Single cell in vivo brain optogenetic stimulation by two-photon excitation fluorescence transfer

Optogenetic manipulation with single-cell resolution can be achieved by two-photon excitation; however, this frequently requires relatively high laser powers or holographic illumination. Here we developed a practical strategy to improve the efficiency of two-photon stimulation by positioning fluorescent proteins or small fluorescent molecules with high two-photon cross-sections in the vicinity of opsins. This generates a highly localized source of endogenous single-photon illumination that can be tailored to match the optimal opsin absorbance. Through neuronal and vascular stimulation in the live mouse brain, we demonstrate the utility of this technique to achieve more efficient opsin stimulation, without loss of cellular resolution. We also provide a theoretical framework for understanding the potential advantages and constrains of this methodology, with suggestions for future improvements. Altogether, this fluorescence transfer illumination method allows experiments difficult to implement in the live brain such as all-optical neural interrogation and control of regional cerebral blood flow.