Optical constraints on two-photon voltage imaging.

Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo , but the relative merits of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized. Here we compare the photophysical and imaging properties of commonly used GEVIs under 1P and 2P excitation. 2P excitation requires ∼10 4 -fold more illumination power per cell to produce comparable photon count rates to 1P excitation, driving a stringent tradeoff between shot noise and tissue photodamage.