Multiphoton tools for hydrogen peroxide imaging in vivo with subcellular resolution

Hydrogen peroxide (H2O2) has been identified recently as a meaningful signaling molecule. The ability to perform imaging of H2O2 in complex biological organisms with a high spatio-temporal resolution requires advancements in both biosensors and in vivo visualization methods. In this work, we investigated the potential of fluorescent protein HyPer7, a bright and sensitive indicator of hydrogen peroxide, for multiphoton visualization in cell cultures, acute brain slices and neocortical neurons of anesthetized mice. Employing an alternating interrogation of the reduced and oxidized forms of HyPer7 enables the recording of H2O2 dynamics at a rate of one frame per second. Taking advantage of two-photon ratiometric readout of HyPer7, we monitored the intracellular H2O2 concentration growth in hippocampal neurons under chemogenetics tools handling in acute brain slices. At the final step, we visualized the HyPer7-expressed neurons at depths up to 600µm below the brain surface of the mouse under anesthesia by two- and three-photon excitation microscopy. Autofluorescence, light scattering and out-of-focus background signal reduce the depth limit for HyPer7-imaging by two-photon microscopy, however, these challenges may be overcome by means of three-photon excitation fluorescence by the pulses of the short-wavelength infrared region.