Significance
Two-photon optogenetics combines nonlinear excitation with noninvasive activation of neurons to enable the 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.
Aim
We aimed to assess how the optical point spread function (OPSF) contributes to the spatial precision of two-photon photostimulation in neurobiology.
Approach
We altered the axial spread of the OPSF of the photostimulation beam using a spatial light modulator. Subsequently, calcium imaging was used to monitor the axial spatial precision of two-photon photostimulation of layer 2 neurons in the mouse neocortex.
Results
We found that optical resolution is not always the limiting factor of the spatial precision of two-photon optogenetic photostimulation and, by doing so, reveal the key factors that must be improved to achieve maximal precision.
Conclusions
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 in which neural circuits operate.
