Three-dimensional light microscopy, optimized staining, and automated image analysis of cell nuclei in thick tissue slices

The number, size, shape and three-dimensional (3D) distribution of cell nuclei in thick tissue samples is often critical to our understanding of tissue level organization and function. Examples include the analysis of neurons in brain tissue as a function of development, electrical activity, and exposure to toxins as well as the development of whole embryos. The sample is stained with a fluorescent dye selective for nucleic acid, and observed with a confocal light microscope. The classic Feulgen stain, although primarily an absorption stain, is often used for this purpose. However, it is unreliable for fluorescence due to the mixture of compounds usually present in Fuchsin. We have optimized a procedure using acriflavine as the Schiff reagent for staining thick (100 - 500 μ m) brain slices. In addition, Ancin and Roysam have developed software for automatically analyzing 3D objects, which are classified by volume, shape, intensity and relative 3D spatial locations.