ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01514.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA commonly used three‐dimensional reconstruction method in confocal scanning laser microscopy (CSLM) involves the generation of stereo views by stacking optical sections. Under certain conditions the resulting stereo pairs exhibit inferior quality with respect to contrast and depth perception. A method based on digital image processing is described in which the individual images are enhanced prior to reconstruction, thereby increasing the number of usable optical sections by a factor of up to five. Furthermore, we introduce a new contrast enhancement transformation based upon local statistics and a grey‐level probability density function that provides improved depth perception. Digital image processing methods map a discrete grey scale onto a continuous, unbounded interval. Inasmuch as a closed, discrete grey scale is required for computer display purposes, we present an appropriate mapping function derived from an entropy criter

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01515.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThree‐dimensional (3‐D) images of osteocyte lacunae were examined on a confocal microscope. Both geostatistical and morphological processing techniques were used to improve and to analyse them. By a geostatistical approach, this study aims at improving 3‐D confocal images before any further image processing. Optimized linear filters, which take account of the second‐order statistics and the 3‐D structure of the data, allow for the removal of imperfections such as noise and/or blur due to the axial convolution, and interpolate voxels on a face‐centred cubic grid from an initial cubic grid. An application of this technique to 3‐D biological images is demonstrated. In a second step, a 3‐D binary image is digitized and cleaned with 3‐D morphological filters. The standard 3‐D measurements cannot be applied in this case, since all osteocytes cut the border of the field. For this reason a 3‐D Boolean model has been adjusted, from which it is possible to derive all useful information on the repartition and the morphol

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01516.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYMicroscopic resolution can be characterized byK= x NA/δ, where x is the distance between two objects, or the interval of a grating, just resolved with light of wavelength λ and an objective of aperture NA. Using a computer simulation of imaging the followingKvalues were obtained on the Sparrow resolution criterion for line and grating objects and various imaging methods (figures for the Rayleigh criterion, which assumes a finite contrast‐sensitivity of the light detector, are in parentheses). Several results appear to be novel. Due to limitations discussed in the text some data are only approximate.With a grating objectKis 1.0 (1.0) for axial coherent illumination, 0.5 (0.5) for coherent illumination at an obliquity NAobiwhich just enters the objective aperture, 0.5 (about 0.53) for incoherent illumination, 0.5 (about 0.52) for illumination with a condenser aperture NAcequal to NA, 0.5 (about 0.515) for transmitted‐light confocal scanning, and 0.25 (about 0.38) for fluorescent confocal scanning. If the object consists of two parallel linesKis about 0.68 (0.71) for axial coherent illumination, about 0.44 (0.5) for incoherent illumination, 0.375 (about 0.48) for optimal partially coherent illumination in which NAcmay exceed NA, 0.44 (0.48) for transmitted‐light confocal scanning, and 0.32 (0.41) for fluorescent confocal scanning.For inter‐object distances of 1, 1.5 and 2 wavelengths, respectively, NAcvalues of about 0.69, 0.5 and 0.375 gave optimal contrast and resolution irrespective of NA. The practice of setting NActo about two‐thirds of the NA of a high‐power objective is supported by the fact that a condenser aperture of about 0.69 gives excellent or optimum resolution and contrast for most inter‐object distances and objective apertures tested, although with some distances and apertures reducing NAcimproved contrast slightly.The rule (sometimes attributed to Abbe) that resolving power is proportional to the mean of NA and NAcis correct for oblique coherent illumination in the case of a grating object, provided NAobldoes not exceed NA. In the case of two isolated objects the rule is only approximately correct, but applies even if NAoblis greater than NA. Coherent light at an obliquity of 0.5λ/x introduces a half‐wavelength phase difference between two objects and permits their resolution (with perhaps an incorrect apparent inter‐object distance) even with objective apertures approaching zero.In confocal scanning the width of the scanning spots has only a moderate effect on resolution, and two objects can sometimes be resolved with scanning spots wider than the inter‐object distance provided the lens apertures are neither t

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01517.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYGlycol methacrylate (GMA) sections of animal tissues were stained with a group of twenty‐seven reagents of very varied chemical characteristics. The artefactual background staining of the resin was found to be dependent on the hydrophilic/lipophilic character of the staining reagent, as estimated from the logarithm of its octanol‐water partition coefficient (logP). Intense background staining occurred with lipophilic stains, whose logP>2. In keeping with this, use of GMA semi‐permeable membranes for enzyme histochemistry failed to give staining when using a lipophilic substrate, probably because the substrate was trapped in the membrane. An analysis of other routine histochemical stains—in terms of the probable occurrence of high resin background staining and low tissue sensitivity—is made. A numerical guide is provided to help avoid artefacts resulting from hydrophobic and size effects. Note: small, hydrophilic reagents (logP<0; molecular weight2; 550

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01518.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA new cryo‐jet freezing apparatus is described that is easy to use and gives good results using a propane‐butene mixture (3: 1). Our use of the freezer in the study of mouse spinal cord explant cultures is discussed. At the tissue surface, the quality of tissue preservation from freezing, followed by freeze substitution, rivals that of conventional electron microscopic methods. Certain intracellular structures are better visualized using our methods. There is no evidence of the tissue being distorted by the cryogen jet when the freezer is operated correctly. A new freeze substitution device is also discus

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01519.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe interpretation of electron microscope autoradiographs concerns the estimate of the relative concentration of a radiolabelled molecule in subcellular compartments. Because of the radiation spread, an autoradiographic grain underlying a subcellular compartment cannot be directly assigned to the latter. Observed data must therefore be corrected for cross‐fire (CF). We studied the efficiency of several methods designed for the CF correction when membrane components possess a high concentration of labelling, resulting in a high proportion of CF. Grains generated from simulated sources were subjected to the CF method, to an original method based on the simulated annealing optimization algorithm, and to the expectation‐maximization algorithm. The expectation‐maximization algorithm appears to be clearly superior to the two other methods. Nevertheless, the variances of the estimates were higher than expected. Since no analytical expression of the estimates is available, an objective comparison of the labelling in different compartments is not possible with a standard test. Consequently, the interpretation of autoradiographs in electron microscopy is still an open pr

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01520.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYExtended electron energy‐loss fine structure (EXELFS) and selected‐area electron diffraction (SAED) techniques have both been applied to the study of the crystalline structure of Pd clusters of average diameters ranging from bulk to 24 Å. The combined use of these techniques gives complementary information about the crystalline structure of Pd clusters. Both techniques show the same lattice parameter expansion, about 4% for the smallest Pd cluster, with respect to the bulk. The EXELFS analysis performed on the Pd‐M4,5edge shows a sizeable increase of structural disorder in the smallest cluster. SAED gives additional information about the Pd bulk sample, showing the occurrence of crystalline regions about 50 Å in d

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01521.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYEvidence is provided for the formation of surface faceting due to heating in air at 1160 K. For the first time diamond‐shaped facets with angles of 60 and 120° have been observed on the surface of a sample of ultrapure platinum wire in air using scanning tunnelling microscopy. The maximum extension of the facets is about 1000

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01522.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01523.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY