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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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Numerical simulation of image formation in near-field optical microscopy

Numerical simulation of image formation in near-field optica...

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

作者： Baibyrin, Vil B. Volkov, Uryi P. Avetisyan, Uryi A. Krasnikova, Irina V. Bespalova, Natalija V. Saratov State Technical University Politechnitcheskaja 77 Saratov 410007 Russia

Numerical simulation of image formation in near field optical microscopy is needed to understand a relationship between near field images and actual structure of sample since the image can be differs strongly from real structure. In order to estimate the near field image formation, two different approaches are used, namely numerical solution of integral equations with boundary element method or moment method and analytical solution of light scattering problem obtained by assumption of some hypotheses. The first approach results in reasonably complicated algorithm of calculation and often causes a bed conditioned system of linear equations to be formed. The approach based on an analytical solution of scattering problem is more preferable since allow to reduce the algorithm and increase the calculations speed. A new program of simulation of near field image formation for arbitrary sample shape has been developed. The program is based on the results of analytical calculation of near field light diffraction on sinusoidal diffraction grating of arbitrary corrugation amplitude. The sample shape is presented as a number of sinusoidal gratings of various amplitudes by Fourier transformation, for each of the gratings the light scattering amplitude is calculated and the amplitudes are summarized.

关键词： Near field scanning optical microscopy

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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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High resolution spectral self-interference fluorescence microscopy

High resolution spectral self-interference fluorescence micr...

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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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Optical properties of spherical microlens

Optical properties of spherical microlens

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

作者： Volkov, UP Avetisyan, UA Baibyrin, VB Bespalova, NV Saratov State Tech Univ Saratov 410007 Russia

ISBN: (纸本)0819443603

A scanning optical far and near-field microscopes resolution is limited by focused light spot size used to scan a sample. As a focusing element in scanning microscopes are ordinary used lenses with dimensions of rather more than a light wavelength. The focus spot size of such lens is limited by well-known diffraction limit. However, if a lens dimension is comparable to a light wavelength the Fresnel's diffraction formula of focus spot size calculation is not suitable. At present, there are no data about theoretical or experimental studies of the small lenses and even about the ones fabrication methods. This paper is considers the fabrication method of small spherical glass lenses with diameters from I um to 100 um and discusses some theoretical approaches to the focus spot size calculation and it experimental measurement. The lenses fabrication method is based on melting of fine glass powder with oxygen-acetylene burner with subsequent lenses selection under conventional light microscope. To calculate a spot size of the small lens the Mie theory of light scattering in near field by small spherical particles has been used. The light intensity diagrams of sphere lens of various diameters calculated by developed program are presented.

关键词： spherical microlens scanning optical microscopy Mie theory optic

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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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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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Programmable beam-splitter for confocal laser scanning microscopy

Programmable beam-splitter for confocal laser scanning micro...

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

作者： Birk, H Engelhardt, J Storz, R Hartmann, N Bradl, J Ulrich, H Leica Microsyst Heidelberg GMBH D-68165 Mannheim Germany

ISBN: (纸本)0819443603

In confocal microscopy manifold combinations of dyes are used. The entire wavelength range from near-ultraviolet to near-infrared is used to excite these dyes. Their emission is then detected primarily in the visible range. In addition to the number of dyes, the number of possible excitation laser lines is already large and constantly increasing. Due to the almost unlimited number of possible combinations, programmable devices are required. This is true for excitation-modules, beam-splitters and detection modules. Systems using filters with fixed spectral properties can practically fulfill the requirements for only a very small subset of applications. Programmable devices have been realized for excitation using Acousto Optical Tunable Filters (AOTF's). Freely definable spectral detectors are available for a perfect adaptation to the desired emission bands. The missing link for a completely filter-free design is now introduced using an Acousto Optical Beam Splitter (AOBS(TM)).

关键词： multi-spectral fluorescence detection Acousto-Optical Tunable Filter (AOTF) Acousto Optical Beam Splitter (AOBS((TM))) confocal laser scanning

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Standards in digital microscopy

Standards in digital microscopy

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

作者： Kriete, A Shah, S Inst Anat & Cell Biol IPL D-35385 Giessen Germany

This paper discusses some aspects of standardization in digital microscopy. Included are image acquisition, use of image and graphics formats and data protocols in multidimensions, particular image analysis procedures... 详细信息

ISBN: (纸本)0819439398

关键词： Standards microscopy image acquisition image formats image processing

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Inverse scattering with subwavelength resolution in the near-zone

Inverse scattering with subwavelength resolution in the near...

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

作者： Carney, PS Schotland, JC Washington Univ Dept Elect Engn St Louis MO 63130 USA

ISBN: (纸本)0819439398

In classical microscopy and inverse scattering, images are made from far field measurements. It will be shown that the standard Abbe or Rayleigh resolution limits arise as a result of assumptions which do not hold in the near field. Existing technologies such as TIRM and PSTM that exploit these properties of the near field produce two dimensional images which exhibit structure on a subwavelength scale. These images have proven difficult to interpret when the sample presents variations in structure and optical properties simultaneously in three dimensions. We present the analytic singular value decomposition of the linearized scattering operator. This representation leads to a robust inversion algorithm for the inverse scattering problem in the near zone. Numerical implementation of the algorithm is demonstrated using simulated noisy data to produce tomographic images of a three dimensional scatterer.

关键词： near-field optics inverse scattering microscopy tomography

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Point spread functions for particle imaging using inhomogeneous polarization in scanning optical microscopy

Point spread functions for particle imaging using inhomogene...

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

作者： Youngworth, KS Biss, DP Brown, TG Univ Rochester Inst Opt Rochester NY 14627 USA

ISBN: (纸本)0819439398

Polarization control and analysis continue to be a useful tools for microscopy. We have previously addressed the use of spatially inhomogeneous polarization, specifically azimuthal polarization, in scanning optical microscopy. In this paper we describe new techniques for generating both azimuthally and radially polarized cylindrical-vector beams, investigate high numerical aperture focusing, compare the theoretical and experimental point spread functions for small particle imaging, and explore the use of these beams with microscopy.

关键词： radial polarization azimuthal polarization inhomogeneous polarization cylindrical-vector beams axial electric field high numerical aperture focusing scanning optical microscopy point spread function small particle imaging

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