to diagnose skin cancer. This meta-analysis was carried out to assess the accuracy of RCM for the diagnosis of malignant skin tumours. We conducted a systematic literature search of EMBASE, PubMed, the Cochrane Library and Web of Science database for relevant articles in English published up to 24 December 2015. The quality of the included studies was assessed using the QUADAS-2 tool. Statistical analyses were conducted using the software Meta-Disc version 1.4 and STATA version 12.0. A total of 21 studies involving 3108 patients with a total of 3602 lesions were included in the per-lesion analysis. The corresponding pooled results for sensitivity and specificity were 93.6% (95% CI: 0.92-0.95) and 82.7% (95% CI: 0.81-0.84) respectively. Positive likelihood ratio and negative likelihood ratio were 5.84 (95% CI: 4.27-7.98) and 0.08 (95% CI: 0.07-0.10) respectively. Subgroup analysis showed that RCM had a sensitivity of 92.7% (95% CI: 0.90-0.95) and a specificity of 78.3% (95% CI: 0.76-0.81) for detecting melanoma. The pooled sensitivity and specificity of RCM for detecting basal cell carcinoma were 91.7% (95% CI: 0.87-0.95) and 91.3% (95% CI: 0.94-0.96) respectively. RCM is a valid method of identifying malignant skin tumours accurately. PMID:27230832

  20. Computational Dosimetry for Electron Microbeams: Monte-Carlo Track Simulation with Confocal Microscopy

    SciTech Connect

    Miller, John H.; Wilson, W E.; Lynch, D J.; Resat, Marianne S.; Trease, Harold E.

    2001-10-15

    Both in vitro and in vivo experiments show that cells that do not receive energy directly from the radiation field (bystanders) respond to radiation exposure. This effect is most easily demonstrated with radiation fields composed of particles with high linear energy transfer (LET) that traverse only a few cells before they are stopped. Even at a moderate fluence of high-LET radiation only a small fraction of cells in the irradiated population are hit; hence, many bystanders are present. Low-LET radiation tends to generate a homogeneous distribution of dose at the cellular level so that identifying bystanders is much more difficult than in experiments with the same fluence of high-LET radiation. Experiments are underway at several laboratories to characterize bystander responses induced by low-LET radiation. At the Pacific Northwest National Laboratory, experiments of this type are being carried out with an electron microbeam. A cell selected to receive energy directly from the irradiation source is placed over a hole in a mask that covers an electron gun. Monte Carlo simulations by Miller et al.(1) suggest that individual mammalian cells in a confluent monolayer could be targeted for irradiation by 25 to 100 keV electrons with minimal dose leakage to their neighbors. These calculations were based on a simple model of the cellular monolayer in which cells were assumed to be cylindrically symmetric with concentric cytoplasm and nucleus. Radial profiles, the lateral extent of cytoplasm and nucleus as a function of depth into a cell, were obtained from confocal microscopy of HeLa-cell monolayers.

  1. [Revealing the Cell Structure and Formation of Bamboo with Confocal Raman Microscopy].

    PubMed

    Li, Xiao-li; Zhou, Bin-xiong; Zhang, Yi; Yao, Yan-ming; He, Yong

    2016-02-01

    Parenchyma cell (PAC), transition tissue between parenchyma cell and fiber cell (TC) and fibre cell (FC) of bamboo were studied by confocal Raman microscopy in this paper. Partial least squares regression was applied to establish a quantitative differentiation model for the three types of cells. The result showed that the determination coefficients (R²) of calibration and validation were respectively 0.810 and 0.800, and the root mean square error (RMSE) were respectively 0.323 and 0.332. What's more, three raman bands of 1,095, 1,319 and 1,636 cm⁻¹, verified to the characteristic peaks of pectin, hemicellulose and lignin, were found to be the important bands for the differentiation. Subsequently, these three raman bands were used to establish a multiple linear regression (MLR) model, and the determination coefficients (R²) of calibration and validation of the model were respectively 0.644 and 0.643, and the root mean square error (RMSE) were respectively 0.442 and 0.443. This result showed that there existed obvious difference among the three types of cells in these three raman bands. Finally, the raman spectral signal processed by wavelet transform to eliminate baseline were used to chemical imaging analysis. These results showed a rather large microfibril angle between cellulose fibrils and fibre axis, which contributed to higher modulus and hardness of cells. Hemicellulose and cellulose have similar distribution in the raman chemical image, due to the connection of hemicellulose and cellulose microfiber through hydrogen bond and the closely combination under the action of van der Waals force. The cell corners (CC) and compound middle lamella (CML) were heavily lignified, and a gradual decrease of lignification from the outer layer to the inner layer of the three cells indicate that lignification was first occurred at the CC and CML, and the lignification was not fully completed.

  2. A meta-analysis of reflectance confocal microscopy for the diagnosis of malignant skin tumours.

    PubMed

    Xiong, Y D; Ma, S; Li, X; Zhong, X; Duan, C; Chen, Q

    2016-08-01

    Early diagnosis is extremely important for treatment and prognosis of skin cancer. Reflectance confocal microscopy (RCM) is a recently developed technique used to diagnose skin cancer. This meta-analysis was carried out to assess the accuracy of RCM for the diagnosis of malignant skin tumours. We conducted a systematic literature search of EMBASE, PubMed, the Cochrane Library and Web of Science database for relevant articles in English published up to 24 December 2015. The quality of the included studies was assessed using the QUADAS-2 tool. Statistical analyses were conducted using the software Meta-Disc version 1.4 and STATA version 12.0. A total of 21 studies involving 3108 patients with a total of 3602 lesions were included in the per-lesion analysis. The corresponding pooled results for sensitivity and specificity were 93.6% (95% CI: 0.92-0.95) and 82.7% (95% CI: 0.81-0.84) respectively. Positive likelihood ratio and negative likelihood ratio were 5.84 (95% CI: 4.27-7.98) and 0.08 (95% CI: 0.07-0.10) respectively. Subgroup analysis showed that RCM had a sensitivity of 92.7% (95% CI: 0.90-0.95) and a specificity of 78.3% (95% CI: 0.76-0.81) for detecting melanoma. The pooled sensitivity and specificity of RCM for detecting basal cell carcinoma were 91.7% (95% CI: 0.87-0.95) and 91.3% (95% CI: 0.94-0.96) respectively. RCM is a valid method of identifying malignant skin tumours accurately.

  3. Morphological characterization of solar lentigines by in vivo reflectance confocal microscopy: a longitudinal approach.

    PubMed

    Pollefliet, C; Corstjens, H; González, S; Hellemans, L; Declercq, L; Yarosh, D

    2013-04-01

    Solar lentigines are benign hyperpigmented skin lesions. Despite their widespread distribution, knowledge on the mechanisms of development is largely unknown. A clinical study was designed in which solar lentigines were characterized using various non-invasive clinical techniques. A subset of solar lentigines was followed over a 5-year time period. One hundred and twenty-eight solar lentigines were evaluated using in vivo reflectance confocal microscopy (RCM) for the evaluation of the length and density of their dermal papillae as well as the deformation of the alignment pattern of hyperrefractive basal cells. Skin colour, colour contrast, the size of the solar lentigo, epidermal proliferation rate, melanin and haemoglobin content were quantified. RCM imaging of solar lentigines revealed a profound structural deformation of the dermal papillae, as the alignment pattern of hyperrefractive basal cells shifted from a circle in non-lesional skin to an irregular non-circular shape in solar lentigines. There was a rise in the number of dermal papillae, and these dermal papillae were significantly longer. Solar lentigines had increased melanin and haemoglobin levels and a higher rate of epidermal proliferation. For a subset of nineteen solar lentigines, a longitudinal study was set-up in which these measurements were repeated 5 years after the first evaluation. The deformation and the number of the hyperrefractive dermal papillary rings increased significantly over the 5-year time span. The size of the lesion increased, and the skin colour became darker. RCM is a useful non-invasive clinical tool for the characterization of solar lentigines, in particular the compressive deformation of the dermal papillae. This deformation became more severe over a time period of 5 years. To our knowledge, this is the first time that the in vivo time-dependent progression of solar lentigines was supported by RCM images, contributing to an improved understanding of the formation and

  4. Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy

    SciTech Connect

    Talamás-Lara, Daniel; Talamás-Rohana, Patricia; Fragoso-Soriano, Rogelio Jaime; Espinosa-Cantellano, Martha; Chávez-Munguía, Bibiana; González-Robles, Arturo; Martínez-Palomo, Adolfo

    2015-10-01

    Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica. - Highlights: • Striking differences in adhesion to FN between E. histolytica and E. dispar. • A greater degree of cell stiffness in E. histolytica with respect to E. dispar. • E. histolytica but not E. dispar forms regions of close contact with FN. • The actin cytoskeleton is involved in the pathogenicity of E. histolytica.

  5. 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.

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

    PubMed

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

    2015-11-01

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

  7. Advanced chemical imaging and comparison of human and porcine hair follicles for drug delivery by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Franzen, Lutz; Mathes, Christiane; Hansen, Steffi; Windbergs, Maike

    2013-06-01

    Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery.

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

    PubMed

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

    2015-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

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

    EPA Science Inventory

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

  11. In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach.

    PubMed

    Pineda Rodó, Albert; Váchová, Libuše; Palková, Zdena

    2012-01-01

    Many essential cellular processes are affected by transmembrane H(+) gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.

