Two-Photon Excitation of Azobenzene Photoswitches for Synthetic Optogenetics

Synthetic optogenetics is an emerging optical technique that enables users to photocontrol molecules, proteins, and cells in vitro and in vivo. This is achieved by use of synthetic chromophores—denoted photoswitches—that undergo light-dependent changes (e.g., isomerization), which are meticulously designed to interact with unique cellular targets, notably proteins. Following light illumination, the changes adopted by photoswitches are harnessed to affect the function of nearby proteins. In most instances, photoswitches absorb visible light, wavelengths of poor tissue penetration, and excessive scatter. These shortcomings impede their use in vivo. To overcome these challenges, photoswitches of red-shifted absorbance have been developed. Notably, this shift in absorbance also increases their compatibility with two-photon excitation (2PE) methods. Here, we provide an overview of recent efforts devoted towards optimizing azobenzene-based photoswitches for 2PE and their current applications.