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Conference on 3-dimensional and multidimensional microscopy: image acquisition and processing ix

作者： Swan, AK Moiseev, L Tong, YJ Lipoff, SH Karl, WC Goldberg, BB Ünlü, MS Boston Univ Dept Elect & Comp Engn Boston MA 02215 USA

ISBN: (纸本)0819443603

We present a new method of fluorescence imaging, which yields nm-scale axial height determination and similar to15 nm axial resolution. The method uses the unique spectral signature of the fluorescent emission intensity well above a reflecting surface to determine vertical position unambiguously. We have demonstrated axial height determination with nm sensitivity by resolving the height difference of fluorescein directly on the surface or ontop of streptavidin. While different positions of fluorophores of different color are determined independently with nm precision, resolving the position of two fluorophores of the same color is a more convoluted problem due to the finite spectral emission widow of the fluorophores. Hence, for physically close (

关键词： fluorescence microscopy self interference spectroscopy

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Optically sectioned images in widefield fluorescence microscopy

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

作者： Wilson, T Neil, MAA Juskaitis, R Univ Oxford Dept Engn Sci Oxford OX1 3PJ England

An analysis is presented of the image formation in widefield fluorescence microscopy using standard light. The region of support of the resulting optical transfer function is discussed.

ISBN: (纸本)0819427004

An analysis is presented of the image formation in widefield fluorescence microscopy using standard light. The region of support of the resulting optical transfer function is discussed.

关键词： Luminescence microscopy Fringe analysis Spatial frequencies image acquisition Microscopes Imaging systems Optical transfer functions Point spread functions Roads

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High-throughput hyperspectral microscopy

High-throughput hyperspectral microscopy

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

作者： Gehm, M. E. Brady, D. J. Duke Univ Fitzpatrick Ctr Photon Durham NC 27708 USA

ISBN: (纸本)0819461326

A hyperspectral imager provides a 3-D data cube in which the spatial information (2-D) of the image is complemented by spectral information (1-D) about each spatial location. A static, high-throughput spectrometer design previously developed by our group can be used as the spectral engine in a high-throughput hyperspectral imager that avoids the Fourier undersampling issues present in previous dispersive designs. We present the theory for both pushbroom and tomographic operation and describe experimental results from our proof-of-concept implementation of a hyperspectral microscope.

关键词： hyperspectral imaging multidimensional microscopy

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three-dimensional reconstruction by focus sensing from conventional 2D image slices

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

作者： Nahm, KB Shin, ES Ryoo, SM Hallym Univ Dept Phys Chunchon 200702 Kangwon South Korea

ISBN: (纸本)0819427004

We introduce an optical means of reconstructing the topology of rough objects observed under the microscope or at a relatively high magnification. First, 2D images were obtained under the conventional optical microscope or under the macroscopic imaging system, focused on differing height of the sample. The depth information was extracted by sensing the focus of each pixel of the conventional image slice. Each sample height associated with the best "focus figure" for each pixel was marked and were later utilized to generate the three dimensional coordinate map.

关键词： surface reconstruction microscopy focus sensing image slice

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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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Mapping organism expression levels at cellular resolution in developing Drosophila

Mapping organism expression levels at cellular resolution in...

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Conference on 3-dimensional and multidimensional microscopy: image acquisition and processing ix

作者： Knowles, DW Keränen, S Biggin, MD Sudar, D Lawrence Berkeley Lab Dept Cell & Mol Biol Berkeley CA 94720 USA

ISBN: (纸本)0819443603

The development of an animal embryo is orchestrated by a network of genetically determined, temporal and spatial gene expression patterns that determine the animals final form. To understand such networks, we are developing novel quantitative optical imaging techniques to map gene expression levels at cellular and sub-cellular resolution within pregastrula Drosophila. Embryos at different stages of development are labeled for total DNA and specific gene products using different fluorophors and imaged in 3D with confocal microscopy. Innovative steps have been made which allow the DNA-image to be automatically segmented to produce a morphological mask of the individual nuclear boundaries. For each stage of development an "average" morphology is chosen to which images from different embryo are compared. The morphological mask is then used to quantify gene-product on a per nuclei basis. What results is an atlas of the relative amount of the specific gene product expressed within the nucleus of every cell in the embryo at the various stages of development. We are creating a quantitative database of transcription factor and target gene expression patterns in wild-type and factor mutant embryos with single cell resolution. Our goal is to uncover the rules determining how patterns of gene expression are generated.

