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

Abstract

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.

Two-photon all-optical electrophysiology for the dissection of larval zebrafish brain functional connectivity

“…These devices indeed, owing to their discontinuous scanning with constant access time, enable random-access modality. This feature empowers AODs with the native capability to perform rapid sequential excitation over multiple sparsely distributed cellular targets, a feature recently sought after also by SLM adopters 75 . Indeed, rapid sequential stimulation enabled by AODs represents an invaluable tool for studies aiming at replicating a physiological neuronal activation pattern.…”

High-throughputin vivosynaptic connectivity mapping of neuronal micro-circuits using two-photon holographic optogenetics and compressive sensing

“…In our experimental conditions we used Δt ≈100 ms (with t SLM ≈40 ms). The use of faster SLM (refresh rate 300Hz) 42 , that will lower t SLM down to ≈ 2-3ms, or the use of approaches for fast (50-90 μs) scanning through multiple holograms 101 , also combined with current deconvolution algorithms 75 , could reduce the sequential time interval to Δt ≈ t ill or Δt ≈ t rec if t rec > t ill .…”

Interrogation of single-neuron functional connectivity in the cortex and hippocampus via fast cross-layer all-optical physiology

“…Future enhancements will involve integrating a meso-objective lens with dual scan engines, thereby expanding the capacity of the CLAOP system for cross-region interrogation of neuronal connectivity (Yu et al 2021). Additionally, temporal focusing has been utilized in optogenetic manipulation to enhance the axial physiological resolution of photostimulation (Faini et al 2023; Pégard et al 2017). Therefore, integrating CLAOP with temporal focusing, which offers more confined axial excitation, will provide more reliable connectivity mapping in densely arranged brain regions, such as hippocampus CA1 and DG.…”