# Two-Photon Excitation of Azobenzene Photoswitches for Synthetic Optogenetics
## 4 Shifting from Visible to Near-Infrared  
Despite progresses made since the very first demonstrations of Synthetic optogenetics by the [PERSON] group in the late 1960s [19; 32], current illumination schemes remain as they were back then, namely rely on the use of visible light, typically near-UV (\(\sim\)400 nm) for _trans-to-cis_ photoisomerization and blue-shifted wavelengths (\(\sim\)500) for _cis-to-trans_[9; 12; 15; 17; 18; 24; 33; 34]. Aside the potential cytotoxicity of these wavelengths [35; 36], visible light is not well-suited for deep tissue penetration due to strong absorption and scatter from endogenous components [37]. The use of longer, near infrared (NIR; \(>\)700 nm) wavelengths is advantageous as these are less absorbed by biological tissues and water [38], induce less photodamage [36], and are less prone to scatter. Synthetic optogenetics is slow to adapt NIR illumination owing to the very poor absorbance of these low energy wavelengths by the azobenzene core, a common challenge with other optogenetic tools [39], not to mention the relatively little information extant for NIR absorption by photoswitches ([40; 41] and see below). In fact, though the _-cis_ azobenzene was first discovered almost a century ago (see [42]), the debate regarding the exact mechanism for photoisomerization, and how different factors affect it, remains lively to this day [43; 44]. Following excitation, the azobenzene molecule proceeds from S0 to S1 and S2 states, with distinct absorption bands for each transition (n-\(\pi^{\star}\) transition excite azobenzene compounds to S1 state and \(\pi\)-\(\pi^{\star}\) transition leads to S2 state) [45; 46]. However, many different factors can have dramatic influences on the these, for instance substitutions on the phenyl rings, solvent properties, temperature, to name the more common factors. However, it is much less known by which of the proposed mechanisms the isomerization of azobenzene proceeds, namely rotation, inversion, concerted inversion, and inversion assisted rotation (for more details see [44]). These uncertainties arise from different approaches used, or different experimental settings. For instance, whereas non-viscous polar solvents favor a rotation mechanism, viscous non-polar favor inversion [44]. It has also been shown that the use of different wavelengths can affect the isomerization mechanism. In fact, these differences are also seen with other compounds such as fluorescent proteins [47; 48] and extant data of how wavelengths affect isomerization mostly pertains to one-photon excitation of azobenzene compounds [49]. This actuality makes it very difficult to infer the mechanisms for NIR excitation as the absorption properties between these different illumination schemes are substantially different [41]. For instance, use of 1-photon for isomerization shows that the transition from _trans_-to-_cis_ path develops triexponentially with times of 0.3, 3, and 16 ps (where the first two reflect population relaxation and the third corresponds to the final relaxation to ground state [49]) and these are not affected by solvent viscosity rather by wavelengths at early (t\(\sim\)1 ps) and late (t\(\sim\)100 ps) times but show similar photoisomerization behavior on a 10 ps time scale [50]. Some of these hurdles can be overcome by using, at least, two NIR photons; each with half the energy of needed, to be absorbed in a single event (within \(\sim\)10\({}^{-18}\) s) in order to reach the molecule's excited state [51]. For instance, if a photoswitch/chromophore efficiently absorbs near-UV light at 400 nm, in principle it should be possible to excite it by two simultaneous photons at approximately 800 nm each [41]. This technique is denoted two-photon excitation (2 PE) [52]. However, it is important to note that 2 PE is a third-order nonlinear process that depends not only on the absorption cross-section (\(\sigma\)2) of the molecule, but also on the concentration of incident photons.