关键词： confocal imaging image segmentation image registration quantitative fluorescence analysis regulatory gene networks Drosophila

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image estimation accounting for point-spread function depth variation in three-dimensional fluorescence microscopy

Image estimation accounting for point-spread function depth ...

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

作者： Preza, C Conchello, JA Washington Univ Dept Elect Engn St Louis MO 63130 USA

ISBN: (纸本)0819447641

An approximate model for optical-sectioning microscopy describing depth-varying imaging is developed. The model incorporates changes in the point-spread function due to refractive index mismatch between the immersion medium and the specimen. which causes spherical aberration that worsens with increasing depth tinder the coverslip. Comparison of model predictions to measured images from a bead phantom shows that the approximate model captures the main features in the data. The model presented in this paper is the first step towards depth-variant image estimation for optical-sectioning microscopy. An expectation maximization algorithm for maximum-likelihood restoration based on this model is also presented.

关键词： 3D microscopy depth-variant imaging model maximum likelihood estimation

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2-D and 3-D deconvolution of confocal fluorescence images by maximum likelihood estimation

2-D and 3-D deconvolution of confocal fluorescence images by...

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three-dimensional and multidimensional microscopy: image acquisition and processing ix

作者： Larson, Jeffrey M. Nikon Instruments Inc. Melville NY 11747 United States

Deconvolution of confocal fluorescence images by maximum likelihood estimation (MLE) was investigated for its ability to increase the information content in the images. 3-D MLE algorithms, applied to confocal image stacks, increase lateral and axial resolution and result in a finer optical section. Contrast, especially at edges, is enhanced, improving the documentation quality of high magnification images beyond that possible by confocal microscopy alone. Axial smear associated with spherical aberration was not removed by deconvolution and a lateral thinning artifact was introduced. Single confocal images can be rapidly deconvolved by 2-D MLE by applying a two-dimensional point spread function and treating them as images of planar objects. The 2-D algorithm can also deconvolve a maximum projection of a stack. The method works best when there is a minimal overlap of fluorescent structures. The projection is treated as a two dimensional object. Intensity information excluded by the projection operation cannot be recovered. Deconvolution of images acquired with the pinhole opened to increase sensitivity closely matches images acquired through an optimal opening, although in 2-D MLE, colocalization cannot be distinguished from overlap and the integrity of quantitative data cannot be guaranteed. Properly applied, MLE deconvolution increases the useful information content of confocal images.

关键词： image processing

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image enhancement for a low cost TEM acquisition system.

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

作者： Randall, G Fernandez, A Trujillo-Cenoz, O Apelbaum, G Bertalmio, M Vazquez, L Malmierca, F Morelli, P Univ Republ Fac Ingn Inst Ingn Elect Montevideo Uruguay

This paper describes a method for the improvement of biological images acquired using a Transmission Electronic Microscope (TEM). Several techniques are presented that deal with noise reduction, artifact removal and n... 详细信息

ISBN: (纸本)0819427004

关键词： Transmission Electronic microscopy image processing non-uniform illumination

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Depolarization by high-aperture focusing

Depolarization by high-aperture focusing

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Conference on 3-dimensional and multidimensional microscopy: image acquisition and processing ix

作者： Bahlmann, K Hell, SW MIT Dept Mech Engn Cambridge MA 02139 USA

ISBN: (纸本)0819443603

We propose and demonstrate a method employing ferroelectric monomolecular layers, by which it is possible to precisely measure the planar light field polarization in the focus of a lens. This method allowed us to establish for the first time to our knowledge, the perpendicularly oriented field that is anticipated at high apertures. For a numerical aperture 1.4 oil immersion lens illuminated with linearly polarized plane waves, the integral of the modulus square of the perpendicular component amounts to (1.51 (r)0.2)% of that of the initial polarization. It is experimentally proven that depolarization decreases with decreasing aperture angle and increases when using annular apertures. Annuli formed by a central obstruction with a diameter of 89% of that of the entrance pupil raise the integral to 5.5%. This compares well with the value of 5.8% predicted by electromagnetic focusing theory;however, the depolarization is also due to imperfections connected with focusing by refraction. Besides fluorescence microscopy and single molecule spectroscopy, the measured intensity of the depolarized component in the focal plane is relevant to all forms of light spectroscopy combining strong focusing with polarization analysis.

关键词： Luminescence Polarization Molecules Objectives microscopy Single molecule spectroscopy Refraction Electromagnetism Wavefronts Optical microscopy

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