title: Quantifying exocytosis by combination of membrane capacitance measurements and total internal reflection fluorescence microscopy in chromaffin cells.
abstract: 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.
authors.affiliation: Universität des Saarlandes, Physiologisches Institut, Homburg, Saar, Germany. ute.becherer@uks.eu
authors.forename: Ute
authors.initials: U
authors.lastname: Becherer
authors.forename: Mathias
authors.initials: M
authors.lastname: Pasche
authors.forename: Shahira
authors.initials: S
authors.lastname: Nofal
authors.forename: Detlef
authors.initials: D
authors.lastname: Hof
authors.forename: Ulf
authors.initials: U
authors.lastname: Matti
authors.forename: Jens
authors.initials: J
authors.lastname: Rettig
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology*; D025802:Electric Capacitance; D005089:Exocytosis / Q000502:physiology*; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D022142:Secretory Vesicles / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein