Quantifying Exocytosis by Combination of Membrane Capacitance Measurements and Total Internal Reflection Fluorescence Microscopy in Chromaffin Cells

• DOI: 10.1371/journal.pone.0000505
• Publication Date: 2007-06-05T18:33:11Z
• Authors: Ute Becherer; Mathias Pasche; Shahira Nofal; Detlef Hof; Ulf Matti; Jens Rettig
• Keywords: 0303 health sciences; 571; Neuroscience/Neuronal and Glial Cell Biology; Science; Chromaffin Cells; Secretory Vesicles; Q; Cell Membrane; R; Electric Capacitance; Exocytosis; Luminescent Proteins; 03 medical and health sciences; Cell Biology/Membranes and Sorting; Microscopy, Fluorescence; Neuroscience/Neuronal Signaling Mechanisms; Medicine; Animals; Cattle; Cells, Cultured; Research Article; Red Fluorescent Protein

Total internal reflection fluorescence microscopy (TIRF-Microscopy) allows the observation of individual secretory vesicles in real-time during exocytosis. In contrast to electrophysiological methods, such as membrane capacitance recording or carbon fiber amperometry, TIRF-Microscopy also enables the observation of vesicles as they reside close to the plasma membrane prior to fusion. However, TIRF-Microscopy is limited to the visualization of vesicles that are located near the membrane attached to the glass coverslip on which the cell grows. This has raised concerns as to whether exocytosis measured with TIRF-Microscopy is comparable to global secretion of the cell measured with membrane capacitance recording. Here we address this concern by combining TIRF-Microscopy and membrane capacitance recording to quantify exocytosis from adrenal chromaffin cells. We found that secretion measured with TIRF-Microscopy is representative of the overall secretion of the cells, thereby validating for the first time the TIRF method as a measure of secretion. Furthermore, the combination of these two techniques provides a new tool for investigating the molecular mechanism of synaptic transmission with combined electrophysiological and imaging techniques.