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Optical sectioning using finite-sized, multiple aperture arrays in fluorescence direct-view microscopy;experimental results

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 236-240页

作者： Smith, P.J. Taylor, C. McCabe, E.M. Trinity Coll Dublin Dublin Ireland

ISBN: (纸本)0819430757;9780819430755

Confocal fluorescence imaging is widely used, particularly for biological applications, and also notably in direct-view microscopes. Recent work has compared the use of coherent and incoherent illumination sources on the optical sectioning characteristics of fluorescence direct-view microscopes. However this detailed comparison has been done in theory. This paper addresses the experimental aspects of using coherent light sources in fluorescence imaging using a range of finite-sized, multiple-aperture arrays. The experimental difficulties of choosing a suitable uniform, flat, fluorescent plane with a high quantum efficiency are considered. Axial response curves obtained with a fluorescent laser dye sample are presented.

关键词： Optical microscopy

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High resolution real-time full-field interference microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 13-20页

作者： Dubois, Arnaud Lebec, Martial Beaurepaire, Emmanuel Leveque, Sandrine Boccara, Claude Ecole Superieure de Physique et Chimie Industrielles Paris France

ISBN: (纸本)0819430757;9780819430755

We have built an interference microscope that produces in real-time images of cross-section slices located at adjustable depths inside 3-D objects. The microscope is based on a Michelson-type polarization interferometer. A light emitting diode (LED), used as an optical source at λ = 840 nm with short coherence length, provides optical sectioning ability with better than 10 μm resolution in the depth dimension. By using high numerical aperture objective lenses (NA = 0.95), the depth resolution can be improved to better than 1 μm, in good agreement with theory. images can be produced at the rate of 50 per second using a multiplexed lock-in detection and MMX assembler-optimized calculation routines. Cross-section images inside an onion and at different depths in a multilayer silicon integrated circuit are presented.

关键词： Interferometers

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Segmentation for the optical micrograph of fungi by using spectral transmittance and neural network

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 288-297页

作者： Takeya, Masaru Tsumura, Norimichi Haneishi, Hideaki Miyake, Yoichi Chiba Univ Chiba Japan

ISBN: (纸本)0819430757;9780819430755

Transmittance spectra of optical micrograph of fungi were estimated from seven band images based on the Wiener estimation method. The optical microscope adjusted with monochrome digital camera (Kodak DCS420m, 1536 × 1024 pixels) with seven band filters was used for image acquisition. Transmittance spectra of 16 color transparencies were measured by spectral photometer in advance, and the seven band images of color transparency were taken by microscopic imaging system. A matrix for Wiener estimation was calculated using these measured transmittance spectra and camera responses. As the result of Wiener estimation, normalized root mean square error (NRMSE) between the original and estimated transmittance spectra of 16 color transparencies was 0.035. Wiener estimation method was applied to estimate the spectral transmittance of five species which belong to one genus of fungi. Transmittance spectra of fungi were calculated from camera responses in each pixel and the above estimation matrix. The estimated transmittance spectra were used for segmentation of conidia and hyphae in fungal microscopic image to identify them. Competitive learning in neural network was applied to the segmentation from spectral microscopic image. It was found that segmentation based on spectral transmittance was more accurate than the segmentation based on RGB values.

关键词： image segmentation

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Estimating spectral models for multidimensional data mapping.

Estimating spectral models for multidimensional data mapping...

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

作者： Charonov, S Saadi, A Kokota, A Manfait, M Univ Reims UFR Pharm Unite Median F-51096 Reims France

ISBN: (纸本)0819430757

The basic procedure of spectral image analysis is mapping, i.e., generation of conventional images using specific spectral features from multidimensional data set. Commonly, it is based on decomposition of the original spectra into a sum of model spectra which are usually attributed to chemically/physically different components. However, the problem is complicated when the model spectra are not clearly definable or difficult and even impossible to obtain experimentally. Here we describe a method for estimating spectral models based on the elimination of physical meaningless solutions obtained by PCA (Principal Component Analysis). The method includes the following steps: segmentation of the original spectral image using a white-light image;generation of primary factors for each segmented region by MDF ( maximum distance factors) algorithm;extending the factor space to satisfy non-negative score requirements;generation of model limits by rotation of factor matrix in conditions of non-negative intensity values;calculation of the common model as intersection regions in the score space. The precision of the method depends on the spectral variations in the original data. When these variations are significant the models could be obtained precisely and then attributed to possible sample components. This method was validated in different research fields : to study the distribution of anticancer drugs in single living cells, to characterize phenolic species in wheat walls.

关键词： spectral image analysis factor analysis data mapping single living cells wheat grains

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Transparent three-dimensional microstructures for trapping of living cells

Transparent three-dimensional microstructures for trapping o...

