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检索条件"任意字段=Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIX"

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Fluorescence Fluctuation Analysis of Mixed Chromophores from a Line-Scanning Hyperspectral Imaging System

Fluorescence Fluctuation Analysis of Mixed Chromophores from...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVII

作者： Davis, Ryan W. Aaron, Jesse S. Rempe, Susan L. Timlin, Jerilyn A. Sandia Natl Labs 7011 East Ave Livermore CA 94550 USA Sandia Natl Labs Albuquerque NM 87123 USA

ISBN: (纸本)9780819479662

Fluorescence fluctuation analysis of dilute biomolecules can provide a powerful method for fast and accurate determination of diffusion dynamics, local concentrations, and aggregation states in complex environments. However, spectral overlap among multiple exogenous and endogenous fluorescent species, photobleaching, and background inhomogeneities can compromise quantitative accuracy and constrain useful biological implementation of this analytical strategy in real systems. In order to better understand these limitations and expand the utility of fluctuation correlation methods, spatiotemporal fluorescence correlation analysis was performed on spectrally resolved line scanned images of modeled and real data from mixed fluorescent nanospheres in a synthetic gel matrix. It was found that collecting images at a pixel sampling regime optimal for spectral imaging provides a method for calibration and subsequent temporal correlation analysis which is insensitive to spectral mixing, spatial inhomogeneity, and photobleaching. In these analyses, preprocessing with multivariate curve resolution (MCR) provided the local concentrations of each spectral component in the images, thus facilitating correlation analysis of each component individually. This approach allowed quantitative removal of background signals and showed dramatically improved quantitative results compared to a hypothetical system employing idealized filters and multi-parameter fitting routines.

关键词： Fluorescence correlation spectroscopy hyperspectral imaging spatiotemporal image correlation spectroscopy fluorescence fluctuation multivariate curve resolution line-scan imaging autofluorescence photobleaching

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Determination of direction-independent optical path-length distribution of cells using rotational-diversity transmitted-light differential interference contrast (DIC) images

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Conference on 3-dimensional and multidimensional microscopy - image acquisition and processing V

作者： Preza, C van Munster, EB Aten, JA Snyder, DL Rosenberger, FU Washington Univ Inst Biomed Comp St Louis MO 63130 USA

ISBN: (纸本)0819427004

Two different phase-estimation methods that have been developed for the computation of the optical path-length (OPL) distribution of a specimen from DIC images are compared in this paper. The first method is based on the Wiener filter approach and uses only a single DIC image for the determination of the OPL distribution. The second phase-estimation method is based on the conjugate-gradient optimization method and estimates the OPL distribution using rotational-diversity DIC images;i.e. multiple DIC images obtained by rotating the specimen. For this study, 24 different DIC images of a single bovine spermatozoa head were acquired by rotating the cell by approximately 15 degrees between images. The images were registered and aligned using fiducial marks, and then processed with both methods. Results obtained with the filtering method were found to be dependent on the orientation of the cell with respect to the shear direction. Comparison of the integrated optical path length (IOPL) computed with the the filtering method and the rotational-diversity method using two, four and eight DIC images at different rotation angles showed that the IOPL estimated with the rotational-diversity method is less dependent on the rotation angle, even when only two images separated by 90-degree cell rotation are used for the phase estimation. Our results show that the use of rotational-diversity images in the determination of the OPL distribution is very beneficial because it overcomes the directional dependence of DIC imaging.

关键词： computational Nomarski DIC microscopy phase estimation optical path length

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Practical optical quality assessment and correction of a nonlinear microscope

Practical optical quality assessment and correction of a non...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVII

作者： Aviles-Espinosa, Rodrigo Andilla, Jordi Porcar-Guezenec, Rafael Olarte, Omar Santos, Susana I. C. O. Levecq, Xavier Artigas, David Loza-Alvarez, Pablo ICFO Inst Photon Sci Mediterranean Technol PkAv Canal Olimp S-N Castelldefels 08860 Barcelona Spain Imagine Opt F-91400 Orsay France Univ Politecn Cataluna Barcelona 08034 Spain

