Ultrafast light targeting for high-throughput precise control of neuronal networks.

Two-photon, single-cell resolution optogenetics based on holographic light-targeting approaches enables the generation of precise spatiotemporal neuronal activity patterns and thus a broad range of experimental applications, such as high throughput connectivity mapping and probing neural codes for perception. Yet, current holographic approaches limit the resolution for tuning the relative spiking time of distinct cells to a few milliseconds, and the achievable number of targets to 100-200, depending on the working depth. To overcome these limitations and expand the capabilities of single-cell optogenetics, we introduce an ultra-fast sequential light targeting (FLiT) optical configuration based on the rapid switching of a temporally focused beam between holograms at kHz rates. We used FLiT to demonstrate two illumination protocols, termed hybrid- and cyclic-illumination, and achieve sub-millisecond control of sequential neuronal activation and high throughput multicell illumination in vitro (mouse organotypic and acute brain slices) and in vivo (zebrafish larvae and mice), while minimizing light-induced thermal rise. These approaches will be important for experiments that require rapid and precise cell stimulation with defined spatio-temporal activity patterns and optical control of large neuronal ensembles.

Descanned fast light targeting (deFLiT) two-photon optogenetics.

Two-photon light-targeting optogenetics allows controlling selected subsets of neurons with near single-cell resolution and high temporal precision. To push forward this approach, we recently proposed a fast light-targeting strategy (FLiT) to rapidly scan multiple holograms tiled on a spatial light modulator (SLM). This allowed generating sub-ms timely-controlled switch of light patterns enabling to reduce the power budget for multi-target excitation and increase the temporal precision for relative spike tuning in a circuit. Here, we modified the optical design of FLiT by including a de-scan unit (deFLiT) to keep the holographic illumination centered at the middle of the objective pupil independently of the position of the tiled hologram on the SLM. This enables enlarging the number of usable holograms and reaching extended on-axis excitation volumes, and therefore increasing even further the power gain and temporal precision of conventional FLiT.