  12. Influences of edges and steep slopes in 3D interference and confocal microscopy

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    particular instrument. In addition, measurement results are compared with those achieved by a Mirau interferometer of NA = 0.55. A commercial confocal microscope with NA of 0.95 serves as a reference instrument for further comparison. Numerical simulations considering diffraction effects are carried out in order to explain the experimental results obtained by the different white and colored light interferometers

  13. Influences of edges and steep slopes in 3D interference and confocal microscopy

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    instrument. In addition, measurement results are compared with those achieved by a Mirau interferometer of NA = 0.55. A commercial confocal microscope with NA of 0.95 serves as a reference instrument for further comparison. Numerical simulations considering diffraction effects are carried out in order to explain the experimental results obtained by the different white and colored light interferometers

  14. Detection of living Sarcoptes scabiei larvae by reflectance mode confocal microscopy in the skin of a patient with crusted scabies

    NASA Astrophysics Data System (ADS)

    Levi, Assi; Mumcuoglu, Kosta Y.; Ingber, Arieh; Enk, Claes D.

    2012-06-01

    Scabies is an intensely pruritic disorder induced by a delayed type hypersensitivity reaction to infestation of the skin by the mite Sarcoptes scabiei. The diagnosis of scabies is established clinically and confirmed by identifying mites or eggs by microscopic examination of scrapings from the skin or by surface microscopy using a dermatoscope. Reflectance-mode confocal microscopy is a novel technique used for noninvasive imaging of skin structures and lesions at a resolution compatible to that of conventional histology. Recently, the technique was employed for the confirmation of the clinical diagnosis of scabies. We demonstrate the first ever documentation of a larva moving freely inside the skin of a patient infected with scabies.

  15. Pulsed vs continuous light accelerated corneal collagen crosslinking: in vivo qualitative investigation by confocal microscopy and corneal OCT

    PubMed Central

    Mazzotta, C; Traversi, C; Caragiuli, S; Rechichi, M

    2014-01-01

    Purpose To assess qualitative corneal changes and penetration of pulsed and continuous light accelerated crosslinking by in vivo confocal microscopy and corneal OCT. Methods A total of 20 patients affected from progressive keratoconus were enrolled in the study. Ten eyes of 10 patients underwent an epithelium-off pulsed-light accelerated corneal collagen crosslinking (PL-ACXL) by the KXL UV-A source (Avedro Inc.) with 8 min (1 s on/1 s off) of UV-A exposure at 30 mW/cm2 and energy dose of 7.2 J/cm2; 10 eyes of 10 patients underwent an epithelium-off continuous-light accelerated corneal collagen crosslinking (CL-ACXL) at 30 mW/cm2 for 4 min. Riboflavin 0.1% dextran-free plus hydroxyl-propyl-methylcellulose solution (VibeX Rapid, Avedro Inc.) was used for a 10-min corneal soaking. Treated eyes were examined by in vivo scanning laser confocal analysis and spectral anterior segment OCT at 1, 3, and 6 months. Results Epithelial stratification and nerves regeneration improved in time, being complete at month 6 in both groups without endothelial damage. Keratocyte apoptosis in PL-ACXL was estimated at a mean depth of ∼200 μm, whereas an uneven demarcation line was detectable by confocal microscopy at a mean depth of 160 μm in CL-ACXL. Conclusion In vivo confocal microscopy and corneal OCT allowed a precise qualitative analysis of the cornea after epithelium-off PL-ACXL and CL-ACXL treatments. Apoptotic effect was higher in pulsed than in continuous light treatments, exceeding 200 μm in corneal stroma. According to different morphological data, the clinical efficacy of ACXL needs to be determined in a long-term follow-up and large cohort of patients. PMID:25060847

  16. 3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

    SciTech Connect

    Fredrich, J.T.

    1999-02-10

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

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

    PubMed

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

    2015-01-01

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

  18. 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

  19. 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

  20. 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

  1. Clinical features and in vivo confocal microscopy assessment in 12 patients with ocular cicatricial pemphigoid

    PubMed Central

    Long, Qin; Zuo, Ya-Gang; Yang, Xue; Gao, Ting-Ting; Liu, Jie; Li, Ying

    2016-01-01

    AIM To describe the clinical features and microstructural characteristics assessed by in vivo confocal microscopy (IVCM) in patients with ocular cicatricial pemphigoid (OCP). METHODS A descriptive, uncontrolled case series study. Patients diagnosed with OCP were examined by clinical history, slit-lamp biomicroscopy features and IVCM images. The results of direct immunofluorescence (DIF) biopsies and indirect immunofluorescence (IIF) were also recorded. Local and systemic immunosuppressive therapy were administered and adjusted according to response. RESULTS A total of 12 consecutive OCP patients (7 male, 5 female; mean age 60.42±10.39y) were recruited. All patients exhibited bilateral progressive conjunctival scarring and recurrent chronic conjunctivitis was the most frequent clinical pattern. The mean duration of symptoms prior to diagnosis of OCP was 2.95±2.85y (range: 5mo to 10y). The Foster classification varied from stage I to IV and 20 eyes (83%) were within or greater than Foster stage III on presentation. Two of the 12 patients (17%) demonstrated positive DIF; 3 of the 12 (25%) patients reported positive IIF. The mean duration of the follow-up period was 20.17±11.88mo (range: 6 to 48mo). IVCM showed variable degrees of abnormality in the conjuctiva-cornea and conjuctival scarring was detected in all the involved eyes. Corneal stromal cell activation and dendritic cell infiltration presented as ocular surface inflammation, ocular surface keratinization along with the destroyed Vogt palisades was noted in eyes with potential limbal stem cell deficiency. After treatment, remission of ocular surface inflammation was achieved in all the patients, 18 eyes (75%) remained stable, 6 eyes (25%) had recurrent conjunctivitis and cicatrization in 2 eyes (8%) was progressing. CONCLUSION As an autoimmune disease, OCP manifests as variable degrees of clinical and laboratory abnormalities with both local and systemic immunosuppressive treatment playing important roles

  2. Reflectance confocal microscopy of oral epithelial tissue using an electrically tunable lens

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

    We present the use of a commercially available electrically tunable lens to achieve axial scanning in a reflectance confocal microscope. Over a 255 μm axial scan range, the lateral and axial resolutions varied from 1-2 μm and 4-14 μm, respectively, dependent on the variable focal length of the tunable lens. Confocal imaging was performed on normal human biopsies from the oral cavity ex vivo. Sub-cellular morphologic features were seen throughout the depth of the epithelium while axially scanning using the focus tunable lens.

  3. Resolution and signal-to-noise ratio improvement in confocal fluorescence microscopy using array detection and maximum-likelihood processing

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    Confocal fluorescence microscopy (CFM) is widely used in biological sciences because of its enhanced 3D resolution that allows image sectioning and removal of out-of-focus blur. This is achieved by rejection of the light outside a detection pinhole in a plane confocal with the illuminated object. In this paper, an alternative detection arrangement is examined in which the entire detection/image plane is recorded using an array detector rather than a pinhole detector. Using this recorded data an attempt is then made to recover the object from the whole set of recorded photon array data; in this paper maximum-likelihood estimation has been applied. The recovered object estimates are shown (through computer simulation) to have good resolution, image sectioning and signal-to-noise ratio compared with conventional pinhole CFM images.

  4. Spontaneous confocal Raman microscopy--a tool to study the uptake of nanoparticles and carbon nanotubes into cells

    NASA Astrophysics Data System (ADS)

    Romero, Gabriela; Rojas, Elena; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio Enrique

    2011-06-01

    Confocal Raman microscopy as a label-free technique was applied to study the uptake and internalization of poly(lactide- co-glycolide) (PLGA) nanoparticles (NPs) and carbon nanotubes (CNTs) into hepatocarcinoma human HepG2 cells. Spontaneous confocal Raman spectra was recorded from the cells exposed to oxidized CNTs and to PLGA NPs. The Raman spectra showed bands arising from the cellular environment: lipids, proteins, nucleic acids, as well as bands characteristic for either PLGA NPs or CNTs. The simultaneous generation of Raman bands from the cell and nanomaterials from the same spot proves internalization, and also indicates the cellular region, where the nanomaterial is located. For PLGA NPs, it was found that they preferentially co-localized with lipid bodies, while the oxidized CNTs are located in the cytoplasm.

  5. Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

    EPA Science Inventory

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

  6. Non destructive analysis of the wax layer of apple (Malus domestica Borkh.) by means of confocal laser scanning microscopy.

    PubMed

    Veraverbeke, E A; Van Bruaene, N; Van Oostveldt, P; Nicolaï, B M

    2001-08-01

    Confocal laser scanning microscopy (CLSM) was used to non-destructively analyse the changes in the structure and thickness of the cuticle during storage of apples (Malus domestica Borkh.). Interpretation of the confocal images was performed by comparison with scanning electron microscopy and environmental scanning electron microscopy images. The natural reflectance of the wax and the auto-fluorescence of the underlying cells made it possible with CLSM to distinguish the wax from the underlying layers without any pretreatment of the fruit. The thickness of the consecutive layers (wax, cutin, cells) could be estimated from measurements of the reflection and fluorescence intensities as a function of the number of pixels. The mean wax-layer thickness measured in this way amounted to 2.58 microm, 3.41 microm or 4.14 microm for the cultivars Jonagold, Jonagored and Elstar, respectively. Changes in the wax structure and cells of the same important Belgian apple cultivars as mentioned above were monitored during nine months of storage in ultra low oxygen and after exposure to ambient conditions. The changes in the wax ultrastructure and cell morphology are likely related to water losses and specific protection of the apple cultivars against water losses during storage and shelf life.

