Understanding the Ca2+-dependent Fluorescence Change in Red Genetically Encoded Ca2+ Indicators

Genetically encoded Ca 2+ indicators (GECIs) are widely used to illuminate dynamic Ca 2+ signaling activity in living cells and tissues. Various fluorescence colors of GECIs are available, including red. Red GECIs are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a circularly permuted red fluorescent protein fused to a Ca 2+ sensing domain, calmodulin and a calmodulin-binding peptide. A conformational change in the sensing domain upon binding Ca 2+ causes a change in the fluorescence intensity of the fluorescent protein. Three factors could contribute to this change in fluorescence: 1) a shift in the protonation state of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient for one-photon excitation or the two-photon cross section for two-photon excitation. We conducted a systematic study of the photophysical properties of a select cohort of red GECIs in their Ca 2+ -free and Ca 2+ -saturated states to determine which factors are most important for the Ca 2+ -dependent change in fluorescence. In total, we analyzed nine red GECIs, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We found that these red GECIs could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases the magnitude of the change in fluorescence was different depending on one-photon excitation or two-photon excitation by up to a factor of two.