19] Measurement of secretion in confocal microscopy

Abstract

The chapter presents a discussion on measurement of secretion in confocal microscopy. The chapter describes the measurement of exocytosis and the paracellular pathway in rat salivary glands, using confocal microscopy combined with the fluorescent tracer technique. Salivary acini, the secretory end piece of this gland, are composed of highly polarized acinar cells capable of secreting enzymes and fluid; the secretion is regulated distinctively under different autonomic receptor control. Methods of cell dissociation, selection of fluorescent tracers, and analytical procedures are described. Secretion by exocytosis involves the cycles of fusion and the removal of secretory granule membranes at the cell surface (exocytosis-removal cycle). The chapter describes the visualization of the exocytosis-removal cycle of single secretory granules using fluid-phase fluorescent tracers. Practically, cell dissociation is the commendable way to image cells with high resolution. For the measurement of paracellular pathway (tight junctional permeability), fluid-phase fluorescent tracers of varying molecular weights are used. The combined use of different fluorescent tracers is possible when the emission wavelengths of tracers are discriminated distinctively by the detector. The basic structure of salivary acini is described first to evaluate properly the results of experiments.