Two-photon optogenetics.

The use of optogenetics, the technology that combines genetic and optical methods to monitor and control the activity of specific cell populations, is now widely adopted in neuroscience. The development of optogenetic tools, such as natural photosensitive ion channels and pumps or calcium- and voltage-sensitive proteins, has been growing tremendously during the past 10 years, thanks to the improvement of their performances in terms of facilitating light stimulation. To this aim, efficient illumination methods are also needed. The most common way to photostimulate optogenetic tools has been, so far, widefield illumination with visible light. However, the necessity of addressing the complexity of brain architecture has recently imposed switching to the use of two-photon excitation, which provides a better spatial specificity and deeper penetration in scattering tissue. Two-photon excitation is still challenging, due to intrinsic characteristics of optogenetic tools (e.g., the low conductivity of light-sensitive channels), and efficient illumination methods are therefore essential for advancing in this domain. Here, we present a review on the existing two-photon optical approaches for photoactivation of optogenetic tools, and future perspectives for the widespread implementation of these techniques. Copyright Â