Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches
with near-infrared light has been recently demonstrated, but their practical use in neuronal
tissue to photostimulate individual neurons with three-dimensional precision has been
hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and secondly, the short cis state lifetime of
the two-photon responsive azo switches. Here we report the rational design based on theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high
two-photon absorption cross section and slow thermal back-isomerization. These compounds provide optimized and sustained two-photon neuronal stimulation both in lightscattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse
intensities that are comparable to those achieved under one-photon excitation. This finding
opens the way to use both genetically targeted and pharmacologically selective azobenzene
photoswitches to dissect intact neuronal circuits in three dimensions.Peer Reviewe