In recent decades, optogenetics has been transforming neuroscience research, enabling neuroscientists to drive and read neural circuits. The recent development in illumination approaches combined with two-photon (2P) excitation, either sequential or parallel, has opened the route for brain circuit manipulation with single-cell resolution and millisecond temporal precision. Yet, the high excitation power required for multi-target photostimulation, especially under 2P illumination, raises questions about the induced local heating inside samples. Here, we present and experimentally validate a theoretical model that makes it possible to simulate 3D light propagation and heat diffusion in optically scattering samples at high spatial and temporal resolution under the illumination configurations most commonly used to perform 2P optogenetics: single- and multi-spot holographic illumination and spiral laser scanning. By investigating the effects of photostimulation repetition rate, spot spacing, and illumination dependence of heat diffusion, we found conditions that make it possible to design a multi-target 2P optogenetics experiment with minimal sample heating. Picot et al. model light and heat diffusion under the typical illumination conditions used for single- and multi-target two-photon optogenetics and compare the heat distribution under parallel and spiral scanning illumination. They experimentally validate the model using the temperature-dependent fluorescence of erbium-ytterbium co-doped glass particles.
Picot, A., Dominguez, S., Liu, C., Chen, I. W., Tanese, D., Ronzitti, E., … Emiliani, V. (2018). Temperature Rise under Two-Photon Optogenetic Brain Stimulation. Cell Reports, 24(5), 1243-1253.e5. https://doi.org/10.1016/j.celrep.2018.06.119