  7. Latest advances in confocal microscopy of skin cancers toward guiding patient care: a Mohs surgeon's review and perspective (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nehal, Kishwer S.; Rajadhyaksha, Milind

    2016-02-01

    Latest advances in confocal microscopy of skin cancers toward guiding patient care: a Mohs surgeon's review and perspective About 350 publications worldwide have reported the ability of reflectance confocal microscopy (RCM) imaging to detect melanocytic skin lesions in vivo with specificity of 84-88% and sensitivity of 71-92%, and non-melanocytic skin lesions with specificity of 85-97% and sensitivity 100-92%. Lentigo maligna melanoma can be detected with sensitivity of 93% and specificity 82%. While the sensitivity is comparable to that of dermoscopy, the specificity is 2X superior, especially for lightly- and non-pigmented lesions. Dermoscopy combined with RCM imaging is proving to be both highly sensitive and highly specific. Recent studies have reported that the ratio of equivocal (i.e., would have been biopsied) lesions to detected melanomas dropped by ~2X when guided by dermoscopy and RCM imaging, compared to that with dermoscopy alone. Dermoscopy combined with RCM imaging is now being implemented to guide noninvasive diagnosis (to rule out malignancy and biopsy) and to also guide treatment, with promising initial impact: thus far, about 3,000 patients have been saved from biopsies of benign lesions. These are currently under follow-up monitoring. With fluorescence confocal microscopy (FCM) mosaicing, residual basal cell carcinomas can be detected in Mohs surgically excised fresh tissue ex vivo, with sensitivity of 94-97% and specificity 89-94%. FCM mosaicing is now being implemented for guiding Mohs surgery. To date, about 600 Mohs procedures have been performed, guided with mosaicing, and with pathology being performed in parallel to confirm the final outcome. These latest advances demonstrate the promising ability of RCM and FCM to guide patient care.

  8. The Inferior Whorl For Detecting Diabetic Peripheral Neuropathy Using Corneal Confocal Microscopy

    PubMed Central

    Petropoulos, Ioannis N.; Ferdousi, Maryam; Marshall, Andrew; Alam, Uazman; Ponirakis, Georgios; Azmi, Shazli; Fadavi, Hassan; Efron, Nathan; Tavakoli, Mitra; Malik, Rayaz A.

    2015-01-01

    Purpose. In vivo corneal confocal microscopy (CCM) is increasingly used as a surrogate endpoint in studies of diabetic polyneuropathy (DPN). However, it is not clear whether imaging the central cornea provides optimal diagnostic utility for DPN. Therefore, we compared nerve morphology in the central cornea and the inferior whorl, a more distal and densely innervated area located inferior and nasal to the central cornea. Methods. A total of 53 subjects with type 1/type 2 diabetes and 15 age-matched control subjects underwent detailed assessment of neuropathic symptoms (NPS), deficits (neuropathy disability score [NDS]), quantitative sensory testing (vibration perception threshold [VPT], cold and warm threshold [CT/WT], and cold- and heat-induced pain [CIP/HIP]), and electrophysiology (sural and peroneal nerve conduction velocity [SSNCV/PMNCV], and sural and peroneal nerve amplitude [SSNA/PMNA]) to diagnose patients with (DPN+) and without (DPN−) neuropathy. Corneal nerve fiber density (CNFD) and length (CNFL) in the central cornea, and inferior whorl length (IWL) were quantified. Results. Comparing control subjects to DPN− and DPN+ patients, there was a significant increase in NDS (0 vs. 2.6 ± 2.3 vs. 3.3 ± 2.7, P < 0.01), VPT (V; 5.4 ± 3.0 vs. 10.6 ± 10.3 vs. 17.7 ± 11.8, P < 0.01), WT (°C; 37.7 ± 3.5 vs. 39.1 ± 5.1 vs. 41.7 ± 4.7, P < 0.05), and a significant decrease in SSNCV (m/s; 50.2 ± 5.4 vs. 48.4 ± 5.0 vs. 39.5 ± 10.6, P < 0.05), CNFD (fibers/mm2; 37.8 ± 4.9 vs. 29.7 ± 7.7 vs. 27.1 ± 9.9, P < 0.01), CNFL (mm/mm2; 27.5 ± 3.6 vs. 24.4 ± 7.8 vs. 20.7 ± 7.1, P < 0.01), and IWL (mm/mm2; 35.1 ± 6.5 vs. 26.2 ± 10.5 vs. 23.6 ± 11.4, P < 0.05). For the diagnosis of DPN, CNFD, CNFL, and IWL achieved an area under the curve (AUC) of 0.75, 0.74, and 0.70, respectively, and a combination of IWL-CNFD achieved an AUC of 0.76. Conclusions. The parameters of CNFD, CNFL, and IWL have a comparable ability to diagnose patients with DPN. However, IWL

  9. Detection of latent fingerprints using high-resolution 3D confocal microscopy in non-planar acquisition scenarios

    NASA Astrophysics Data System (ADS)

    Kirst, Stefan; Vielhauer, Claus

    2015-03-01

    In digitized forensics the support of investigators in any manner is one of the main goals. Using conservative lifting methods, the detection of traces is done manually. For non-destructive contactless methods, the necessity for detecting traces is obvious for further biometric analysis. High resolutional 3D confocal laser scanning microscopy (CLSM) grants the possibility for a detection by segmentation approach with improved detection results. Optimal scan results with CLSM are achieved on surfaces orthogonal to the sensor, which is not always possible due to environmental circumstances or the surface's shape. This introduces additional noise, outliers and a lack of contrast, making a detection of traces even harder. Prior work showed the possibility of determining angle-independent classification models for the detection of latent fingerprints (LFP). Enhancing this approach, we introduce a larger feature space containing a variety of statistical-, roughness-, color-, edge-directivity-, histogram-, Gabor-, gradient- and Tamura features based on raw data and gray-level co-occurrence matrices (GLCM) using high resolutional data. Our test set consists of eight different surfaces for the detection of LFP in four different acquisition angles with a total of 1920 single scans. For each surface and angles in steps of 10, we capture samples from five donors to introduce variance by a variety of sweat compositions and application influences such as pressure or differences in ridge thickness. By analyzing the present test set with our approach, we intend to determine angle- and substrate-dependent classification models to determine optimal surface specific acquisition setups and also classification models for a general detection purpose for both, angles and substrates. The results on overall models with classification rates up to 75.15% (kappa 0.50) already show a positive tendency regarding the usability of the proposed methods for LFP detection on varying surfaces in non

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

    PubMed

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

    2014-01-01

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

  11. Aquagenic wrinkling of the palms: morphological changes in reflectance confocal microscopy and high-definition optical coherence tomography.

    PubMed

    Bielicky, Lea; Braun-Falco, Markus; Ruzicka, Thomas; Maier, Tanja

    2015-01-01

    Aquagenic wrinkling of the palms (AWP) is a rare condition, which is characterized by appearance of whitish papules and plaques, and an excessive wrinkling and swelling of the palmar skin after exposure to water. In most cases, young women are affected, and an association of AWP with cystic fibrosis (CF) has been surmised. We report on two cases of AWP, which were not related to CF, in whom we used two innovative imaging techniques, namely high-definition optical coherence tomography and reflectance confocal microscopy, to show in vivo skin changes occurring after exposure of the skin to tap water in comparison to the findings in a healthy control person.

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

    NASA Astrophysics Data System (ADS)

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

    2007-03-01

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

  13. 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

  14. 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.

  15. A machine learning method for identifying morphological patterns in reflectance confocal microscopy mosaics of melanocytic skin lesions in-vivo

    NASA Astrophysics Data System (ADS)

    Kose, Kivanc; Alessi-Fox, Christi; Gill, Melissa; Dy, Jennifer G.; Brooks, Dana H.; Rajadhyaksha, Milind

    2016-02-01

    We present a machine learning algorithm that can imitate the clinicians qualitative and visual process of analyzing reflectance confocal microscopy (RCM) mosaics at the dermal epidermal junction (DEJ) of skin. We divide the mosaics into localized areas of processing, and capture the textural appearance of each area using dense Speeded Up Robust Feature (SURF). Using these features, we train a support vector machine (SVM) classifier that can distinguish between meshwork, ring, clod, aspecific and background patterns in benign conditions and melanomas. Preliminary results on 20 RCM mosaics labeled by expert readers show classification with 55 - 81% sensitivity and 81 - 89% specificity in distinguishing these patterns.

  16. Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance

    NASA Astrophysics Data System (ADS)

    Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; Dimarzio, Charles; Rajadhyaksha, Milind

    2011-07-01

    Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology.

  17. Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance.

    PubMed

    Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; DiMarzio, Charles; Rajadhyaksha, Milind

    2011-07-01

    Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology.

  18. Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence.

    PubMed

    Beilharz, Michael; De Nardo's, Dominic; Latz, Eicke; Franklin, Bernardo S

    2016-01-01

    Inflammasome assembly results in the formation of a large intracellular protein scaffold driven by the oligomerization of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). Following inflammasome activation, ASC polymerizes to form a large singular structure termed the ASC "speck," which is crucial for recruitment of caspase-1 and its inflammatory activity. Hence, due to the considerably large size of these structures, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide two detailed protocols for imaging ASC specks: by (1) live-cell imaging of monocyte/macrophage cell lines expressing a fluorescently tagged version of ASC and (2) immunofluorescence of endogenous ASC in cell lines and human immune cells. In addition, we outline a protocol for increasing the specificity of ASC antibodies for use in immunofluorescence. PMID:27221487

  19. 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.

  20. Three dimensional confocal microscopy of fluorescent microspheres: imaging and size determination

    NASA Astrophysics Data System (ADS)

    Rakovich, Y. P.; Yang, L.; Taylor, C. M.; Dunbar, L. A.; Mac Raighne, Aaron; Donegan, J. F.; McCabe, Eithne M.

