3-DIMENSIONAL IMAGING AND IMAGE-ANALYSIS OF HIPPOCAMPAL-NEURONS - CONFOCAL AND DIGITALLY ENHANCED WIDE-FIELD MICROSCOPY

The microscopy of biological specimens has traditionally been a two-di mensional imaging method for analyzing what are in reality three-dimen sional (3-D) objects. This has been a major limitation of the applicat ion of one of science's most widely used tools. Nowhere has this limit ation been more acute than in neurobiology, which is dominated by the necessity of understanding both large- and small-scale 3-D anatomy. Fo rtunately, recent advances in optical instrumentation and computationa l methods have provided the means for retrieving the third dimension, making full 3-D microscopic imaging possible. Optical designs have con centrated on the confocal imaging mode while computational methods hav e made 3-D imaging possible with wide field microscopes using deconvol ution methods. This work presents a brief review of these methods, esp ecially as applied to neurobiology, and data using both approaches. Sp ecimens several hundred micrometers thick can be sampled allowing esse ntially intact; neurons to be imaged. These neurons or selected compon ents can be contrasted with either fluorescent, absorption, or reflect ion stains. Image analysis in 3-D is as important as visualization in 3-D. Automated methods of cell counting and analysis by nuclear detect ion as well as tracing of individual neurons are presented. (C) 1994 W iley-Liss, Inc.