The biophysical limitations of existing optogenetic tools restrict the scale, speed, and precision of neural control. Using structure-guided mutagenesis, we have engineered high-potency opsins that retain fast kinetics, enabling large-scale, spatiotemporally precise control of neuronal populations. These novel opsins have been extensively benchmarked against existing optogenetic tools and characterized under two-photon illumination, providing a valuable resource for selecting the optimal opsin for patterned optogenetics experiments.

By integrating these engineered opsins with optimized holographic photostimulation, we achieved simultaneous activation of hundreds of spatially defined neurons and nearly doubled this number through rapid temporal interleaving of holograms. This breakthrough significantly enhances the capabilities of optogenetics for studying neural circuits and behavior.

Keywords: optogenetics, high-potency opsins, fast-kinetics opsins, neural control, holographic photostimulation, two-photon optogenetics, patterned illumination, neurotechnology, neural circuits, brain research, precision neuroscience.