HIGH-EFFICIENCY TWO-PHOTON IN VIVO ALL-OPTICAL NEURONAL INTERROGATION USING ACOUSTO-OPTIC TECHNIQUES

In vivo manipulation of neural circuits with single cell resolution is necessary to uncover the mechanisms underlying many brain functions. State-of-the-art parallel illumination techniques, based on digital holography, enable splitting the excitation light across multiple neurons. Yet, this approach implies the need to accumulate light energy over time to reach the action potential threshold. We reasoned that an ultrafast sequential approach would provide higher peak energy levels per neuron, thus improving activation efficiency due to the relationship between opsin excitation probability and peak energy under two-photon (2P) excitation. To validate this idea, we used “ULoVE”, a random-access acousto-optic technique previously developed for high efficiency 2P voltage optical recording (Villette et al. 2019). ULoVE generates a local sampling volume, matching the dimensions of a neuron, which can be moved from neuron to neuron every 70µs. We leveraged this method to stimulate the excitatory opsin ChRmine, bicistronically expressed with GCaMP6m. Combining simultaneous imaging and stimulation, we could trigger action potentials illuminating neurons for 70µs, using as little as 7µJ of total excitation energy, representing a ~7-fold reduction of the amount of energy per spike compared to state-of-the-art holographic approaches (Marshel et al. 2019; Bounds et al. 2023). Moreover, by tuning the number of pulses and peak energy delivered, we demonstrated the responses elicited scaled quadratically with the energy per pulse. The ULoVE-based approach opens the possibility to stimulate hundreds of neurons within tens of milliseconds, overcoming current techniques’ limit (tens of neurons with similar illumination power over similar timeframe).