CALL FOR PAPERS Methods in Cell Physiology FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets

Kuznetsov, Andrey, Vytautas P. Bindokas, Jeremy D. Marks, and Louis H. Philipson. FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets. Am J Physiol Cell Physiol 289: C224–C229, 2005. First published March 9, 2005; doi:10.1152/ajpcell.00004.2005.—Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (Vm) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from Vm 70 to 50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in Vm. When Vm was measured simultaneously with intracellular Ca , all active cells showed tight coupling of Vm to islet cell Ca changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans.