Genetically engineered Ca2+ indicators (GECIs) as a tool to investigate submembrane Ca2+ microdomains in beta cells

Background and aims It has been argued that the relevant Ca2+-concentration for fusion of the insulin granules only exists within a microdomain around plasma membrane Ca2+-channels. The genetically engineered Ca2+ indicator (GECI) GCaMP6m-XC fused to the granule membrane protein phogrin has been shown to report changes of the bulk cytosolic Ca2+-concentration ([Ca2+]i). To measure Ca2+ in the microdomain with this indicator, the following questions had to be answered: Can phogrin-GCaMP6m-XC be used with TIRF microscopy? Does the fusion of the GECI to a membrane protein affect Ca2+-measurement and granule structure?