# Two-Photon Excitation of Azobenzene Photoswitches for Synthetic Optogenetics
## 7 Future Prospects  
There is growing interest in synthetic optogenetic tools in fields such as in neuroscience [8, 12, 23], heart physiology [86, 87, 88] and, intuitively, vision restoration [89, 90, 91, 92]. Thus, we expect these to motivate further developments of the strategy in the upcoming years, in particular towards progression of the technique towards enhancing multiphoton absorption. Synthetic optogenetic tools can be combined with electrophysiological recordings and optical tools (e.g., Ca\({}^{2+}\)-probes [78]), making it highly tailored for in vivo use in different animal models in an all-optical manner. We also suggest that several photoswitches might even make it to the clinic [93], in particular photoswitches that target native proteins (e.g., [28, 29]) or those applicable in the blood [74]. In addition, synthetic optogenetic tools have shown promise for studying (and maybe treating) [PERSON]'s disease-related receptors [94], inhibiting cellular division in cancer [95], not to mention controlling cellular excitability, highly relevant for brain diseases such as epilepsy [96].  
Here, we have briefly summarized several of the newest developments in the field of synthetic optogenetics, more precisely efforts devoted towards rendering photoswitches absorbent of longer, red-shifted wavelengths and their ability to undergo 2 PE. These properties are highly desired for making the method more operational in vivo. Despite this motivation, it remains that most laboratories focus on designing unique photoswitches for 1P-applications. We believe that this stems from limited information on nonlinear multiphoton properties of photoswitches and the difficulties in determining, _a priori_, their multiphoton absorption properties [47, 67], relaxation rates and whether optimization (derivatization) of 1P-functional photoswitches towards 2 PE will not render them inoperative.  
It appears that bistable photoswitches provide an optimal starting point for designing next-generation 2 PE-compatible photoswitches. The slow relaxing photoswitches with light-harvesting antennae and symmetrical azo-cores display high 2P-absorption cross section that should allow for robust photoactivation of optical actuators in vivo. However, they will require further modifications to increase their solubility in aqueous solution prior use with cells. The bistable \(MAG_{2p,F}^{slow}\) exhibits slightly better features. Though of lower 2P cross-section (\(<\)70 GM) and almost no shift in 1P absorption maxima, its lower thermal relaxation allows for larger responses. The effect of this, and other, photoswitches could be further improved by additional means such as use of higher 2P-laser intensities (keeping in mind that higher intensities may be harmful to cells [52]), increase in the expression of the optical actuators [97, 98] and, importantly, use of parallel excitation methods for simultaneous activation of larger regions of interest in all axes [55, 57, 78, 99].  
In summary, we see current hurdles as wonderful incentives and opportunities for designing better photoswitches and protein actuators. Indeed, many labs around the world are working intensely to address these issues. Consequently, we anticipate that the potential of synthetic optogenetics towards in vivo use and the clinic will be realized within the next few years.  
**Author Contributions**: Conceptualization, S.B. and S.K.; methodology, S.B.; data curation, S.B. and S.K.; writing--review and editing, S.B. and S.K.; visualization, S.B.; supervision, S.B.; funding acquisition, S.B. The research submitted is in partial fulfilment for a Doctoral degree for S.K. All authors have read and agreed to the published version of the manuscript.  
**Funding**: This research was funded by the Israel Science Foundation (ISF), grant number 1096/17 (S.B.).  
**Conflicts of Interest**: The authors declare no conflict of interest.