Three-dimensional spatiotemporal focusing of holographic patterns (Conference Presentation)

Genetically encoded light-sensitive channels and reporters enable both neuronal activity optical control and read-out. Full explotation of these optogenetic tools requires single-cell scale methods to pattern light into neural tissue. Computer Generated Holography (CGH) can powerfully enhance optogenetic stimulation by efficiently shaping light onto multiple cellular targets. However, a linear proportionality between lateral shape area and axial extent degrades axial precision for cases demanding extended lateral patterning i.e., to cover entire soma of multiple cells. To address this limitation, we previously combined CGH with temporal focusing (TF) to stretch laser pulses outside of the focal plane, which combined with two-photon’s nonlinear fluorescence dependence, axially confines fluorescence regardless of lateral extent. However, this configuration restricts nonlinear excitation to a single spatiotemporal focal plane: which is the objective focal plane. Here we demonstrate a novel scheme enabling generation of spatiotemporally focused pattern generation in three dimensions. We demonstrate that this approach enables simultaneous photoconversion of tens of zebrafish larvae spinal cord neurons occupying separate axial planes.