Intensity attenuation of Three-Dimensional, confocal fluorescence images of thick tissue
The confocal scanning laser microscope (CSLM) provides three-dimensional (3-D) images from fixed tissues. These images are obtained by stacking consecutive confocal planes through the depth of a specimen. We have developed software for quantitative analysis of these data sets and have applied these methods to cell counting. A major issue in this analysis is the relative intensity of signal measured from like objects through the depth of the specimen.The rat hippocampus was chosen to test the attenuation of the fluorescence signal allowing us to make observations in areas of higher density-pyramidal cell layer-and lower density -extra-pyramidal regions. Paraformaldehyde fixed specimens were stained with Feulgen-Schiffs acriflavine. This stain was selected for its fluorescent properties and its high DNA specificity. Specimens were mounted in media with different glycerol concentrations. Significant attenuation was observed using a 50:50 mixture of glycerol and buffer, while 100% glycerol provided significantly less attenuation. All data presented here were collected using sections mounted in 100% glycerol. 3-D images were collected from 50-, 75-, and 100-μm thick sections with a 40x oil objective, having an (x,y) resolution of 2 pixels//xm and a distance of 1 fim between optical sections. Fig. 1 is a projection of a 100-μm section of the rat hippocampus illustrating pyramidal and extra-pyramidal areas.