Enhanced‐contrast two‐photon optogenetic pH sensing and pH ‐resolved brain imaging

Abstract We present experiments on cell cultures and brain slices that demonstrate two‐photon optogenetic pH sensing and pH‐resolved brain imaging using a laser driver whose spectrum is carefully tailored to provide the maximum contrast of a ratiometric two‐photon fluorescence readout from a high‐brightness genetically encoded yellow‐fluorescent‐protein‐based sensor, SypHer3s. Two spectrally isolated components of this laser field are set to induce two‐photon‐excited fluorescence (2PEF) by driving SypHer3s through one of two excitation pathways—via either the protonated or deprotonated states of its chromophore. With the spectrum of the laser field accurately adjusted for a maximum contrast of these two 2PEF signals, the ratio of their intensities is shown to provide a remarkably broad dynamic range for pH measurements, enabling high‐contrast optogenetic deep‐brain pH sensing and pH‐resolved 2PEF imaging within a vast class of biological systems, ranging from cell cultures to the living brain.