In this paper we present a three-dimensional visualization technique for multi-channel volume data. The technique simulates the physical process of fluorescence, hence its name: achromatic multi-channel simulated fluorescent process (umSFP). The data set is simulated as 3D distribution of different fluorescent dyes, where each channel is represented by a particular type of dye. Apart from the spatial density map, no additional characteristics about the data set have to be defined; no image segmentation is needed prior to visualization. The degree of interaction among the channels in the fluorescence process can be adapted to optimally render specific structures in the image. 3D multi-channel data can be obtained by a three-dimensional imaging device that is able to measure a number of physical quantities at a given location within a specimen. The fluorescence principle, the algorithm, and its implementation are presented. We have used the technique to investigate the relative spatial arrangement of blood vessels and astrocytes in the cat retina. The two components have been stained with different fluorescence dyes and recorded in a confocal light microscope to form a twochannel 3D data set. o 1993 Wiley-Liss, Inc.

The distribution of astrocytes in the vascularized retina of pigs, rats and cats was investigated by confocal microscopy and computer-assisted image processing. In whole mounts, immunocytochemical identification was done by staining astrocytes for glial fibrillary acidic protein (GFAP), and blood vessels for alpha-smooth muscle actin or collagen IV. Double-staining was followed through consecutive optical sections and made it possible to precisely align the two markers in the inner retina. The resulting computer-assisted image reconstructions revealed asymmetric ensheathment of blood vessels by GFAP-positive fibres. The ultrastructural basis for this asymmetry, as studied by electron microscopy, was found to be different in pigs and cats. In the pig, astrocytes firmly ensheathed the vessel circumference, but glial filaments were much more abundant on the vitreal and lateral than on the scleral side. By contrast, in the cat astrocytes were generally confined to regions occupied by axonal bundles and constituted only part of the vascular glia limitans, else formed by Müller cells. Moreover, our observations unambiguously showed that individual astrocytes maintained simultaneous contact with axons and blood vessels and lined the vitreous body. The physical links provided by astrocytes suggest that they are able to function as central communicating elements between ganglion cells, the vasculature and the vitreous body.

Confocal light microscopy has found its place among the standard analytical tools in cell and molecular biology. When combined with techniques such as immunofluorescence or fluorescent in situ hybridization, the spatial distribution of individual biological components can be traced within cells and tissues and, under certain circumstances, even with living samples. In this article, advanced 3D visualization techniques have been applied to analyze the distribution of myofibrillar proteins in cultured adult rat cardiomyocytes. By combining confocal immunofluorescence microscopy with specially designed three-dimensional visualization, we have obtained images which are similar to those obtained with the scanning electron microscope. The subcellular distribution of proteins expressed after transfection of cDNA is monitored in the cultured heart cells. The expressed proteins are distinguished from their endogenous counterparts by the use of an epitope tagging technique. The described methods are suitable to specifically monitor the behavior of several closely related isoprotein mutants in cell or tissue preparations.

No abstract

No abstract