Confocal Microscopy and Three-Dimensional Image Analysis Methods for Recognition and Fluorometric Measurements: An Application to Inhibitory Glycine Receptor.

Abstract

Confocal scanning laser microscopy (CSLM) and 3D image analysis have been used for recognition and fluorometric measurements of fluorescent spots in specimens. The methods have been applied to clusters of chloride-dependent glycine receptors on the surface membranes of neurons in the central nervous system. The receptors were labeled with a specific monoclonal antibody and by indirect immunofluorescence. These glycine-activated chloride channels form aggregates that appear as spots with high fluorescence intensity and more or less regular contour, each corresponding to a postsynaptic density. A CSLM recorded the fluorescence within the specimens and described it with digital volumes. Digital 3D image analysis was used for recognition of spots in these volumes and for measurements of their geometric and photometric properties. When these methods were applied to neurons from the goldfish reticulata, the results indicated that the concentration of receptor is relatively stable from one cluster to another but their size and the total number of receptive molecules vary from one synaptic contact to the next. Results of this kind are important in modeling the integrative properties of central neurons.