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three-dimensional and multidimensional microscopy: image acquisition processing VII

作者： Boutet de Monvel, J. Le Calvez, S. Ulfendahl, M. Karolinska Hospital Stockholm Sweden

image restoration algorithms provide efficient tools for recovering part of the information lost in the imaging process of a microscope. We describe recent progress in the application of deconvolution to confocal microscopy. The point spread function of a Biorad-MRC1024 confocal microscope was measured under various imaging conditions, and used to process 3D-confocal images acquired in an intact preparation of the inner ear developed at Karolinska Institutet. Using these experiments we investigate the application of deionizing methods based on wavelet analysis as a natural regularization of the deconvolution process. Within the Bayesian approach to image restoration, we compare wavelet denoising with the use of a maximum entropy constraint as another natural regularization method. Numerical experiments performed with test images show a clear advantage of the wavelet deionizing approach, allowing to `cool down' the image with respect to the signal, while suppressing much of the fine-scale artefacts appearing during deconvolution due to the presence of noise, incomplete knowledge of the point spread function, or undersampling problems. We further describe a natural development of this approach, which consists of performing the Bayesian inference directly in the wavelet domain.

关键词： Medical imaging

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Method for intracellular imaging of ion concentrations, using confocal microscopy and fluorophore lifetimes

Method for intracellular imaging of ion concentrations, usin...

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three-dimensional and multidimensional microscopy: image acquisition processing VII

作者： Carlsson, Kjell Liljeborg, Anders Andersson, Ronnie M. Brismar, Hjalmar Royal Inst of Technology Stockholm Sweden

There exist a number of fluorescent probes whose lifetimes change in response to ion concentrations (for example H+ and Ca2+) in the surrounding medium. We describe a technique for utilizing this property in a confocal scanning laser microscope. The technique is based on intensity-modulated laser illumination of the specimen, and phase-sensitive lock-in detection of the fluorescent light. In this way we get a lifetime-dependent output signal which, after calibration, can provide information concerning ion concentrations. In the current study we have used a pH sensitive fluorophore, SNAFL-2, to study the performance of this technique. We find that the sensitivity is such that a pH difference of 0.1 units can easily be detected in an 8-bit digital image. Noise measurements show that under realistic conditions we can expect a pixel-to-pixel standard deviation of approximately one to two pH units.

关键词： Medical imaging

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Dynamic image correlation spectroscopy (ICS) and two-color image cross-correlation spectroscopy (ICCS): Concepts and application

Dynamic image correlation spectroscopy (ICS) and two-color i...

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three-dimensional and multidimensional microscopy: image acquisition processing VII

作者： Wiseman, Paul W. Squier, Jeff A. Wilson, Kent R. Univ of California at San Diego La Jolla United States

The interactions of macromolecules in space and time are known to be important for the regulation of many biochemical reactions. image correlation spectroscopy (ICS) was recently introduced as an imaging analog of fluorescence correlation aspectroscopy (FCS) optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. We present two novel developments of dynamic ICS that will greatly enhance our abilities to measure molecular interactions as a function of time and space in living cells. We illustrate the use of a rapid scan two-photon microscope system to collect image series at high time resolution (30 frames/s) for dynamic ICS analysis. Secondly, we demonstrate the implementation of two-color image cross-correlation spectroscopy (ICCS) with a CLSM using multiple wavelength excitation, and with two-photon excitation of samples containing two different fluorescent species. Cross-correlation analysis allows the degree of co-localization of two different fluorophores to be measured directly. By performing two-color ICCS, we can monitor the interactions of non-identical labeled macromolecules as a function of time and space. We describe the experimental setup for both methods and illustrate the application for measurements of the diffusion coefficients of singly and doubly labeled fluorescent microspheres in aqueous solutions.

关键词： Medical imaging

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Laser phase microscopy and functional imaging of living human cancer cells during the cell cycle

Laser phase microscopy and functional imaging of living huma...