    2005-06-01

    In this paper, we experimentally studied both the bright-field and fluorescence images of microspheres by conventional and confocal scanning polarization microscopes. A qualitative analysis have been given to show a physical picture on the imaging of the microspheres. Emission spectra from melamine formaldehyde microspheres stained with Ethidium Bromide or covered by thin shell of CdTe nanocrystals have been experimentally studied. We adopted analytical expressions describing the resonance spacing in order to determine the size of the microspheres.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. Confocal Microscopy and Image Analysis Indicates a Region-Specific Relation between Active Caspases and Cytoplasm in Ejaculated and Epididymal Sperm

    PubMed Central

    García Vazquez, Susana; Aragón Martínez, Andrés; Flores-Alonso, Juan Carlos

    2012-01-01

    Previously, it was suggested a relation between the presence of apoptosis markers with cytoplasm in mammalian sperm. In this work, flow cytometry, confocal microscopy and image analysis were used to analyze the relationship between active caspase-3 and -7 and intracellular esterases expression in ejaculated or epididymal ram sperm. Sperm obtained from ejaculates from the caput, corpus, or cauda of the epididymis were treated with an inhibitor of active caspase-3 and -7 and a marker of cytoplasmic esterases. Additionally, ejaculated sperm were incubated for one, two, or three hours before evaluation for active caspases. Sperm subpopulations positive for active caspases and/or intracellular esterases were detected by flow cytometry and confocal microscopy; however, image analysis of confocal images showed that the correlation between active caspases and cytoplasmic esterases in sperm is region-specific. Lower values of Spearman correlation coefficients were found when whole sperm or head sperm was analyzed; however, a high correlation was observed for midpiece sperm. Incubation of sperm for two or three hours promoted the autoactivation of caspases. It has been suggested that the presence of apoptotic markers in sperm are related with a process of abortive apoptosis and with errors during spermiogenesis. Our results permit us suggest that the origin of the relationship between active caspases and cytoplasmic esterases is due to differentiation errors occurring during spermiogenesis because the percentages of sperm with active caspases are not different in the caput, corpus, or cauda of the epididymis. In this study we demonstrate that existing sperm subpopulations can express active caspases and intracellular esterases and that the correlation between these molecules is high in midpiece sperm. PMID:22530029

  4. 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.

  5. Evidence for ciliary pigment localization in colored ciliates and implications for their photosensory transduction chain: a confocal microscopy study.

    PubMed

    Colombetti, Giuliano; Checcucci, Giovanni; Lucia, Sabina; Usai, Cesare; Ramoino, Paola; Bianchini, Paolo; Pesce, Mattia; Vicidomini, Giuseppe; Diaspro, Alberto

    2007-12-01

    In this study we report for the first time the localization of a photoreceptor pigment in the cilia of the colored heterotrich ciliates Blepharisma japonicum red and blue form, Fabrea salina, and Stentor coeruleus, as result of a confocal microscopy investigation. Optical sectioning confocal microscopy has been used for studying the spatial distribution of the pigment in the cell body, surprisingly showing that, besides its expected presence in the cortical region immediately below the cell membrane, it is located in the cilia too. In order to ascertain possible differences in the pigment fluorescence properties along the cell body, we have measured emission spectra from different parts of it (anterior, posterior, and cilia). Our results clearly indicate that in all cases the spectra are the same, within experimental errors. Finally, we have evaluated the pigment relative fluorescence efficiency of these ciliates. In an ordered scale from lower to greater efficiency, we have S. coeruleus, B. japonicum blue, B. japonicum red, and F. salina. The possible implications of our findings for the process of photosensory transduction are discussed. PMID:17661390

  6. Characterization of Pad-Wafer Contact and Surface Topography in Chemical Mechanical Planarization Using Laser Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Sun, Ting; Zhuang, Yun; Borucki, Leonard; Philipossian, Ara

    2010-06-01

    In this study, an optical method using laser confocal microscopy was developed to measure the surface contact area and topography of pads under a dry and static condition. A custom-made pad sample holder with a sapphire window and a miniature load cell was used to collect pad surface contact images at controlled loads. By extracting the black spots in the collected images, pad contact area and contact summit density were obtained. The analysis of a post polishing pad sample (8,289×921 µm2) showed that the contact area increased from 0.026 to 0.045% when the pressure increased from 2 to 4 psi and increased further to 0.059% when the pressure increased to 6 psi. The contact summit density also exhibited a linear increase with the applied pressure. The above results were consistent with the Greenwood and Williamson theory, which predicted a linear relationship between pad contact area and contact summit density. Laser confocal microscopy was also used to measure pad surface topography by establishing probability density functions (PDFs) of pad surface height.

  7. Characterization of Pad-Wafer Contact and Surface Topography in Chemical Mechanical Planarization Using Laser Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Ting Sun,; Yun Zhuang,; Leonard Borucki,; Ara Philipossian,

    2010-06-01

    In this study, an optical method using laser confocal microscopy was developed to measure the surface contact area and topography of pads under a dry and static condition. A custom-made pad sample holder with a sapphire window and a miniature load cell was used to collect pad surface contact images at controlled loads. By extracting the black spots in the collected images, pad contact area and contact summit density were obtained. The analysis of a post polishing pad sample (8{,}289× 921 μm2) showed that the contact area increased from 0.026 to 0.045% when the pressure increased from 2 to 4 psi and increased further to 0.059% when the pressure increased to 6 psi. The contact summit density also exhibited a linear increase with the applied pressure. The above results were consistent with the Greenwood and Williamson theory, which predicted a linear relationship between pad contact area and contact summit density. Laser confocal microscopy was also used to measure pad surface topography by establishing probability density functions (PDFs) of pad surface height.

  8. Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells

    NASA Astrophysics Data System (ADS)

    Rehman, Shagufta; Brown-Steinke, Kathleen; Palmer, Lisa; Periasamy, Ammasi

    2015-03-01

    Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.

  9. Effects of Long-Term Antiglaucoma Eye Drops on Conjunctival Structures: An In Vivo Confocal Microscopy Study

    PubMed Central

    Zhu, Wenqing; Kong, Xiangmei; Xu, Jianjiang; Sun, Xinghuai

    2015-01-01

    Purpose. The study was aimed at comparing the long-term effects of different antiglaucoma eye drops on conjunctival structures using laser scanning confocal microscopy. Methods. Eighty patients diagnosed with primary open-angle glaucoma and twenty healthy volunteers were included in this study. The participants were divided into 5 groups according to the different medications. The lachrymal film break-up time, Schirmer's I test, and Ocular Surface Disease Index Questionnaire were performed in all subjects. The confocal microscopy was used to observe the basal epithelial cell density (ECD), goblet cell density (GCD), dendritic cell density (DCD), and subepithelial collagen fiber diameter (SFD). Results. Statistically significant differences were found among the control group and the antiglaucoma therapy groups in the values of three clinical data (P < 0.05). The GCD, DCD, and SFD showed significant differences in all glaucoma groups when compared to the control (P < 0.001). Moreover, the prostaglandin group differed from the other antiglaucoma therapy groups in the GCD and SFD (P < 0.05). Conclusions. Our study confirmed the significant differences in the conjunctival structures based on the effects of antiglaucoma medications. Less pronounced changes were found in the patients treated with prostaglandin analogue than in the other kinds of antiglaucoma therapies. PMID:26171239

  10. Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

    NASA Astrophysics Data System (ADS)

    Mitra, Debasis; Boutchko, Rostyslav; Ray, Judhajeet; Nilsen-Hamilton, Marit

    2015-03-01

    In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.

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

    PubMed Central

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

    2016-01-01

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

  12. Effects of Long-Term Antiglaucoma Eye Drops on Conjunctival Structures: An In Vivo Confocal Microscopy Study.

    PubMed

    Zhu, Wenqing; Kong, Xiangmei; Xu, Jianjiang; Sun, Xinghuai

    2015-01-01

    Purpose. The study was aimed at comparing the long-term effects of different antiglaucoma eye drops on conjunctival structures using laser scanning confocal microscopy. Methods. Eighty patients diagnosed with primary open-angle glaucoma and twenty healthy volunteers were included in this study. The participants were divided into 5 groups according to the different medications. The lachrymal film break-up time, Schirmer's I test, and Ocular Surface Disease Index Questionnaire were performed in all subjects. The confocal microscopy was used to observe the basal epithelial cell density (ECD), goblet cell density (GCD), dendritic cell density (DCD), and subepithelial collagen fiber diameter (SFD). Results. Statistically significant differences were found among the control group and the antiglaucoma therapy groups in the values of three clinical data (P < 0.05). The GCD, DCD, and SFD showed significant differences in all glaucoma groups when compared to the control (P < 0.001). Moreover, the prostaglandin group differed from the other antiglaucoma therapy groups in the GCD and SFD (P < 0.05). Conclusions. Our study confirmed the significant differences in the conjunctival structures based on the effects of antiglaucoma medications. Less pronounced changes were found in the patients treated with prostaglandin analogue than in the other kinds of antiglaucoma therapies.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  14. Optical monitoring of oxygen tension in cortical microvessels with confocal microscopy

    PubMed Central

    Yaseen, Mohammad A.; Srinivasan, Vivek J.; Sakadžić, Sava; Wu, Weicheng; Ruvinskaya, Svetlana; Vinogradov, Sergei A.; Boas, DavidA

    2010-01-01

    Evaluating cerebral oxygenation is of critical importance for the understanding of brain function and several neuropathologies. Although several techniques exist for measuring cerebral oxygenation in vivo, the most widely accepted techniques offer limited spatial resolution. We have developed a confocal imaging system for minimally invasive measurement of oxygen tension (pO2) in cerebral microvessels with high spatial and temporal resolution. The system relies on the phosphorescence quenching method using exogenous porphyrin-based dendritic oxygen probes. Here we present high-resolution phosphorescence images of cortical microvasculature and temporal pO2 profiles from multiple locations in response to varied fraction of inspired oxygen and functional activation. PMID:20052157

  15. Image analysis of modified Hamming aperture: application on confocal microscopy and holography

    NASA Astrophysics Data System (ADS)

    Hamed, A. M.; Alsaeed, T. A.

