Scanning Microscopy


This paper describes a general method for visualizing individual cells in intact tissue in three dimensions. The method involves immunostaining intact tissue to label specific cells, "optical sectioning" the stained tissue by laser scanning confocal microscopy, computationally reconstructing a three dimensional image data set from the digitized confocal optical sections, delineating isosurfaces of specific intensity within the reconstructed image by a "marching cubes" algorithm to generate polygon meshes defining boundaries of cells, and displaying individual cells, identified as three dimensional objects enclosed by contiguous polygon meshes, using computer graphics techniques. Each of the components of this method has been described previously in conjunction with other applications. However the combination of these techniques to visualize a variety of different individual cell types in three dimensions in intact tissue represents a new approach.

To illustrate the application of this method, we have visualized three different glial cell types in mouse CNS tissue. Oligodendrocytes, specifically stained with antibody to myelin basic protein, were used as an example of cells labelled with an internal membrane antigen. Astrocytes, specifically stained with antibody to glial fibrillary acidic protein, were used as an example of cells labelled with a cytoplasmic antigen. Microglia, specifically stained with Mac.1 antibody, were used as an example of cells labelled with an external membrane antigen. The images that are generated contain remarkably detailed volumetric and textural information that is not obtainable by conventional imaging techniques.

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