Three-Dimensional Reconstruction of Fluorescence-Labeled Neurons in the Lamprey Central Nervous System, Using a Confocal Laser Scanning Microscope

Abstract

Confocal microscopy is a particularly powerful analysis tool for the study of the three-dimensional (3-D) morphology of cells with a complex shape, such as nerve cells. The combination of specific fluorescence labeling of functionally characterized neurons and confocal laser scanning microscopy (CLSM) opens the possibility of directly correlating the 3-D morphology with the function of the neuron under study. This paper describes the application of the CLSM technique to the study of the 3-D structure of identified neurons in the vertebrate central nervous system, utilizing whole-mount or thick-section preparations of the CNS of the lamprey, a lower vertebrate. Features of the CLSM technique particularly relevant to 3-D analysis are discussed, including aspects of image processing and presentation of 3-D images, as well as multiple fluorophore detection. The procedures used for fluorescence labeling, tissue preparation, and scanning of the tissue volume are also described, along with hints for a successful analysis and avoidance of pitfalls.