Imaging a target of Ca2+ signalling: dense core granule exocytosis viewed by total internal reflection fluorescence microscopy.
Ca2+ ions are the most ubiquitous second messenger found in all cells, and play a significant role in controlling regulated secretion from neurons, endocrine, neuroendocrine and exocrine cells. Here, we describe microscopic techniques to image regulated secretion, a target of Ca2+ signalling. The first of these, total internal reflection fluorescence (TIRF), is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation (<150 nm). It is thus particularly useful for studies of regulated hormone secretion. A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope. We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained. A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included.
Unit of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, UCL 55.30 Avenue Hippocrate 55, B-1200 Brussels, Belgium.
Magalie A
MA
Ravier
Takashi
T
Tsuboi
Guy A
GA
Rutter
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D005456:Fluorescent Dyes; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D016716:PC12 Cells; D051381:Rats
D005456:Fluorescent Dyes; D002118:Calcium