    2015-06-01

    Digital truncated and obstructed Hamming apertures are used in confocal laser scanning microscope (CLSM) to improve the resolution. The resultant point spread function (RPSF) and the coherent transfer function of the defined microscope, using the modified Hamming apertures, are computed. The mammographic images are used in the CLSM to test resolution of the investigated apertures. The image is computed from the modulus square of the convolution product of the RPSF and the complex amplitude of the object. A comparison with the images obtained in case of circular uniform apertures is investigated. Another application on holography allows the recognition of the modulated Hamming apertures.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

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

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

    PubMed

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

    2000-01-01

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

  19. Optical coherence tomography and confocal microscopy investigations of dental structures and restoration materials

    NASA Astrophysics Data System (ADS)

    Negrutiu, Meda L.; Sinescu, Cosmin; Rominu, Mihai; Hughes, Michael; Dobre, George; Podoleanu, Adrian G.

    2009-02-01

    Nowadays, optical tomographic techniques are of particular importance in the medical imaging field, because these techniques can provide non-invasive diagnostic images. The present study evaluates the potential of en-face optical coherence tomography (OCT) as a possible non-invasive high resolution imaging method in supplying the necessary information on the quality of dental hard tissues and defects of dental restorative materials. Teeth after several treatment methods are imaged in order to asses the material defects and micro-leakage of tooth-filling interface as well as to evaluate the quality of dental hard tissue. C-scan and B-scan OCT images as well as confocal images are acquired from a large range of samples. Cracks and voids in the dental structures as well as gaps between the dental interfaces and material defects are clearly exposed. The advantages of the OCT method consist in non-invasiveness and high resolution.

  20. 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.

  1. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-12-01

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

  4. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    SciTech Connect

    Al-Gubory, Kais H. . E-mail: kais.algubory@jouy.inra.fr

    2005-11-01

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  6. Velocity profile of thin film flows measured using a confocal microscopy particle image velocimetry system with simultaneous multi depth position

    NASA Astrophysics Data System (ADS)

    Kikuchi, K.; Mochizuki, O.

    2015-02-01

    In this paper, we report a technique for simultaneously visualizing flows near walls at nano-depth positions. To achieve such a high interval of depth gradient, we developed a tilted observation technique in a particle image velocimetry (PIV) system based on confocal microscopy. The focal plane along the bottom of the flow channel was tilted by tilting the micro-channel, enabling depth scanning in the microscopic field of view. Our system is suitable for measuring 3D two-component flow fields. The depth interval was approximately 220 nm over a depth range of 10 μm, depending on the tilt angle of the micro-channel. Applying the proposed system, we visualized the near-wall flow in a drainage film flow under laminar conditions to the depth of approximately 30 μm via vertical scanning from the bottom to the free surface. The velocity gradient was proportional to the distance from the wall, consistent with theoretical predictions. From the measured near-wall velocity gradient, we calculated the wall shear stress. The measurement accuracy was approximately 1.3 times higher in our proposed method than in the conventional confocal micro-PIV method.

  7. In vivo confocal microscopy of the sclerocorneal limbus after limbal stem cell transplantation: Looking for limbal architecture modifications and cytological phenotype correlations

    PubMed Central

    Mastropasqua, Leonardo; Lanzini, Manuela; Nubile, Mario; Colabelli-Gisoldi, Rossella Annamaria; De Carlo, Luca; Pocobelli, Augusto

    2016-01-01

    Purpose To correlate a biomicroscopic evaluation, an in vivo confocal microscopy examination, and impression cytologic findings of the corneal center and sclerocorneal limbus after cultured limbal stem cell transplantation and to test the effectiveness of in vivo confocal microscopy as a diagnostic procedure in ocular surface cell therapy reconstructive surgery. Methods Six eyes of six patients affected by limbal stem cell deficiency after chemical burns underwent ex vivo expanded limbal stem cell transplantation (two eyes) and ex vivo expanded limbal stem cell transplantation with subsequent penetrating keratoplasty (four eyes) to restore corneal transparency. One year after surgery, all patients underwent a biomicroscopic evaluation, central cornea impression cytology to detect cytokeratin 12 (CK12) positivity, and in vivo confocal microscopy of the central cornea and the sclerocorneal limbus to investigate the epithelial cellular morphology, limbal architecture, and corneal inflammation level. Results Impression cytology analysis showed CK12 positivity in five of six cases, in concordance with the biomicroscopic evaluation. Confocal microscopy pointed out irregular limbal architecture with the absence of the palisades of Vogt in all cases; the central epithelial morphology presented clear corneal characteristics in three cases and irregular morphology in the remaining three. Conclusions After successful ex vivo expanded limbal stem cell transplantation, in the presence of a complete anatomic architecture subversion, documented by support of in vivo confocal microscopy, the sclerocorneal limbus seemed to maintain its primary function. In vivo confocal microscopy confirmed the procedure was a non-invasive, efficacious diagnostic ocular surface procedure in the case of cell therapy reconstructive surgery. PMID:27440993

  8. Assessment of possibilities of ceramic biomaterial fracture surface reconstruction using laser confocal microscopy and long working distance objective lenses.

    PubMed

    Stach, Sebastian; Sapota, Wiktoria; Wróbel, Zygmunt; Ţălu, Ştefan

    2016-05-01

    A numerical description of fracture is an important step in the search of the correlation between specific micromechanisms of decohesion and material characteristics designated with the use of fracture mechanics methods. This issue is essential for the proper orientation of the search for basic relationships between chemical composition, technology, structure, and properties of materials. It often happens that fracture surfaces are well developed, which can significantly hinder or even prevent the measurement and reconstruction of the tested material surface geometry. In this article, comparative measurements of a biomaterial surface were performed using laser confocal microscopy. To this end, short working distance lenses dedicated to a focused UV laser beam and long working distance objective lenses were used. The article includes a quantitative comparative analysis and interpretation of the obtained results.

  9. Interaction of maize zein with wheat gluten in composite dough and bread as determined by confocal laser scanning microscopy.

    PubMed

    Bugusu, Betry A; Rajwa, Bartlomiej; Hamaker, Bruce R

    2002-01-01

    Protein body-free maize zein, when mixed at 35 degrees C (above its glass transition temperature range), significantly (p < 0.01) improved the rheological and leavening properties of sorghum-wheat composite flour dough, resulting in improved loaf volume. Confocal laser scanning microscopy was used to observe the structure of zein fibrils and the interaction between zein and gluten proteins in the composite dough and bread systems. Autofluorescence and immunolocalization techniques were used to locate gluten and zein, respectively. Optical sections were collected every 0.4 microm through the samples and digitally processed to produce reconstructed three-dimensional images. Results showed that zein fibrils form an outer layer that intermittently coats the gluten networks, thereby strengthening them. This type of microstructure is able to withstand the pressure exerted by gas cell expansion during yeast fermentation to increase loaf volume.

  10. Analysis of micro-structural relaxation phenomena in laser-modified fused silica using confocal Raman microscopy

    SciTech Connect

    Matthews, M; Vignes, R; Cooke, J; Yang, S; Stolken, J

    2009-12-15

    Fused silica micro-structural changes associated with localized 10.6 {micro}m CO{sub 2} laser heating are reported. Spatially-resolved shifts in the high-frequency asymmetric stretch transverse-optic (TO) phonon mode of SiO{sub 2} were measured using confocal Raman microscopy, allowing construction of axial fictive temperature (T{sub f}) maps for various laser heating conditions. A Fourier conduction-based finite element model was employed to compute on-axis temperature-time histories, and, in conjunction with a Tool-Narayanaswamy form for structural relaxation, used to fit T{sub f}(z) profiles to extract relaxation parameters. Good agreement between the calculated and measured T{sub f} was found, yielding reasonable values for relaxation time and activation enthalpy in the laser-modified silica.

  11. Studying DEHP migration in plasticized PVC used for blood bags by coupling Raman confocal microscopy to UV spectroscopy.

    PubMed

    Al Salloum, H; Saunier, J; Tfayli, A; Yagoubi, N

    2016-04-01

    Plasticized PVC is widely used to make medical devices such as tubing, perfusion bags and blood bags. By using confocal Raman microscopy on a PVC sheet plasticized with around 40% of di-(2-ethylhexyl)phthalate (DEHP), we propose a simple and sensitive approach to studying and understanding the diffusion of plasticizers from polymers into the surrounding media. Moreover, we sought to correlate our findings to standard measurements conducted by UV spectroscopy. This study showed differences in the concentration gradient observed due to the diffusion of the plasticizer inside a PVC sheet. We can thus follow the critical DEHP ratios that can impact the diffusion process. Water and ethanol were chosen as storage media: in ethanol, the lowest concentration of DEHP was observed at the surface resulting in the formation of a less plasticized layer near the interface; unlike ethanol, PVC sheets stored in water showed a greater concentration of DEHP on the film surface as an exudation of DEHP onto the surface.

  12. Micro- and nanodomain imaging in uniaxial ferroelectrics: Joint application of optical, confocal Raman, and piezoelectric force microscopy

    SciTech Connect

    Shur, V. Ya. Zelenovskiy, P. S.

    2014-08-14

    The application of the most effective methods of the domain visualization in model uniaxial ferroelectrics of lithium niobate (LN) and lithium tantalate (LT) family, and relaxor strontium-barium niobate (SBN) have been reviewed in this paper. We have demonstrated the synergetic effect of joint usage of optical, confocal Raman, and piezoelectric force microscopies which provide extracting of the unique information about formation of the micro- and nanodomain structures. The methods have been applied for investigation of various types of domain structures with increasing complexity: (1) periodical domain structure in LN and LT, (2) nanodomain structures in LN, LT, and SBN, (3) nanodomain structures in LN with modified surface layer, (4) dendrite domain structure in LN. The self-assembled appearance of quasi-regular nanodomain structures in highly non-equilibrium switching conditions has been considered.

