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Enhanced‐contrast two‐photon optogenetic pH sensing and pH ‐resolved brain imaging
Author(s) -
Chebotarev Artem S.,
Pochechuev Matvei S.,
Lanin Aleksandr A.,
Kelmanson Ilya V.,
Kotova Daria A.,
Fetisova Elena S.,
Panova Anastasiya S.,
Bilan Dmitry S.,
Fedotov Andrei B.,
Belousov Vsevolod V.,
Zheltikov Aleksei M.
Publication year - 2021
Publication title -
journal of biophotonics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.877
H-Index - 66
eISSN - 1864-0648
pISSN - 1864-063X
DOI - 10.1002/jbio.202000301
Subject(s) - optogenetics , fluorescence , chromophore , fluorescence lifetime imaging microscopy , laser , two photon excitation microscopy , chemistry , contrast (vision) , biophysics , materials science , nuclear magnetic resonance , photochemistry , optics , physics , biology , neuroscience
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.