Optical resolution is not the limiting factor for spatial precision of two-photon optogenetic photostimulation

Two-photon optogenetics combines nonlinear excitation with noninvasive activation of neurons to enable manipulation of neural circuits with a high degree of spatial precision. Combined with two-photon population calcium imaging, these approaches comprise a flexible platform for all-optical interrogation of neural circuits. However, a multitude of optical and biological factors dictate the exact precision of this approach in vivo, where it is most usefully applied. Here, we carefully assessed how the optical factors determine the spatial precision of activation. We found that optical resolution is not the limiting factor of the spatial precision of two-photon optogenetic photostimulation, and by doing so, reveal the key factors to improve to achieve maximal precision. Our results enable future work to focus on the optimal factors by providing key insight from controlled experiments in a manner not previously reported. This research can be applied to advance the state-of-the-art of all-optical interrogation, extending the toolkit for neuroscience research to achieve spatiotemporal precision at the crucial levels at which neural circuits operate. ### Competing Interest Statement The authors have declared no competing interest.