ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03538.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYBecause of shadowing, multiple scatter and polarization effects, the interpretation of images of gratings with fine periods, isolated deep structures, and multiple scattering volume objects is seriously complicated. In this paper a review of the methods used to model such effects is presented. Periodic surface relief gratings are of particular current importance because of the possibility of producing calibration samples using them. Several examples which illustrate electromagnetic volume effects are examined. General trends which help in validating the use of Fourier‐transform‐based scalar transmittance theory are then indicated. The angular spectrum approach, which can be used, together with a scatter function generated using the rigorous electromagnetic theory, to calculate coherent, partially coherent and confocal images of volume objects, is also discus

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03539.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYA method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three‐dimensional (3‐D) DNA image cytometry is described. The method is based on studying the stack of 2‐D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin‐fixed paraffin‐embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3‐D DNA imag

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03540.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYNear‐field optics (NFO) opens the door to light microscopy techniques with resolutions well beyond the diffraction limit. The richness of optical investigations is now applicable on a near‐molecular level. Among the novel scanning near‐field optical microscopy (SNOM) schemes, the most prominent representatives are aperture SNOM and scanning tunnelling optical microscopy (STOM or PSTM).New experimental and theoretical work has to be performed to study the phenomena specific to NFO. One such example is the angular dependence of light emission in aperture SNOM. The detection of radiation at angles greater than the critical angle of total internal reflection αc= arcsin(n−1), wherenis the sample refractive index, can represent a microscopy scheme that combines the respective advantages of both aperture SNOM and STOM. Recent experiments have demonstrated the expected exponential dependence of light intensity on gap width (for fixed emission angle α>αc). The decay length as a function of α is in agreement with the Fresnel description of the evanescent field when total reflection occurs at an interface. These investigations were additionally motivated by calculations based on the multiple multi

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03541.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYThis paper describes an objective and automatic method for detection and correction of sectioning deformations in digitized micrographs, as well as an evaluation of the method applied to light and electron microscopic images of semi‐thin and ultra‐thin serial sections from brain cortex. The detection is based on matching of image subregions and the deformation model is bi‐linear, i.e. two first‐order polynomials are used for modelling compression/expansion in perpendicular directions. The procedure is applicable to prealigned serial two‐dimensional sections and is primarily aimed at three‐dimensional reconstruction of tissue samples consisting of a large number of cells with random distribution and

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03542.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYThe imaging performance in single‐photon (1‐p) and two‐photon (2‐p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three‐dimensional (3‐D) point spread function and the 3‐D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2‐p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3‐D optical transfer function has the strongest response along th

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03543.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYScanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three‐dimensional picture of the rock microfractography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock‐forming components.The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three‐dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures.These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water‐conducting open fissures, and the rock‐forming minerals provide a means of modelling the ‘radionuclide retardation mechanism’, a security factor in their definitive storage i

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03544.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYThis study demonstrates the use of Voronoi tessellation procedures to obtain quantitative morphological data for chromosome territories in the cell nucleus. As a model system, chromosomes 7 and X were visualized in human female amniotic fluid cell nuclei by chromosomalin situsuppression hybridization with chromosome‐specific composite probes. Light optical serial sections of 18 nuclei were obtained with a confocal scanning laser fluorescence microscope. A three‐dimensional (3‐D) tessellation of the image volumes defined by the stack of serial sections was then performed. For this purpose a Voronoi diagram, which consists of convex polyhedra structured in a graph environment, was built for each nucleus. The chromosome territories were extracted by applying the Delaunay graph, the dual of the Voronoi diagram, which describes the neighbourhood in the Voronoi diagram. The chromosome territories were then described by three morphological parameters, i.e. volume, surface area and a roundness factor (shape factor). The complete evaluation of a nucleus, including the calculation of the Voronoi diagram, 3‐D visualization of extracted territories using computer graphic methods and parameterization was carried out on a Silicon Graphics workstation and was generally completed within 5 min. The geometric information obtained by this procedure revealed that both X‐ and 7‐chromosome territories were similar in volume. Roundness factors indicated a pronounced variability in interphase shape for both pairs of chromosomes. Surface estimates showed a significant difference between the two X‐territories but not between chromosome

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03545.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYBased on the principle of laser‐feedback interferometry (LFI), a laser‐feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ∼ 1 nm and a lateral (x, y) resolution of ∼ 200 nm when used with a high‐numerical‐aperture oil‐immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re‐entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back‐reflected light gives information about an object's position and reflectivity. Using a 1‐mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256‐pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ∼ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back‐reflected light originating from small refractive‐index changes (>0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point‐by‐point by growing or placing a cell suspension on a higher‐reflecting substrate (glass or a silicon wafer). Advantages of the laser‐feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single‐axis interferometric design; the confocal property of the laser‐feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re‐enters the laser meeting the stringent frequency, spatial (TEM00), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use o

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03546.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SUMMARYA theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non‐steady‐state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determinat

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03547.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY