Confocal light microscopy of brain cells and tissue: Image analysis & quantitation

The brain is an inherently three-dimensional (3-D) structure with tissue re gions that occupy large complex volumes and cells that have complicated mor phologies, e.g. branching fields of dendrites and axons extending in all th ree dimensions. The linear dimensions range from micrometers to tens of mil limeters. The confocal light microscope can image relatively large volumes of tissue, and is therefore an ideal imaging tool for studying the brain an d its cells. We have used histochemical and immunohistochemical labels to d elineate structure at the tissue, cellular, and subcellular levels. The 3-D images are analyzed by custom software that automatically segments and cou nts nuclei over large tissue regions, automatically montages any number of 3-D images, and traces branching structures such as neuronal processes or b lood vessels. Examples of nuclear detection in the rat hippocampus, where; the cell density is high and nuclear images are close together, and of trac ings of the dendritic field of a dye-filled neuron from the visual cortex o f the kitten are shown. Complete penetration of thick tissue sections by la rge immunolabeling molecular complexes is demonstrated in tissue double-lab eled for glial fibrillary protein and laminin. The ability to correlate mul tiple labels in 3-D space is another strong point for confocal light micros copy. The influence of deconvolution on the quality of 3-D images is also d emonstrated.