# Two-Photon Excitation of Azobenzene Photoswitches for Synthetic Optogenetics
## 5 Two-Photon Compatible MAG-Based Photoswitches  
One of the most commonly employed tethered photoswitch is MAG [9] (Figures 1b and 2). MAG, similar to MAL-AZO-QA (see above), has an azobenzene-core (A), flanked by a cysteine-reactive maleimide (M), but instead of a channel blocker bears a glutamate molecule (G). This photoswitch is therefore intended for glutamate-binding proteins. The MAG photoswitch has been employed to activate glutamate receptors (e.g., LiGluR, LiGluN, mGluRs) in its _-cis_ form, not to mention to antagonize a glycine-binding receptor (the GluN1a-subunit [26]). Akin to other azobenzene-based photoswitches, MAG undergoes very efficient _-trans_ to _-cis_ photoisomerization when irradiated with near-UV light (MAG\({}_{0}\); \(\lambda_{\text{trans-cis}}\) 340\(-\)400 nm), and reverts from _-cis_ back to _-trans_ by thermal relaxation or, significantly more rapidly, by blue-green wavelengths (MAG\({}_{0}\); \(\lambda_{\text{cis-trans}}\) 440\(-\)580 nm). Nevertheless, with regards to 2 PE, MAG exhibits very poor absorption in the red-to-NIR range (\(\sim\)700-1400 nm) [41]. In the case of first generation MAGs (MAG0 [26; 63; 64]), endowed with a symmetrically-substituted azobenzene (Figure 2 and Table 1), the 2P-absorption cross section is low (\(\sigma\)2 = 10 GM at 820 nm [41]), albeit on the order of magnitude of a common fluorescent protein such as eGFP (\(\sigma\)2 = 30 GM at 927 nm [47]). This, along the lower expression and density of channels at membrane of neurons, results in very little or no capacity to photocontrol cellular activity when 2 PE is employed [41]. This prompted the design of second-generation MAGs with increased 2P-absorption cross sections. More precisely, this required lowering the energetic barrier of _trans-to-cis_ isomerization by specifically adjusting and shifting MAG's spectral properties towards'red'-er wavelengths. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Photonwidth** & **Type** & \begin{tabular}{c} **17 and 27 Peak** \\ **Absorption (nm)** \\ \end{tabular}  & \begin{tabular}{c} **27-absorption** \\ **Conse Section (CM)** \\ \end{tabular}  & \begin{tabular}{c} **Two Photon** \\ **Insight** \\ \end{tabular}  & **Cell Type** & **Application** & **Ref** \\ \hline LAMGG & PTL & 17-230 nm & \ \begin{tabular}{c} 127-27 mammalian \\ **100 nm** \\ \end{tabular}  &
\begin{tabular}{c} 27-base sampling \\ and \(\pm\) 200 ML at 400 nm \\ and \(\pm\) 200 ML at 4 One strategy employed involved breaking the symmetry around the azobenzene core (below in Figure 3 and Table 1) [74]. This was obtained by creating a push-pull system, where one benzene ring of the azo-core was decorated with an electron donating group, while the second azo-unit was supplemented with an electron withdrawing group. These led to an asymmetric azobenzene with a significantly red-shifted absorbance [21; 68; 74; 75; 76; 77]. This strategy was employed in one of the very first 2P-compatible PTLs, where the MAG photoswitch was redesigned to include an asymmetric aminoazobenzene core (tertiary amine in the 4'-position) to act as a strong electron-donating group, denoted MAG2p (Figure 3) [65]. Indeed, this photoswitch exhibited a red-shifted 1P-absorption spectrum peaking at 420 nm; \(\sim\)60 nm red-shifted compared to the parent MAG. Notably, this photoswitch could be activated by 2 PE (Table 1). In a subsequent report, the authors estimated its 2P-absorption cross section and suggest it to be -five times higher than that of the parent photoswitch (MAG2p, \(\sigma\)2 = 56 GM [72] at 850 nm, Table 1). The authors go on to show that, when coupled to a glutamate receptor (LiGluR [63]) optimal 2P-responses are obtained at 900 nm. Another unique feature of this photoswitch is that it is no longer bi-stable, rather spontaneously reverts back to -_trans_ in the dark. Notably, decrease in the thermal stability of the -_cis_ isomer by red-shifting of absorbance of azobenzene is a well-described phenomenon [74].