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

Abstract

The brain is an inherently three-dimensional (3-D) structure with tissue regions that occupy large complex volumes and cells that have complicated morphologies, e.g. branching fields of dendrites and axons extending in all three dimensions. The linear dimensions range from micrometers to tens of millimeters. The confocal light microscope can image relatively large volumes of tissue, and is therefore an ideal imaging tool for studying the brain and its cells. We have used histochemical and immunohistochemical labels to delineate structure at the tissue, cellular, and subcellular levels. The 3-D images are analyzed by custom software that automatically segments and counts nuclei over large tissue regions, automatically montages any number of 3-D images, and traces branching structures such as neuronal processes or blood vessels. Examples of nuclear detection in the rat hippocampus, where the cell density is high and nuclear images are close together, and of tracings of the dendritic field of a dye-filled neuron from the visual cortex of the kitten are shown. Complete penetration of thick tissue sections by large immunolabeling molecular complexes is demonstrated in tissue double-labeled for glial fibrillary protein and laminin. The ability to correlate multiple labels in 3-D space is another strong point for confocal light microscopy. The influence of deconvolution on the quality of 3-D images is also demonstrated.