Optical constraints on two-photon voltage imaging.

Significance:Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits and limitations of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized. Aim:We consider the optical and biophysical constraints particular to 1P and 2P voltage imaging and compare the imaging properties of commonly used GEVIs under 1P and 2P excitation. Approach:We measure brightness and voltage sensitivity of voltage indicators from commonly used classes under 1P and 2P illumination. We also measure the decrease in fluorescence as a function of depth in mouse brain. We develop a simple model of the number of measurable cells as a function of reporter properties, imaging parameters, and desired signal-to-noise ratio (SNR). We then discuss how the performance of voltage imaging would be affected by sensor improvements and by recently introduced advanced imaging modalities. Results:Compared to 1P excitation, 2P excitation requires ~104-fold more illumination power per cell to produce similar photon count rates. For voltage imaging with JEDI-2P in mouse cortex with a target SNR of 10 (spike height:baseline shot noise), a measurement bandwidth of 1 kHz, a thermally limited laser power of 200 mW, and an imaging depth of > 300 μm, 2P voltage imaging using an 80 MHz source can record from no more 12 cells simultaneously. Conclusions:Due to the stringent photon-count requirements of voltage imaging and the modest voltage sensitivity of existing reporters, 2P voltage imaging in vivo faces a stringent tradeoff between shot noise and tissue photodamage. 2P imaging of hundreds of neurons with high SNR at depth > 300 μm will require either major improvements in 2P GEVIs or qualitatively new approaches to imaging.