  13. Intracellular localization analysis of npAu-PpIX in HeLa cells using specific dyes and confocal microscopy

    NASA Astrophysics Data System (ADS)

    Roblero-Bartolón, Victoria Gabriela; Maldonado-Alvarado, Elizabeth; Galván-Mendoza, José Iván; Ramón-Gallegos, Eva

    2012-10-01

    Cervical carcinoma (CC) represents the second leading cause of cancer death in Mexican women. No conventional treatments are being developed such as photodynamic therapy (PDT), involving the simultaneous presence of a photosensitizer (Ps), light of a specific wavelength and tissue oxygen. On the other hand, it has seen that the use of gold nanoparticles coupled to protoporphyrin IX increases the effectiveness of PDT. The aim of this study was to determine the site of accumulation of the conjugate npAu-PpIX in cells of cervical cancer by the use of specific dyes and confocal microscopy. The results indicate that the gold nanoparticles coupled to protoporphyrin IX are accumulated in both the cytoplasm and nucleus of HeLa cells.

  14. In Vivo Confocal Microscopy of the Human Cornea in the Assessment of Peripheral Neuropathy and Systemic Diseases

    PubMed Central

    Wang, Ellen F.; Misra, Stuti L.; Patel, Dipika V.

    2015-01-01

    In vivo confocal microscopy (IVCM) of the living human cornea offers the ability to perform repeated imaging without tissue damage. Studies using corneal IVCM have led to significant contributions to scientific and clinical knowledge of the living cornea in health and pathological states. Recently the application of corneal IVCM beyond ophthalmology to wider clinical and research fields has been demonstrated. Abnormalities of the corneal subbasal nerve plexus have been associated with many forms of peripheral neuropathy and Langerhans cells correlate with systemic inflammatory states. There is a rapidly growing evidence base investigating the use of corneal IVCM in many systemic conditions and a well-established evidence base for IVCM imaging of the corneal subbasal plexus in diabetic peripheral neuropathy. This paper reviews the potential use of corneal IVCM in general clinical practice as a noninvasive method of assessing peripheral neuropathies, monitoring inflammatory states and clinical therapeutic response. PMID:26770980

  15. Advantages of microsphere-assisted super-resolution imaging technique over solid immersion lens and confocal microscopies

    NASA Astrophysics Data System (ADS)

    Darafsheh, Arash; Limberopoulos, Nicholaos I.; Derov, John S.; Walker, Dennis E.; Astratov, Vasily N.

    2014-02-01

    We demonstrate a series of advantages of microsphere-assisted imaging over confocal and solid immersion lens microscopies including intrinsic flexibility, better resolution, higher magnification, and longer working distances. We discerned minimal feature sizes of ˜50-60 nm in nanoplasmonic arrays at the illumination wavelength λ = 405 nm. It is demonstrated that liquid-immersed, high-index (n ˜ 1.9-2.1) spheres provide a superior image quality compared to that obtained by spheres with the same index contrast in an air environment. We estimate that using transparent microspheres at deep UV wavelengths of ˜200 nm might make possible imaging of various nanostructures with extraordinary high ˜30 nm resolution.

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

    PubMed

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

    2013-09-01

    For the removal of superficial and nodular basal cell carcinomas (BCCs), laser ablation provides certain advantages relative to other treatment modalities. However, efficacy and reliability tend to be variable because tissue is vaporized such that none is available for subsequent histopathological examination for residual BCC (and to confirm complete removal of tumor). Intra-operative reflectance confocal microscopy (RCM) may provide a means to detect residual tumor directly on the patient and guide ablation. However, optimization of ablation parameters will be necessary to control collateral thermal damage and preserve sufficient viability in the underlying layer of tissue, so as to subsequently allow labeling of nuclear morphology with a contrast agent and imaging of residual BCC. We report the results of a preliminary study of two key parameters (fluence, number of passes) vis-à-vis the feasibility of labeling and RCM imaging in human skin ex vivo, following ablation with an erbium:yttrium aluminum garnet laser.

  17. An innovative approach for investigating the ceramic bracket-enamel interface - optical coherence tomography and confocal microscopy

    NASA Astrophysics Data System (ADS)

    Romînu, Roxana Otilia; Sinescu, Cosmin; Romînu, Mihai; Negrutiu, Meda; Laissue, Philippe; Mihali, Sorin; Cuc, Lavinia; Hughes, Michael; Bradu, Adrian; Podoleanu, Adrian

    2008-09-01

    Bonding has become a routine procedure in several dental specialties - from prosthodontics to conservative dentistry and even orthodontics. In many of these fields it is important to be able to investigate the bonded interfaces to assess their quality. All currently employed investigative methods are invasive, meaning that samples are destroyed in the testing procedure and cannot be used again. We have investigated the interface between human enamel and bonded ceramic brackets non-invasively, introducing a combination of new investigative methods - optical coherence tomography (OCT) and confocal microscopy (CM). Brackets were conventionally bonded on conditioned buccal surfaces of teeth The bonding was assessed using these methods. Three dimensional reconstructions of the detected material defects were developed using manual and semi-automatic segmentation. The results clearly prove that OCT and CM are useful in orthodontic bonding investigations.

  18. 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

  19. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    NASA Astrophysics Data System (ADS)

    Estrela-Lopis, I.; Romero, G.; Rojas, E.; Moya, S. E.; Donath, E.

    2011-07-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  20. Evaluation through in vivo reflectance confocal microscopy of the cutaneous neurogenic inflammatory reaction induced by capsaicin in human subjects

    NASA Astrophysics Data System (ADS)

    Căruntu, Constantin; Boda, Daniel

    2012-08-01

    We perform an in vivo analysis of the effects of capsaicin on cutaneous microvascularization. A total of 29 healthy subjects are administered a solution of capsaicin (CAP group) or a vehicle solution (nonCAP group) on the dorsal side of the nondominant hand. The evaluation is performed using in vivo reflectance confocal microscopy (RCM). Ten minutes after administration, the area of the section, the perimeter, and the Feret's diameter of the capillaries in the dermal papillae become significantly larger in the CAP group as against the nonCAP group, and this difference is maintained until the conclusion of the experiment. In vivo RCM allows the investigation of cutaneous vascular reactions induced by capsaicin. As such, this method may constitute an useful technique both for research and clinical practice.

  1. Reflectance Confocal Microscopy Features of Focal Dermal Mucinosis Differ from Those Described for Basal Cell Carcinoma: Report of Two Cases.

    PubMed

    Fraga-Braghiroli, Naiara Abreu; Merati, Miesha; Rabinovitz, Harold; Swanson, David; Scope, Alon

    2015-01-01

    The purpose of this study was to describe the reflectance confocal microscopy (RCM) features of focal dermal mucinosis (FDM). The entity clinically and dermatoscopically mimics other diagnostic entities, most notably nonpigmented basal cell carcinoma. We describe two cases that highlight the dermatoscopic, RCM and histopathological attributes of FDM. RCM features such as dermal foci of dense collagen bundles oriented in the same direction, foci of haphazardly oriented thin collagen fibers separated by dark structureless areas and the absence of dark silhouettes and tumor islands are clues for FDM diagnosis. The FDM cases described here present consistent and particular RCM findings that appear to correlate well with the histopathological features of FDM. Therefore, RCM is a promising technology in diagnosing skin lesions and it use can avoid invasive procedures. PMID:26302951

  2. In vivo quantitation of injected circulating tumor cells from great saphenous vein based on video-rate confocal microscopy

    PubMed Central

    Seo, Howon; Hwang, Yoonha; Choe, Kibaek; Kim, Pilhan

    2015-01-01

    The number of circulating tumor cell (CTC) in the peripheral blood of cancer patients can be a valuable biomarker for cancer diagnosis and treatment monitoring. In this study, we implemented a custom-design video-rate confocal microscopy system in capable of direct visualization of fast flowing CTC at great saphenous vein (GSV) of a live animal model in vivo. Continuous acquisition of video-rate images at GSV revealed the highly dynamic time-dependent changes in the number of intravenously injected circulating tumor cells. By extracting a calibration factor through the hemocytometric analysis of intravenously injected long-circulating red blood cells, we established a novel quantitation method for CTC in whole body blood in vivo. PMID:26114035

  3. In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell.

    PubMed

    Smith-Palmer, Truis; Lin, Sicheng; Oguejiofor, Ikenna; Leng, Tianyang; Pustam, Amanda; Yang, Jin; Graham, Lori L; Wyeth, Russell C; Bishop, Cory D; DeMont, M Edwin; Pink, David

    2016-02-01

    Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h. PMID:26903564

  4. In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell.

    PubMed

    Smith-Palmer, Truis; Lin, Sicheng; Oguejiofor, Ikenna; Leng, Tianyang; Pustam, Amanda; Yang, Jin; Graham, Lori L; Wyeth, Russell C; Bishop, Cory D; DeMont, M Edwin; Pink, David

    2016-02-01

    Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h.

  5. Confocal microscopy of epithelial and langerhans cells of the cornea in patients using travoprost drops containing two different preservatives.