ISBN: (纸本)9780819479662

Nonlinear microscopy (NLM) has covered the requirement for higher contrast and resolution compared with other microscopy techniques, however, the optical quality of this imaging apparatus and the sample structure can compromise its capabilities. Here, we show that the imaging capabilities of a NLM can be affected by the aberrations produced by the setup optical elements alignment, the materials from which they are fabricated and more importantly by the sample. To overcome this, a Shack-Hartman Wavefront sensing scheme has been implemented for characterizing: a) the whole NLM setup and, b) the sample induced aberrations. The first part includes all the aberrations introduced by the optical elements, starting from the laser and until the microscope objective. Having these information, aberrations can be compensated in a closed-loop configuration resulting in the system calibration. Then the remaining aberrations (microscope objective and sample) are recorded. This is done employing the sample nonlinear fluorescence signal collected at one point (keeping the excitation beam static) in the imaging plane. Given that this emission is an incoherent process, it can be considered as a point source. Therefore its wavefront will contain the sample and the objective aberrations. Using the wavefront sensor the information is recorded and passed to the deformable mirror which will compensate the aberrations in a "single shot" (open-loop configuration). This compared with other adaptive optics strategies (i.e. iterative algorithms) results in a reduced sample exposure, and greatly decreases sample damage. Importantly the application of both corrections (system and sample) enables a significant signal intensity and contrast improvement.

关键词： Adaptive Optics Nonlinear microscopy Aberration compensation

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Pupil Engineering for a Confocal Reflectance Line-Scanning Microscope

Pupil Engineering for a Confocal Reflectance Line-Scanning M...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Patel, Yogesh G. Rajadhyaksha, Milind DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana Bldg360 Huntington Ave Boston MA 02115 USA Northeastern Univ Dept Mech & Ind Engn Boston MA 02115 USA Serv Dermatol Dept Med Memorial Sloan Kettering Canc Ctr New York NY 10022 USA

ISBN: (纸本)9780819484413

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

关键词： confocal microscopy Fourier optics point-scanning line-scanning full-pupil divided-pupil

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White matter segmentation by estimating tissue optical attenuation from volumetric OCT massive histology of whole rodent brains 24

White matter segmentation by estimating tissue optical atten...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXIV

作者： Lefebvre, Joel Castonguay, Alexandre Lesage, Frederic Ecole Polytech Montreal CP 6079Succ Ctr Ville Montreal PQ H3C 3A7 Canada Inst Cardiol Montreal 5000 Rue Belanger Est Montreal PQ H1T 1C8 Canada

ISBN: (数字)9781510605824

ISBN: (纸本)9781510605817;9781510605824

A whole rodent brain was imaged using an automated massive histology setup and an Optical Coherence Tomography (OCT) microscope. Thousands of OCT volumetric tiles were acquired, each covering a size of about 2.5x2.5x0.8 mm3 with a sampling resolution of 4.9x4.9x6.5 microns. This paper shows the techniques for reconstruction, attenuation compensation and segmentation of the sliced brains. The tile positions within the mosaic were evaluated using a displacement model of the motorized stage and pairwise coregistration. Volume blending was then performed by solving the 3D Laplace equation, and consecutive slices were assembled using the cross-correlation of their 2D image gradient. This reconstruction algorithm resulted in a 3D map of optical reflectivity for the whole brain at micrometric resolution. OCT tissue slices were then used to estimate the local attenuation coefficient based on a single scattering photon model. The attenuation map obtained exhibits a high contrast for all white matter fibres, regardless of their orientation. The tissue optical attenuation from the intrinsic OCT reflectivity contributes to better white matter tissue segmentation. The combined 3D maps of reflectivity and attenuation is a step toward the study of white matter at a microscopic scale for the whole brain in small animals.

关键词： Massive histology Volume reconstruction image processing Optical Coherence Tomography Attenuation Coefficient White matter Segmentation

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Confocal laser microscopy of the in vivo human skin revisited.

Confocal laser microscopy of the <i>in vivo</i> human skin r...

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Conference on 3-dimensional and multidimensional microscopy - image acquisition and processing VI

作者： Corcuff, P Hadjur, C Chaussepied, C Toledo-Crow, R LOreal Labs Rech Avancee F-93601 Aulnay Sous Bois France

ISBN: (纸本)0819430757

A new generation of confocal laser microscope, designed to image the human skin in vivo, improves the resolution, contrast and spectroscopic facilities as compared to the previous Tandem Scanning Microscope (TSM) prototype. The new device has been built with a Oz module (Noran) equipped with the skin contact device, assuming a perfect stability of skin images in the horizontal plane. The Z displacement of the objective lens, mounted directly on the Ch module, is assumed by a piezo motor with a course of 350 mu m. Moreover, the Ch module has been suspended on articulated arms to reach any part of the human body. The power of the Argon/Krypton laser source has been limited to 2 mW to secure safety and provides three visible wavelength : 488, 568 and 647 nm. The facility of instantly checking wavelength during in depth exploration of the skin optimizes the resolution and contrast of images as compared with the white light used in the TSM. Consequently, better image quality of the epidermis is obtained in the blue region with unexpected details of corneocytes and keratinocytes. The papillary dermis comprising the vascular network is advantageously observed with the red light. The fluorescence channel detector gives additional information on the penetration of fluorescent probes through the skin barrier. Optical sections are digitized (512 x 480 x 8 bit) at video rate, providing easy and fast measurements of the thickness of epidermal layers. The Silicon Graphics workstation generates a transparent volume of living human skin in less than 5 minutes. This powerful and convivial new design for imaging the in vivo human skin opens up new promises in skin research and in vivo skin pharmacology.

