# Two-Photon Excitation of Azobenzene Photoswitches for Synthetic Optogenetics
## 5 Two-Photon Compatible MAG-Based Photoswitches
The functional outcome of the thermal relaxation of the photoswitch is a rapid cessation of channel activation, seen as a decrease in current once illumination stops (\(\tau_{\text{off}}\) = 150 ms [67]; \(\sim\)10,000-fold times faster than thermal relaxation of MAG, \(\tau_{\text{off}}\) = 25 min [75]). A push-pull scheme was similarly employed, very shortly after, by [PERSON] et al. [75]. Here, the authors have modified MAG to include an asymmetric azobenzene-core with a tertiary amine at the 4'-position as the electron-donor, but an acetamido-group at the 4'-position, as the weak electron withdrawing group (Figure 3). These modifications resulted in a larger shift in peak 1P-absorption (\(\sim\)100 nm) of the -_trans_ isomer (\(\lambda_{\text{peak}}\) = 460 nm), therefore denoted MAG\({}_{460}\) (Figure 3) [75]. Expectedly, this modification reduced the stability of the -_cis_ isomer (MAG\({}_{460}\), \(\tau_{\text{off}}\) = 0.71 s) [75]. Importantly, this shift led to an 8-fold increase in the photoswitch's 2P-absorption cross section (_trans_-MAG\({}_{460}\), \(\sigma\)2 = 80 GM at 850 nm) [41]. We have later employed this photoswitch to successfully activate LiGluR [41]. Though the photocurrents obtained by 2 PE were of smaller amplitude than when using 1P-illumination, they were sufficient to evoke action potential firing in cultured neurons by parallel excitation methods (digital-holography; Table 1) [41; 78]. Lastly, we also employed a fluorescent reporter as readout for Ca\({}^{2+}\)-activity (R-GECO) and find it to provide reliable responses (\(\Delta F\)/\(F\)), demonstrating that this method is also suitable for all-optical interrogation of cells [78]. Another interesting strategy that has been employed to sensitize the MAG photoswitch towards NIR wavelengths is by adding a light-harvesting molecule (i.e., antenna). Two such photoswitches were designed under the name of maleimide-azobenzene-glutamate antenna, or MAGA. The first--MAGA2p [65]--consisted of the same asymmetric core as us found in MAG2p along an added light-harvesting naphthalene-derivative (with a high 2P-absorption cross section; \(\sigma\)2 \(\approx\) 200 GM at 780 nm [79]) (Figure 3). The antenna was incorporated to sensitize the _trans_-azobenzene by resonant electronic energy transfer (RET) (Figure 3). This photoswitch could therefore be photoactivated by two distinct mechanisms: direct excitation of the push-pull azo-core by 2 PE and RET by the photosensitizing antenna. This photoswitch displayed low thermal stability, with rapid relaxation of the currents (\(\tau_{\text{off}}\) = 265 ms [65]) that, when excited using a laser-scanning method, would not allow efficient activation of the channel or robust cellular responses. In a subsequent improved version, MAGA ligand-2 (Figure 3) [66], the authors maintained an antenna for harvesting light, albeit swapped it by a pyrene molecule (\(\sigma_{2}\) = 55 GM), but reverted back to a symmetrical azo-core with the intention to counteract the pyrene's lower 2P-absorption cross section (\(\sim\)4-fold lower than naphthalene) by increasing the thermal stability of the photoswitch. Indeed, the resulting photoswitch exhibited a highly stable -_cis_ isomer (MAGA ligand-2, \(\tau_{\text{off}}\) = 2.0 h in DMSO). Thus, the increased thermal stability of the -_cis_ state should make this photoswitch more suitable to accumulate activated photoswitches by scanning imaging methods. However, it is worthy to mention that the photosensitizing antenna employed drastically reduced the solubility of the photoswitch and, likely, its usability in biological experiments. Indeed, so far MAGA ligand-2 was not employed with cells (Table 1). Figure 3: Chemical structure and absorption of exemplary 2P-compatible photoswitches. Left, names of photoswitches; right, one photon (1P) peak absorption (\(\lambda_{\max}\)), also depicted by middle color gradient.