    PubMed

    Marsovszky, László; Resch, Miklós D; Visontai, Zsuzsanna; Németh, János

    2014-07-01

    The recently developed confocal cornea microscopy offers the opportunity to examine pathologies of the cornea and to gain insight into the activity of innate immunity. We aimed to investigate the corneal epithelial and Langerhans cell (LC) densities along with dry eye parameters in primary open-angle glaucoma (POAG) subjects, treated with either of two commercially available travoprost 0.004 % topical medications containing different preservatives. (1: benzalkonium chloride 0.015 % (TravBAK) and 2: polyquaternium-1 (PQ) 0.001 % (TravPQ). Consecutive case series of nineteen POAG patients on TravBAK (mean age: 64.8 ± 13.6 years), nineteen POAG patients on TravPQ (mean age: 66.8 ± 11.3 years) and nineteen age-matched healthy control subjects (63.8 ± 8.2 years). Ocular surface disease index (OSDI), lid parallel conjunctival folds (LIPCOF), Schirmer test (ST) and tear break up time (TBUT) were assessed, and then corneal epithelial and LC densities were investigated with confocal microscopy. Tear production was significantly reduced in both glaucoma patient groups compared to healthy individuals (p < 0.05). TBUT was significantly reduced and epithelial cell densities were significantly greater in patients treated with TravBAK compared to healthy individuals (p < 0.05 for all). LC densities were greater in both glaucoma groups compared to control subjects (p < 0.05 for all). Travoprost therapy may compromise ocular surface. The limited alertness of the corneal immune system found in patients with TravPQ can be considered as indicators of a less disturbed ocular surface and better controlled corneal homeostasis.

  6. The nematode stoma: Homology of cell architecture with improved understanding by confocal microscopy of labeled cell boundaries.

    PubMed

    Jay Burr, A H; Baldwin, James G

    2016-09-01

    Nematode stomas vary widely in the cuticular structures evolved for different feeding strategies, yet the arrangement of the epithelial cell classes that form these structures may be conserved. This article addresses several issues that have impeded the full acceptance of this hypothesis including controversies arising from the structure of the Caenorhabditis elegans stoma. We investigated fluorescent antibody labeling of cell boundaries in conjunction with confocal microscopy as an alternative to transmission electron microscopy (TEM), using MH27 to label apical junctions in C. elegans and two other species. Accurately spaced optical sections collected by the confocal microscope provide a three-dimensional array of pixels (voxels) that, using image-processing software, can be rotated and sectioned at accurately chosen thicknesses and locations. Ribbons of fluorescence clearly identify cell boundaries along the luminal cuticle in C. elegans and Zeldia punctata and less clearly in Bunonema sp. The patterns render cell classes and their relationships readily identifiable. In the C. elegans stoma they correct a misreading of serial TEMs that was not congruent with architecture in other nematodes-the row of marginal cells is now seen to be continuous as in other nematodes, rather than being interrupted by encircling pm1 cells. Also impeding understanding, the reference to certain cell classes as 'epithelial' and others as "muscle" in the C. elegans literature is at variance with muscle expression in most other taxa. For consistent comparison among species, we propose that these cell class descriptors based on function be replaced by topological terms. With these and other confusing concepts and terminology removed, the homology of the cellular architecture among taxa becomes obvious. We provide a corrected description of the cell architecture of the C. elegans stoma and examples of how it is modified in other taxa with different feeding strategies. J. Morphol. 277

  7. In vivo comparative documentation of skin hydration by confocal Raman microscopy, SkinSensor, Skicon, and NovaMeter

    NASA Astrophysics Data System (ADS)

    Zhang, Guojin; Papillon, Aline; Ruvolo, Eduardo, Jr.; Bargo, Paulo R.; Kollias, Nikiforos

    2010-02-01

    The stratum corneum provides a vital physical barrier that protects against external insults and excessive internal water loss. Water activity is thought as a key factor to maintain proper skin barrier integrity via regulating enzyme activities and lipid phase behavior. Consequently, maintenance of an optimal hydration level in SC becomes an important clinical and cosmetic concern. The objective methods to assess SC hydration are based on either electrical or optical measurements. Electrical techniques used in the current study include high frequency conductance (Skicon), impedance (Nova DPM) and DC I-V curve (Skinsensor). Confocal Raman Microscopy was utilized to document water profile versus depth, and this technique is based on inelastic scattering of monochromatic light from different chemical species of skin. Water patches were applied on the 14 subjects' forearm for 20 minutes and 1.5 hrs. Skin hydration levels for individuals were documented by utilizing the mentioned above instruments in vivo. Results show that patterns of water profiles upon the hydration are significantly different among the individuals and these differences may be related to skin barrier function integrity. The intrinsic water content and water absorption upon the hydration were summed corresponding to different depths (3 μm and 15 μm) from the data obtained by confocal Raman microscopy. These results were correlated to the readings from electrical approaches. Superficial (3 μm) but not deeper layer (15 μm) water contents correlated well with the readings from SkinSensor. Neither depth measurements correlate well with the Skicon. There is strong correlation between the data acquired with Skicon and SkinSensor.

  8. Cost-effective approaches for high-resolution bioimaging by time-stretched confocal microscopy at 1μm

    NASA Astrophysics Data System (ADS)

    Wong, Terence T. W.; Qiu, Yi; Lau, Andy K. S.; Xu, JingJiang; Chan, Antony C. S.; Wong, Kenneth K. Y.; Tsia, Kevin K.

    2012-12-01

    Optical imaging based on time-stretch process has recently been proven as a powerful tool for delivering ultra-high frame rate (< 1MHz) which is not achievable by the conventional image sensors. Together with the capability of optical image amplification for overcoming the trade-off between detection sensitivity and speed, this new imaging modality is particularly valuable in high-throughput biomedical diagnostic practice, e.g. imaging flow cytometry. The ultra-high frame rate in time-stretch imaging is attained by two key enabling elements: dispersive fiber providing the time-stretch process via group-velocity-dispersion (GVD), and electronic digitizer. It is well-known that many biophotonic applications favor the spectral window of ~1μm. However, reasonably high GVD (< 0.1 ns/nm) in this range can only be achieved by using specialty single-mode fiber (SMF) at 1μm. Moreover, the ultrafast detection has to rely on the state-of- the-art digitizer with significantly wide-bandwidth and high sampling rate (e.g. <10 GHz, <40 GS/s). These stringent requirements imply the prohibitively high-cost of the system and hinder its practical use in biomedical diagnostics. We here demonstrate two cost-effective approaches for realizing time-stretch confocal microscopy at 1μm: (i) using the standard telecommunication SMF (e.g. SMF28) to act as a few-mode fiber (FMF) at 1μm for the time-stretch process, and (ii) implementing the pixel super-resolution (SR) algorithm to restore the high-resolution (HR) image when using a lower-bandwidth digitizer. By using a FMF (with a GVD of ~ 0.15ns/nm) and a modified pixel-SR algorithm, we can achieve time-stretch confocal microscopy at 1μm with cellular resolution (~ 3μm) at a frame rate 1 MHz.

  9. Confocal microscopy of epithelial and langerhans cells of the cornea in patients using travoprost drops containing two different preservatives.

    PubMed

    Marsovszky, László; Resch, Miklós D; Visontai, Zsuzsanna; Németh, János

    2014-07-01

    The recently developed confocal cornea microscopy offers the opportunity to examine pathologies of the cornea and to gain insight into the activity of innate immunity. We aimed to investigate the corneal epithelial and Langerhans cell (LC) densities along with dry eye parameters in primary open-angle glaucoma (POAG) subjects, treated with either of two commercially available travoprost 0.004 % topical medications containing different preservatives. (1: benzalkonium chloride 0.015 % (TravBAK) and 2: polyquaternium-1 (PQ) 0.001 % (TravPQ). Consecutive case series of nineteen POAG patients on TravBAK (mean age: 64.8 ± 13.6 years), nineteen POAG patients on TravPQ (mean age: 66.8 ± 11.3 years) and nineteen age-matched healthy control subjects (63.8 ± 8.2 years). Ocular surface disease index (OSDI), lid parallel conjunctival folds (LIPCOF), Schirmer test (ST) and tear break up time (TBUT) were assessed, and then corneal epithelial and LC densities were investigated with confocal microscopy. Tear production was significantly reduced in both glaucoma patient groups compared to healthy individuals (p < 0.05). TBUT was significantly reduced and epithelial cell densities were significantly greater in patients treated with TravBAK compared to healthy individuals (p < 0.05 for all). LC densities were greater in both glaucoma groups compared to control subjects (p < 0.05 for all). Travoprost therapy may compromise ocular surface. The limited alertness of the corneal immune system found in patients with TravPQ can be considered as indicators of a less disturbed ocular surface and better controlled corneal homeostasis. PMID:24623372

  10. The nematode stoma: Homology of cell architecture with improved understanding by confocal microscopy of labeled cell boundaries.

    PubMed

    Jay Burr, A H; Baldwin, James G

    2016-09-01

    Nematode stomas vary widely in the cuticular structures evolved for different feeding strategies, yet the arrangement of the epithelial cell classes that form these structures may be conserved. This article addresses several issues that have impeded the full acceptance of this hypothesis including controversies arising from the structure of the Caenorhabditis elegans stoma. We investigated fluorescent antibody labeling of cell boundaries in conjunction with confocal microscopy as an alternative to transmission electron microscopy (TEM), using MH27 to label apical junctions in C. elegans and two other species. Accurately spaced optical sections collected by the confocal microscope provide a three-dimensional array of pixels (voxels) that, using image-processing software, can be rotated and sectioned at accurately chosen thicknesses and locations. Ribbons of fluorescence clearly identify cell boundaries along the luminal cuticle in C. elegans and Zeldia punctata and less clearly in Bunonema sp. The patterns render cell classes and their relationships readily identifiable. In the C. elegans stoma they correct a misreading of serial TEMs that was not congruent with architecture in other nematodes-the row of marginal cells is now seen to be continuous as in other nematodes, rather than being interrupted by encircling pm1 cells. Also impeding understanding, the reference to certain cell classes as 'epithelial' and others as "muscle" in the C. elegans literature is at variance with muscle expression in most other taxa. For consistent comparison among species, we propose that these cell class descriptors based on function be replaced by topological terms. With these and other confusing concepts and terminology removed, the homology of the cellular architecture among taxa becomes obvious. We provide a corrected description of the cell architecture of the C. elegans stoma and examples of how it is modified in other taxa with different feeding strategies. J. Morphol. 277

  11. 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.

