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 and limitations of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized.

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.

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.

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.

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.