关键词： confocal microscopy human skin in vivo imaging fluorescence volume rendering

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Quantification of collagen networks in mammary tumors using TPEF and laser-based tomography 29

Quantification of collagen networks in mammary tumors using ...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing Xxix

作者： Kamin, Hannes Nolte, Lena Maurer, Jochen Bleilevens, Andreas Stickeler, Elmar Johannsmeier, Sonja Heinemann, Dag Ripken, Tammo Laser Zentrum Hannover eV Ind & Biomed Opt Dept Hollerithallee 8 D-30419 Hannover Germany Univ Hosp Aachen Clin Gynecol & Obstet Pauwelstr 30 D-52074 Aachen Germany Lower Saxony Ctr Biomed Engn Implant Res & Dev NI Stadtfelddamm 34 D-30625 Hannover Germany Leibniz Univ Hannover Inst Hort Prod Syst Dept Phytophoton Herrenhauser Str 2 D-30419 Hannover Germany Leibniz Univ Hannover Hannover Ctr Opt Technol HOT Nienburger Str 17 D-30167 Hannover Germany

ISBN: (纸本)9781510648043;9781510648036

The triple-negative breast cancer (tnbc) is an aggressive subtype linked to a poor outcome of established breast cancer therapy. Increasing evidence points to the role of the tumor's extracellular matrix (ECM) as a determinant of its aggressiveness as well as the effectiveness of chemical therapeutics. three-dimensional imaging techniques can be used to unravel ECM architecture. Label-free contrast mechanisms such as second harmonic generation (SHG) avoid falsification and artifacts introduced by the labeling process. Here, we present the complementary use of two-photon excitation microscopy (TPEF) and Scanning Laser Optical Tomography (SLOT) for the investigation and quantification of tumor ECM. Both methods were used to capture fluorescence from antibody-labeled samples as well as the SHG signal from collagen strands in the ECM. SLOT generally allows for the investigation of larger samples of several mm up to a few cm in size. This work shows the capabilities of the tomographic setup compared to established TPEF, and demonstrates their combined use to maximize the information content of the acquired data. The obtained images served as a basis for ECM quantification. 3D-analysis allowed for determination of length, straightness and orientation of the collagen fibers based on fluorescence imaging as well as SHG imaging. The resulting coordinates might be used for synthetic reconstruction of a patient-specific tumor matrix, serving as a scaffold for pre-clinical therapeutic testing. Collagen imaging and quantification as presented here can therefore be employed for both basic and clinical research, paving the way for patient-specific cancer therapy.

关键词： cancer collagen imaging multimodal imaging extracellular matrix 3D imaging image reconstruction tomography second harmonic generation

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Hyperspectral image Correlation for Monitoring Membrane Protein Dynamics in Living Cells

Hyperspectral Image Correlation for Monitoring Membrane Prot...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Davis, Ryan W. Carson, Bryan Jones, Howland D. T. Sinclair, Michael B. Sandia Natl Labs Albuquerque NM 87123 USA

ISBN: (纸本)9780819474308

Temporal image correlation provides a powerful fluorescence technique for measuring several biologically relevant parameters of molecules in living cells. These parameters include, but are not limited to local concentrations, diffusion dynamics, and aggregation states of biomolecules. However, the complex cellular environment presents several limitations, precluding high quantitative accuracy and constraining biological implementation. In order to address these issues, high speed spectral imaging was employed to compare the results of image correlation from spectrally unmixed and virtually implemented fluorescence emission filters. Of particular interest in this study is the impact of cellular autofluorescence, which is ubiquitous in fluorescence imaging of cells and tissues. Using traditional instrumentation, corrections for autofluorescence are commonly estimated as a static offset collected from a separate control specimen. While this may be sufficient in highly homogenous regions of interest, the low analyte concentrations requisite to fluctuation-based methods result in the potential for unbounded error resulting from spectral cross-talk between local autofluorescence inhomogeneities and the fluorescence signal of interest. Thus we demonstrate the importance of accurate autofluorescence characterization and discuss potential corrections using a case study focusing on fluorescence confocal spectral imaging of immune cells before and after stimulation with lipopolysaccheride (LPS). In these experiments, binding of LPS to the membrane receptor, YFP-TLR4, is observed to result in initiation of the immune response characterized by altered receptor diffusion dynamics and apparent heterogeneous aggregation states. In addition to characterizing errors resulting from autofluorescence spectral bleed-through, we present data leading to a deeper understanding of the molecular dynamics of the immune response and suggest hypotheses for future work utilizing hyperspectrally e