  12. Improved volume rendering for the visualization of living cells examined with confocal microscopy

    NASA Astrophysics Data System (ADS)

    Enloe, L. Charity; Griffing, Lawrence R.

    2000-02-01

    This research applies recent advances in 3D isosurface reconstruction to images of test spheres and plant cells growing in suspension culture. Isosurfaces that represent object boundaries are constructed with a Marching Cubes algorithm applied to simple data sets, i.e., fluorescent test beads, and complex data sets, i.e., fluorescent plant cells, acquired with a Zeiss Confocal Laser Scanning Microscope (LSM). The marching cubes algorithm treats each pixel or voxel of the image as a separate entity when performing computations. To test the spatial accuracy of the reconstruction, control data representing the volume of a 25 micrometer test shaper was obtained with the LSM. This volume was then judged on the basis of uniformity and smoothness. Using polygon decimation and smoothing algorithms available through the visualization toolkit, 'voxellated' test spheres and cells were smoothed using several different smoothing algorithms after unessential polygons were eliminated. With these improvements, the shape of subcellular organelles could be modeled at various levels of accuracy. However, in order to accurately reconstruct these complex structures of interest to us, the subcellular organelles of the endosomal system or the endoplasmic reticulum of plant cells, measurements of the accuracy of connectedness of structures need to be developed.

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

    NASA Astrophysics Data System (ADS)

    Rollan, Ana; Ward, Thelma; McHale, Anthony P.

    Photodynamic therapy (PDT), in which tissues may be rendered fatally light-sensitive represents a relatively novel treatment for cancer and other disorders such as cardiovascular disease. It offers significant application to disease control in an isolated environment such as space flight. In studying PDT in the laboratory, low energy lasers such as HeNe lasers are used to activate the photosensitized cellular target. A major problem associated with these studies is that events occurring during actual exposure of the target cells to the system cannot be examined in real time. In this study HeLa cells were photosensitized and photodynamic activation was accomplished using the scanning microbeam from a confocal laser scanning microscope. This form of activation allowed for simultaneous photoactivation and observation and facilitated the recording of events at a microscopic level during photoactivation. Effects of photodynamic activation on the target cells were monitored using the fluorophores rhodamine 123 and ethidium homodimer-1. Potential applications of these forms of analyses to space medicine and cell biology are discussed.

  14. Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement.

    PubMed

    Taylor, D P; Slattery, J; Leopold, A C

    1996-05-01

    The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with Cl-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units. PMID:11539373

  15. Efficacy of photodynamic therapy against larvae of Aedes aegypti: confocal microscopy and fluorescence-lifetime imaging

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

    Recently a few demonstration on the use of Photodynamic Reaction as possibility to eliminate larvae that transmit diseases for men has been successfully demonstrated. This promising tool cannot be vastly used due to many problems, including the lake of investigation concerning the mechanisms of larvae killing as well as security concerning the use of photosensitizers in open environment. In this study, we investigate some of the mechanisms in which porphyrin (Photogem) is incorporated on the Aedes aegypti larvae previously to illumination and killing. Larvae at second instar were exposed to the photosensitizer and after 30 minutes imaged by a confocal fluorescence microscope. It was observed the presence of photosensitizer in the gut and at the digestive tract of the larva. Fluorescence-Lifetime Imaging showed greater photosensitizer concentration in the intestinal wall of the samples, which produces a strong decrease of the Photogem fluorescence lifetime. For Photodynamic Therapy exposition to different light doses and concentrations of porphyrin were employed. Three different light sources (LED, Fluorescent lamp, Sun light) also were tested. Sun light and fluorescent lamp shows close to 100% of mortality after 24 hrs. of illumination. These results indicate the potential use of photodynamic effect against the LARVAE of Aedes aegypti.

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

    PubMed Central

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

    1998-01-01

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

  17. [Lipid Composition of Different Breeds of Milk Fat Globules by Confocal Raman Microscopy].

    PubMed

    Luo, Jie; Wang, Zi-wei; Song, Jun-hong; Pang, Rui-peng; Ren, Fa-zheng

    2016-01-01

    Different breeds of cows affect the form of fat exist in dairy products and the final functionality, which depended mainly on the composition of the milk fat globules(MFG). However, the relationship between the composition and breeds has not been illuminated. In our study, differences in the lipid content and fatty acid composition of native bovine, buffalo and yak MFG were investigated by confocal Raman spectroscopy. The research offers the possibility of acquisition and analysis of the Raman signal without disruption of the structure of fat globule. The results showed that yak MFG had a higher ratio of band intensities at 2 885/2 850 cm(-1), indicating yak MFG tend to have a triglyceride core in a fluid state with a milk fat globule membrane in a crystalline state. The buffalo and yak MFG had a higher level of unsaturation compared to bovine MFG, shown by a higher ratio of band intensities at 1 655/1 744 cm(-1). The results indicate that small MFG of buffalo is more unsaturated than yak, while the large MFG of buffalo is less unsaturated than the yak. Thus, selective use of cream with yak MFG would allow a harder and more costly churning process but lead to a softer butter. Buffalo milk which contains larger MFG is more suitable for cream and MFG membrane separation. PMID:27228754

  18. Image adaptive point-spread function estimation and deconvolution for in vivo confocal microscopy.

    PubMed

    Von Tiedemann, M; Fridberger, A; Ulfendahl, M; Tomo, I; Boutet de Monvel, J; De Monvel, J Boutet

    2006-01-01

    Visualizing deep inside the tissue of a thick biological sample often poses severe constraints on image conditions. Standard restoration techniques (denoising and deconvolution) can then be very useful, allowing one to increase the signal-to-noise ratio and the resolution of the images. In this paper, we consider the problem of obtaining a good determination of the point-spread function (PSF) of a confocal microscope, a prerequisite for applying deconvolution to three-dimensional image stacks acquired with this system. Because of scattering and optical distortion induced by the sample, the PSF has to be acquired anew for each experiment. To tackle this problem, we used a screening approach to estimate the PSF adaptively and automatically from the images. Small PSF-like structures were detected in the images, and a theoretical PSF model reshaped to match the geometric characteristics of these structures. We used numerical experiments to quantify the sensitivity of our detection method, and we demonstrated its usefulness by deconvolving images of the hearing organ acquired in vitro and in vivo.

  19. Quantification of Mesenchymal Stem Cell (MSC) Delivery to a Target Site Using In Vivo Confocal Microscopy

    PubMed Central

    Mortensen, Luke J.; Levy, Oren; Phillips, Joseph P.; Stratton, Tara; Triana, Brian; Ruiz, Juan P.; Gu, Fangqi; Karp, Jeffrey M.; Lin, Charles P.

    2013-01-01

    The ability to deliver cells to appropriate target tissues is a prerequisite for successful cell-based therapy. To optimize cell therapy it is therefore necessary to develop a robust method of in vivo cell delivery quantification. Here we examine Mesenchymal Stem Cells (MSCs) labeled with a series of 4 membrane dyes from which we select the optimal dye combination for pair-wise comparisons of delivery to inflamed tissue in the mouse ear using confocal fluorescence imaging. The use of an optimized dye pair for simultaneous tracking of two cell populations in the same animal enables quantification of a test population that is referenced to an internal control population, thereby eliminating intra-subject variations and variations in injected cell numbers. Consistent results were obtained even when the administered cell number varied by more than an order of magnitude, demonstrating an ability to neutralize one of the largest sources of in vivo experimental error and to greatly reduce the number of cells required to evaluate cell delivery. With this method, we are able to show a small but significant increase in the delivery of cytokine pre-treated MSCs (TNF-α & IFN-γ) compared to control MSCs. Our results suggest future directions for screening cell strategies using our in vivo cell delivery assay, which may be useful to develop methods to maximize cell therapeutic potential. PMID:24205131

  20. Deep high-resolution fluorescence microscopy of full organs: the benefit of ultraminiature confocal miniprobes

    NASA Astrophysics Data System (ADS)

    Schwarz, France; Le Nevez, Arnaud; Genet, Magalie; Osdoit, Anne; Lacombe, François

    2009-02-01

    Background: Confocal Laser Endomicroscopy (CLE) based on ultraminiature miniprobes (Cellvizio®, Mauna Kea Technologies, Paris, France) is able to image the inner microstructure of retroperitoneal full organs punctured during EUS-FNA procedures, such as pancreas, liver or lymph nodes. Therefore, pCLE can provide an easy-to-use and precise adjunct tool to ultrasonographic interventions in order to target suspicious areas for biopsies in EUS-FNA. Material and Methods: Probe-based CLE (pCLE) was performed on ex-vivo surgically resected specimens after topical application of fluorophores in standard 19G and 22G needles. Two prototype miniprobes ("S-probe" 300 microns diameter, field of view 400*280 microns, and "S-probe" 650 microns diameter, field of view 500*600 microns) were then inserted into the needles and enabled visualization of the inner microstructures of uterus, lung, kidney, stomach and esophagus, in both healthy and cancerous conditions. Then, pCLE was performed in-vivo on four pigs during three NOTES and one EUS-FNA procedures after intravenous injection of 2-7mL fluorescein 1-10% using the prototype "S-probe" 350 microns diameter inserted in 19G FNA needles. Liver, pancreas and spleen were imaged. Results: During the ex-vivo experiments, pCLE made it possible to distinguish microstructures, such as alveoli and macrophages in the lungs. During the in-vivo experiments, Cellvizio® video sequences showed hepatic lobules and the portal vein in the liver, and red and white pulp in the spleen. Conclusion: pCLE provides in vivo cellular information about full organs. It has the potential to help target biopsies during EUSFNA, which suffers from a high rate of false negatives, thus increasing its sensitivity.