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three-dimensional and multidimensional microscopy: image acquisition processing VII

作者： Perevedentseva, Elena V. Graschew, Georgi Balanos, Evangelos Dressler, Cathrin Beuthan, Juergen Schlag, Peter Max-Delbrueck-Centrum fuer Molekulare Medizin Berlin Germany

The purpose of the investigation was to elaborate a new method of functional imaging of living tumor cells. Human colon carcinoma cells HCT116 were investigated with a conventional light microscope, confocal laser scanning microscope and with a laser phase microscope (LPM). The LPM is a functional imaging technique providing information about cell morphology which is imposed by the physiological inhomogeneity of the refractive index. The phase of the light wave passing through an object contains quantitative information about the object thickness, the shape, and the spatial distribution of the refractive index varying with morphology and chemical composition inhomogeneity inside the object. The new method of investigation of the cells in different stages of the cell cycle is developed. Every phase image of the investigated cells has been compared with conventional light microscopic and confocal microscopic images of the same cell. The relation between the cell state, their morphological peculiarities and the phase characteristics of the measured cell is determined. Data thus acquired, quantitatively characterizing intra- and intercellular processes during the cell cycle, and the method of measurements can be used to investigate with high optic resolution the mechanisms of different physical, chemical and biomolecular interactions with the tumor cells.

关键词： Medical imaging

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Data compression in microscopy: a comparative study

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 158-168页

作者： Kriete, Andres Klein, Norbert Berger, Lars C. Inst of Anatomy and Cell Biology Giessen Germany

ISBN: (纸本)0819430757;9780819430755

image compression in microscopy is a valuable technique, in particular if applied to multidimensional data. Information-preserving and competitive information-losing compression is applied to microscopical data and the resulting image quality is evaluated on a quantitative basis both in the spatial and frequency domain. Included are image data featuring different signal-to-noise ratios, but also voxel data for volume representations as well as graphical data for surface representations. The effect of compression on 3-D visualization and image management with data bases is included.

关键词： image compression

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three-dimensional super-resolution imaging of single molecules by spectrally selective confocal microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 82-89页

作者： Brakenhoff, G.J. van Oijen, A.M. Kohler, J. Muller, M. Schmidt, J. Univ of Amsterdam Amsterdam Netherlands

ISBN: (纸本)0819430757;9780819430755

It is shown that based on spectrally selective excitation of individual molecules in the focus of a high NA lens together with position sensitive imaging sub-resolution imaging of three-dimensional structures can be realised. The feasibility of the idea is demonstrated with NA=0.55 optics on a model system of pentacene molecules in p-terphenal host matrix.

关键词： Optical microscopy

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

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 146-157页

作者： Khatibi, S. Allansson, L. Gustavsson, T. Blomstrand, F. Hansson, E. Olsson, T. Chalmers Univ of Technology

ISBN: (纸本)0819430757;9780819430755

The use of three-dimensional reconstruction methods to estimate the cell volume of astroglial cells in primary culture was studied. Two-dimensional microscopic images were collected by using an automated image-acquisition system. The images were reconstructed by the Linear Maximum a Posteriori method and the non-linear Maximum Likelihood Expectation Maximization (ML-EM) method. A fast variant of the ML-EM method was also developed. Results of this study show that the ML-EM reconstructed images are adequate for the determination of volume changes in cells or parts thereof.

关键词： image reconstruction

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System for fluorescence lifetime imaging microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 55-64页

作者： van Geest, Lambertus K. Boddeke, Frank R. van Dijk, Pieter W. Kamp, Arjen F. van der Oord, Cornelis J.R. Stoop, Karel W.J. Lambert Instruments Netherlands

ISBN: (纸本)0819430757;9780819430755

An affordable, robust and easy-to-use fluorescence lifetime imaging microscopy (FLIM) workstation that is completely automated and does not need any difficult calibration procedure is introduced. The workstation consists of a standard fluorescence microscope, a modulated excitation light source, a camera, a modulation signal generator and acquisition/processing software. An evaluation of the system and its critical components is presented.

关键词： Optical microscopy

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Developments and applications of confocal theta microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 48-54页

作者： Haar, Frank-Martin Swoger, Jim Stelzer, Ernst H.K. Heidelberg Germany

ISBN: (纸本)0819430757;9780819430755

Confocal theta microscopy improves the resolution of confocal laser scanning microscopes by instrumentally solving the problem of the inferior axial resolution. A technical variation of this microscopy technique, the Single-Lens Theta microscopy (SLTM), is designed to be easily adapted to any common confocal laser scanning microscope. With SLTM, different kinds of other microscopical techniques are possible.

关键词： Optical microscopy

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Imaging modalities in confocal correlation microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 107-109页

作者： Juskaitis, Rimas Neil, Mark A.A. Wilson, Tony Oxford Univ Oxford United Kingdom

ISBN: (纸本)0819430757;9780819430755

We present a novel light efficient technique for obtaining optically sectioned images in wide-field microscopy. The technique is a further development of correlation microscopy and is based on using complementary structured light patterns to illuminate the specimen together with uncomplicated post-processing of the captured images.

关键词： Optical microscopy

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