关键词： Hyperspectral imaging image correlation spectroscopy multivariate analysis multivariate curve resolution MCR fluorescence imaging spectral cross-talk spectral bleed-through autofluorescence membrane receptor TLR4 immune response lipopolysaccharide LPS

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Semi-automated Algorithm for Localization of Dermal/Epidermal Junction in Reflectance Confocal microscopy images of Human Skin

Semi-automated Algorithm for Localization of Dermal/Epiderma...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Kurugol, Sila Dy, Jennifer G. Rajadhyaksha, Milind Gossage, Kirk W. Weissman, Jesse Brooks, Dana H. Northeastern Univ 360 Huntington Av Boston MA 02115 USA Mem Sloan Kettering Canc Ctr Serv Dermatol New York NY 10065 USA Unilever Res Labs Trumbull CT 06611 USA

ISBN: (纸本)9780819484413

The examination of the dermis/epidermis junction (DEJ) is clinically important for skin cancer diagnosis. Reflectance confocal microscopy (RCM) is an emerging tool for detection of skin cancers in vivo. However, visual localization of the DEJ in RCM images, with high accuracy and repeatability, is challenging, especially in fair skin, due to low contrast, heterogeneous structure and high inter-and intra-subject variability. We recently proposed a semi-automated algorithm to localize the DEJ in z-stacks of RCM images of fair skin, based on feature segmentation and classification. Here we extend the algorithm to dark skin. The extended algorithm first decides the skin type and then applies the appropriate DEJ localization method. In dark skin, strong backscatter from the pigment melanin causes the basal cells above the DEJ to appear with high contrast. To locate those high contrast regions, the algorithm operates on small tiles (regions) and finds the peaks of the smoothed average intensity depth profile of each tile. However, for some tiles, due to heterogeneity, multiple peaks in the depth profile exist and the strongest peak might not be the basal layer peak. To select the correct peak, basal cells are represented with a vector of texture features. The peak with most similar features to this feature vector is selected. The results show that the algorithm detected the skin types correctly for all 17 stacks tested (8 fair, 9 dark). The DEJ detection algorithm achieved an average distance from the ground truth DEJ surface of around 4.7 mu m for dark skin and around 7-14 mu m for fair skin.

关键词： confocal reflectance microscopy image analysis skin classification

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Parallel, confocal, and complete spectrum imager for fluorescent detection of high-density microarray

Parallel, confocal, and complete spectrum imager for fluores...

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Conference on 3-dimensional and multidimensional microscopy - image acquisition and processing VI

作者： Bogdanov, V Boyce-Jacino, M Orchid Biocomp Inc Baltimore MD 21224 USA

ISBN: (纸本)0819430757

Confined arrays of biochemical probes deposited on a solid support surface (analytical microarray or "chip") provide an opportunity to analysis multiple reactions simultaneously. Microarrays are increasingly used in genetics, medicine and environment scanning as research and analytical instruments. A power of microarray technology comes from its parallelism which grows with array miniaturization, minimization of reagent volume per reaction site and reaction multiplexing. An optical detector of microarray signals should combine high sensitivity, spatial and spectral resolution. Additionally, low-cost and a high processing rate are needed to transfer microarray technology into biomedical practice. We designed an imager that provides confocal and complete spectrum detection of entire fluorescently-labeled microarray in parallel. imager uses microlens array, non-slit spectral decomposer, and high-sensitive detector (cooled CCD). Two imaging channels provide a simultaneous detection of localization, integrated and spectral intensities for each reaction site in microarray. A dimensional matching between microarray and imager's optics eliminates all in moving parts in instrumentation, enabling highly informative, fast and low-cost microarray detection. We report theory of confocal hyperspectral imaging with microlenses array and experimental data for implementation of developed imager to detect fluorescently labeled microarray with a density similar to 10(3) sites per cm(2).

关键词： microarray technology fluorescence confocal scanning two-dimensional spectroscopy

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