Confocal Fluorescence Microscopy in Three-Dimensional Analysis of Axon Terminal Distribution, Neuronal Connectivity, and Colocalization of Messenger Molecules in Nervous Tissue: Computerized Analysis

Publisher Summary This chapter provides an introduction to confocal fluorescence microscopy and its use in the quantitative analysis of immunofluorescently labeled axons. The chapter describes multicolor fluorescence recording and analysis and discusses some general principles for fluorescence microscopy, the main focus being on issues related to image analysis of nervous tissue subjected to immunofluorescence and/or fluorescent tracer studies. With the introduction of the confocal microscope, a tool can be obtained enabling light microscopic analysis of neuronal connectivity on the axon terminal level in three dimensions. Although confocal scanning laser microscope (CSLM) instruments have now come into general use, this technique has been employed in a number of neurobiological applications, revealing information previously impossible to obtain. The chapter describes the potential of CSLMs in fluorescence microscopy, exemplified by the procedure developed for quantitative axon terminal analysis. Thus, noncritical use of CSLMs in these types of applications may lead to misinterpretations of the data and possibly false conclusions. It can be envisaged that various types of software packages will become available for the CSLM user in the near future, not only providing routines for quantitative 3-D analysis but also helping with a number of the problems encountered in CSLM imaging such as light signal attenuation, point-spread function, and refractive index errors.