Exploring two-photon optogenetics beyond 1100 nm for specific and effective all-optical physiology

Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations. Here, firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in mouse visual cortex to tune our detection algorithm towards a 100% specific identification of action potential-related calcium transients. Secondly, we minimized photostimulation artifacts by using extended-wavelength-spectrum laser sources for optogenetic stimulation. We achieved artifact-free all-optical experiments performing optogenetic stimulation from 1100 nm to 1300 nm. Thirdly, we determined the spectral range for maximizing efficacy until 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective GCaMP6f/C1V1-based all-optical physiology, we increased the translational value of these approaches, e.g., for the development of network-based therapies.