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Single-cell volume estimation by applying three-dimensional reconstruction methods

Single-cell volume estimation by applying three-dimensional ...

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

作者： Khatibi, S Allansson, L Gustavsson, T Blomstrand, F Hansson, E Olsson, T Chalmers Dept Signals & Syst Gothenburg Sweden

ISBN: (纸本)0819430757

We have studied three-dimensional reconstruction methods to estimate the cell volume of astroglial cells in primary culture. The studies are based on fluorescence imaging and optical sectioning. An automated image-acquisition system was developed to collect two-dimensional microscopic images. images were reconstructed by the Linear Maximum a Posteriori method and the non-linear Maximum Likelihood Expectation Maximization (ML-EM) method. In addition, because of the high computational demand of the ML-EM algorithm, we have developed a fast variant of this method. 1) Advanced image analysis techniques were applied for accurate and automated cell volume determination. 2) The sensitivity and accuracy of the reconstruction methods were evaluated by using fluorescent micro-beads with known diameter. The algorithms were applied to fura-2-labeled astroglial cells in primary culture exposed to hypo- or hyper-osmotic stress. The results showed that the ML-EM reconstructed images are adequate for the determination of volume changes in cells or parts thereof.

关键词： 3D reconstruction ML-EM algorithm volume estimation fluorescence imaging astroglial primary cultures

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

Optical sectioning using finite-sized, multiple aperture arr...

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

作者： Smith, PJ Taylor, C McCabe, EM Univ Dublin Trinity Coll Dept Phys Dublin 2 Ireland

ISBN: (纸本)0819430757

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 adresses 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： fluorescence microscopy direct-view microscopy

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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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High resolution real-time full-field interference microscopy

High resolution real-time full-field interference microscopy

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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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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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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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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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Confocal photoluminescence microscopy in II-VI materials: annealing and degradation dynamics

Confocal photoluminescence microscopy in II-VI materials: an...

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

作者： McCabe, EM Jordan, C Fewer, DT Donegan, JF Taniguchi, S Hino, T Nakano, K Ishibashi, A Uusimaa, P Pessa, M Univ Dublin Trinity Coll Dublin 2 Ireland

ISBN: (纸本)0819430757

Confocal photoluminescence imaging is an important tool in the investigation of recombination in semiconductors and in the characterisation of material growth. This characterisation is particularly important for II-VI wide band-gap semiconductors where the potential for blue-green lasers is being explored currently. To achieve room-temperature cw operation of these lasers over the multi-thousand hours necessary for commercialisation, extremely low defect densities are required. The confocal microscope is used in this work to image photoluminescence from II-VI materials to characterise the defect formation and propagation within the quantum well region of the material. This imaging approach permits the degradation to be monitored in real time and over a large area in samples with low defect densities. The additional advantages of this set-up over a conventional microscope are, of course, the higher lateral resolution and narrow depth of field associated with a confocal microscope. While considerable effort has been focused on the degradation in these II-VI semiconductors, we have recently observed that annealing can occur simultaneously in the same sample when the material is exposed to intense optical excitation. images of annealing and degradation of a range of II-VI samples will be presented to highlight these observations.

关键词： confocal microscopy photoluminescence imaging semiconductors II-VI materials blue green lasers

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Feature extraction and 3D reconstruction of Nomarski DIC microscope images using local energy and neural net techniques

Feature extraction and 3D reconstruction of Nomarski DIC mic...

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

作者： Kyan, M Guan, L Arison, M Cogswell, C Univ Sydney Dept Elect Engn Sydney NSW 2006 Australia

ISBN: (纸本)0819430757

The Nomarski differential interference contrast (DIC) mode is commonly used for imaging translucent biological specimens and it exhibits several major advantages over other phase contrast techniques, including a boost in high spatial frequencies in the region of focus. However, DIC images (unlike confocal) are limited by the presence of low spatial frequency blur and also by a differential shading gradient at feature boundaries, which make normal confocal visualisation techniques unsuitable for feature extraction or for 3D volume rendering of focus-series datasets. To remedy this problem, we employ a neural network technique based on competitive learning, known as Kohonen's self-organising feature map (SOFM), to perform segmentation, using a collection of statistics (know as features) defining the image. Our past investigation showed that standard features such as the localised mean and variance of pixel intensities provided reasonable extraction of objects such asmitotic chromosomes, but surface detail was only moderately resolved. In this current work, local energy is investigated as an alternative image statistic based on phase congruency in the image. This, along with different combinations of other image statistics, is applied in a SOFM, producing 3D images exhibiting vast improvement in the level of detail and clearly isolating the chromosomes from the background.

关键词： differential interference contrast DIC feature extraction feature space image segmentation self organising feature map SOFM phase congruency local energy Morlet wavelet

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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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