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

作者： Otte, K Zimmer, K Zeitschel, U Hirsch, D Reichenbach, A Bigl, F Bigl, V Inst Oberflachenmodifizierung EV D-04318 Leipzig Germany

ISBN: (纸本)0819430757

The simultaneous manipulation and observation of morphological features of single living cells along with the recording of functional changes related to cellular metabolism, interaction and communication in real time are of growing interest. With the advent of atomic force microscopy (AFM) structural studies of native cells became possible which in combination with adequate light microscopy give a much better resolution than light microscopy alone. However, the motion of the cell, the softness of the cell membrane and the two-dimensional growth of cells in culture limit applicability and resolution of this technique. A good mechanical fixation of living cells in a structure could be achieved by embedding cells into partially covered grooves produced in Si/SiO2. However, for additionally optical microscopy studies a transparent material is essential. In this study we present the fabrication of different transparent three-dimensional structures in a three-layer system (Si3N4, SiO2, Si3N4) on quartz specially sized for the trapping of living neural cells under physiological conditions. For the fabrication of the structures we utilized a combination of e-beam lithography (EBL), laser ablation, reactive ion beam etching (RIBE) and different wet etching techniques. The structures consist of a nano-net of Si3N4 covering a micro-cavity in SiO2 confining enough to prevent the cell body from escaping, but not so constraining that it hinders normal growth and development. Another type of presented structures consists of cavities which are connected by covered micro-channels, hence, an observation of cell-cell interactions is also possible. Advantages of these microstructures are the trapping of cells, the stabilization of the cell membrane and the precise placement of the cells for a multitude of biological investigations.

关键词： three-dimensional microstructure cell manipulation SiO2 Si3N4 AFM wet etching ion beam etching laser ablation

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Measurement of the chirp dependent fluorescence response: a window to excited state population dynamics

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 40-47页

作者： Brakenhoff, G.J. Buist, Arjan H. Mueller, Michiel Squier, Jeff A. Bardeen, Chris J. Yakovlev, Vladislav V. Wilson, Kent R. Univ of Amsterdam Amsterdam Netherlands

ISBN: (纸本)0819430757;9780819430755

High intensity chirped pulses can be used for probing microscopic chemical environments through the use of a particular choice of dye, for instance SNAFL2. The basis for this technique is that the excited state populations can be manipulated through control over the temporal order of the excitation frequencies in the excitation pulse - i.e. chirp - with the outcoming fluorescence as the reporting parameter. A chirp dependent fluorescence response can also be observed in larger molecular systems with more degrees of freedom like for instance green fluorescent proteins. In preparation for application of the technique to microscopy we use a facility permitting observation of this phenomenon in various dyes with high sensitivity. High power, 30 fs pulses from an OPA, tunable from 400 nm to 1.5 micron are used. These pulses with a repetition rate of 1 kHz are sufficiently intense that a relatively large sample region can be excited to saturation from which then a sub-region with uniform excitation conditions can be selected for signal collection.

关键词： Optical microscopy

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Spectral imaging of histological and cytological specimens

Spectral imaging of histological and cytological specimens

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

作者： Rothmann, C Malik, Z Bar Ilan Univ Dept Life Sci IL-52900 Ramat Gan Israel

ISBN: (纸本)0819430757

Evaluation of cell morphology by bright field microscopy is the pillar of histopathological diagnosis. The need for quantitative and objective parameters for diagnosis has given rise to the development of morphometric methods. The development of spectral imaging for biological and medical applications introduced both fields to large amounts of information extracted from a single image. Spectroscopic analysis is based on the ability of a stained histological specimen to absorb, reflect, or emit photons in ways characteristic to its interactions with specific dyes. Spectral information obtained from a histological specimen is stored in a cube whose appellate signifies the two spatial dimensions of a flat sample (x and y) and the third dimension, the spectrum representing the light intensity for every wavelength. The spectral information stored in the cube can be further processed by morphometric analysis and quantitative procedures. Such a procedure is spectral-similarity mapping (SSM), which enables the demarcation of areas occupied by the same type of material. SSM constructs new images of the specimen, revealing areas with similar stain-macromolecule characteristics and enhancing subcellular features. Spectral imaging combined with SSM reveals nuclear organization through the differentiation stages as well as in apoptotic and necrotic conditions and identifies specifically the nucleoli domains.

关键词： spectral-imaging image processing morphometry hormone receptors optical density

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High resolution real-time full-field interference microscopy

High resolution real-time full-field interference microscopy

引用

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

作者： Dubois, A Lebec, M Beaurepaire, E Lévêque, S Boccara, C Ecole Super Phys & Chim Ind Lab Opt CNRS Unite Propre Rech A0005 F-75005 Paris France

ISBN: (纸本)0819430757

We have built an interference microscope that produces in real-time images of cross-section slices located at adjustable depths inside 3-D objects. The microscope is based on a Michelson-type polarization interferometer. A light emitting diode (LED), used as an optical source at lambda = 840 nm with short coherence length, provides optical sectioning ability with better than 10 mu m resolution in the depth dimension. By using high numerical aperture objective lenses (NA = 0.95), the depth resolution can be improved to better than 1 mu m, in good agreement with theory. images can be produced at the rate of 50 per second using a multiplexed lock-in detection and MMX assembler-optimized calculation routines. Cross-section images inside an onion and at different depths in a multilayer silicon integrated circuit are presented.

关键词： interference microscopy optical coherence tomography CCD camera

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Confocal laser microscopy of the in vivo human skin revisited

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 73-81页

作者： Corcuff, Pierre Hadjur, Christophe Chaussepied, Celine Toledo-Crow, Ricardo L'Oreal Aulnay-sous-bois France

ISBN: (纸本)0819430757;9780819430755

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 Oz module, is assumed by a piezo motor with a course of 350 μm. Moreover, the Oz 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 (512×480×8 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： Optical microscopy

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

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 298-307页

作者： Bogdanov, Valery Boyce-Jacino, Michael Orchid Biocomputer Inc Baltimore United States

ISBN: (纸本)0819430757;9780819430755

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 approx. 103 sites per cm2.

关键词： image sensors

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