Optical constraints on two-photon voltage imaging

Abstract

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 ∼104-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. ### Competing Interest Statement The authors have declared no competing interest.