ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03271.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03272.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SUMMARYA problem in three‐dimensional imaging using a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. A new method is proposed to correct for these effects. The approach, valid for weak attenuation, consists of multiplying the measured fluorescence intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the fast Fourier transform. Analytical and numerical estimates are given for the degree of attenuation under which the method is valid, and the method is applied to various test images. A real CSLM image is restored. Finally, the method is compared with a recent iterative method with regard to numerical accuracy and computational efficienc

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03273.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SUMMARYOptical modifications to a confocal scanning laser microscope are described which allow simultaneous fluorescence imaging of living specimens excited by ultraviolet (UV)‐ and visible‐wavelength light. Modifications to a Bio‐Rad MRC 600 Lasersharp confocal microscope include the introduction of UV‐path‐specific lenses and a specially designed UV transmitting eyepiece and tube lens. Upon UV excitation these modifications provide similar resolution and field flatness when compared with visible confocal microscopy. The UV‐path‐specific optics could be adjusted to correct for varying amounts of longitudinal chromatic aberration in commercially available objectives. Eyepiece and tube lenses were chromatically corrected for UV through visible wavelengths to minimize lateral chromatic error. With these modifications, UV‐wavelength light may be used to excite ratioing dyes to quantify intracellular ion concentrations, or as an energy source to release caged compounds in a spatially restricted volume, while simultaneously imaging with dyes excited by visible

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03274.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SUMMARYThis paper describes a new method for data representation and visualization in four dimensions (three dimensions plus time). Sequential volumes, exhibiting morphological activity, are acquired non‐invasively with a confocal scanning laser microscope, where each data set corresponds to a time sample. A pipelined processing includes packing of volumes and specific volume rendering techniques. Subsequent processing in HIS (hue, intensity, saturation) colour space combines functional, packed images with shaded three‐dimensional views. As a result, even subtle changes in morphology become visible and computational time is saved. Experimental findings obtained from investigations of synaptic plasticity in cultured retinal tissue are repor

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03275.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SUMMARYHigh‐pressure scanning electron microscopy (HPSEM) is a promising new family of techniques. The present knowledge of these techniques is reviewed and a new set of criteria developed for optimizing signal detection in HPSEM with a view to preserving specimen integrity. For this purpose, amplification of contrast signals generated in HPSEM was examined by computing the effect of ionization over a range of pressures and biasing fields, routinely used for this technique. The influence of secondary ionization due to ion impact was included in the calculations. To check the calculated results, the experiments were performed in the HPSEM apparatus in a nitrogen atmosphere. A divergence between the experimental values and the calculated values was found. This was removed by taking into account the effect of recombination of charge carriers. Inductive currents generated in the HPSEM environment are transient and do not affect the conclusions of this study. The gas pressure, biasing fields and beam current for preserving specimen integrity and obtaining good micrographs were selected from the data. Experimental measurements of noise are reported, and criteria for optimizing the signal‐to‐noise ratio for performing HPSEM are discussed. The application of these criteria has shown that specimen current detection can be more advantageous than other methods and configurations of detection and was capable of obtaining high/medium‐resolution micr

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03276.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SUMMARYUsing a high‐intensity synchrotron X‐ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D‐periodicity and fibril diameter.The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low‐temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast.Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical‐point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X‐ray pattern. Perhaps the most drastic effect was caused by post‐fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D‐periodicity which occurred during later stage

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03277.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SUMMARYGlucose embedding is a simple and highly effective method for preparing biological macromolecules for high‐resolution electron microscopy. The investigation of conditions that can trap the M‐state intermediate in the bacteriorhodopsin (bR) photocycle has revealed, however, that when glucose‐embedded bR is prepared at ambient humidity, it does not fully retain the capability to execute a proper photocycle. However, ‘native’ photocycle properties are returned after glucose‐embedded samples are equilibrated at 81% relative humidity. Equilibration at relative humidities significantly higher than 81% causes glucose to dissolve in its own water of hydration, resulting in samples that may be too thick to be suitable for electron microscopy. The results obtained with bR indicate that caution should be taken with other biological specimens, and it cannot be assumed that glucose‐embedded biological macromolecules retain completely their native, hydrated structure, even when high‐resolution electron diffraction patterns are obtained. Equilibration of such samples at high humidity may generally be a worthwhile precaution when using the glucose‐em

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03278.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SUMMARYAll transmission electron microscopy (TEM) studies require the use of grids to support thin films or sections of specimens within the microscope. These grids are presumed to remain inert during sample loading and analysis, an assumption which becomes questionable when on‐the‐grid processing methods are employed to probe the response of a specimen to a particular stimulus, such as temperature increase. Uniform dispersions of nanoclusters were observed to develop on and undergo chemical complexation with thin cast films of a poly(siloxaneimide) copolymer supported on various copper grids during thermal annealing at temperatures of up to 200°C. The thermal conditions which promoted nanocluster formation were investigated. Experiments designed to elucidate the mechanism by which these nanoclusters develop revealed that cuprous oxide (Cu2O) from the copper support grids is deposited on these imide‐bearing films from the vapour

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03279.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SUMMARYThe scan speed limit of atomic force microscopes has been calculated. It is determined by the spring constant of the cantileverk, its effective massm, the damping constantDof the cantilever in the surrounding medium and the stiffness of the sample. Techniques to measurek, k/mandD/mare described. In liquids the damping constant and the effective mass of the cantilever increase. A consequence of this is that the transfer function always depends on the scan speed when imaging in liquids. The practical scan speed limit for atomic resolution in vacuum is 0·1 μm/s while in water it increases to about 2 μm/s due to the additional damping of cantilever movements. Sample stiffness or damping of cantilever movements by the sample increase these limits. For soft biological materials imaged in water at a desired resolution of 1 nm the scan speed should not exceed 2 μ

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1993.tb03280.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY