# Two-Photon Excitation of Azobenzene Photoswitches for Synthetic Optogenetics
## 1 Introduction  
Photoreceptors are protein light-detectors that allow the cell/organism to respond to its environment [1]. Most, if not all, photoreceptors are a two-component system: a genetically-encoded protein coupled to a chromophore. Following photon-absorption, chromophores undergo unique alterations (for instance in geometry or charge [2; 3]), resulting in the modulation of the protein partner to which each chromophore is bound to. In most instances, this process is completely reversible, whereby removal of light (i.e., return to darkness) facilitates the relaxation of the chromophore to its initial, thermally stable state. This also restores the function of the protein back to as it were prior illumination. These features, explicitly non-invasive reversible control over proteins, have made it possible to develop fantastic genetically-encoded optical tools--denoted 'optogenetic' tools--for the manipulation of defined subsets of cells [4]. Many tools today are based on naturally-occurring photoreceptors, for instance opsins [5; 6; 7; 8]. However, the need to photocontrol other signaling mechanisms, proteins or molecules, have pushed forward the development of complementary strategies, explicitly Synthetic optogenetics [9; 10]. Synthetic optogenetics relies on the use of non-natural, synthetic chromophores--photoswitches--to relay photon absorption to modulation of structure and function of their target molecule (Figure 1a). Photoswitches do not require a unique chromophore binding pocket or protein domains as in the case of chromophores (e.g., [11]). Instead, photoswitches can be directly conjugated to various locations on their target molecule with use of different genetic or chemical handles (see below) [9; 12]. However, unlike chromophores that can be synthesized by various cells (even if the cells are not intrinsically light-sensitive), photoswitches need to be supplemented to the preparation. This is both a blessing and a curse. Blessing--since the preparation remains naive until the photoswitchis added. Curse--it adds another layer of complexity to the experiment, especially in vivo, such as injection prior experimentation. Photoswitches can be custom-tailored to photomodulate a large variety of effectors. For instance, some have been fashioned to mechanically restructure membrane lipids or DNA molecules [13; 14; 15], to act as light-activated forceps [16] or to include pharmacological agents (e.g., pore-blockers) to block, agonize, or antagonize receptors, ion channels, or enzymes (Figure 1b) (also see reviews [9; 17; 18]). It is noteworthy to mention that although this method dates back to the late 1960s (Figure 2) [19], it has particularly gained momentum in early 2000 owing to critical advances in chemistry, biology, and imaging methods. Here, we focus on the most recent efforts devoted to push it to the next level, namely towards multiphoton activation of photoswitches for in vivo applications in opaque preparations. Figure 1: Synthetic optogenetics. (**a**) Cartoon depiction of a photoswitchable glutamate receptor (space filling based on PDB: 3 KG2). For simplicity, only two of four subunits are presented. In addition, only one subunit is shown to tether the photoswitch (color-coded shapes). The photoswitch consists of a ligand (purple rectangle), an azobenzene-core (red hexagon) and a maleimide for attachment (green circle). In the dark, the photoswitch is in _-trans_ and channel is closed (leftward cartoon, light blue). Photoisomerization of the photoswitch to _-cis_ by red-shifted light (_lv_) allows insertion of the glutamate headgroup (purple rectangle) into its binding pocket inducing channel opening (rightward cartoon, green receptor; dashed arrow depicts conductance of ions). (**b**) Chemical structure of an exemplary photoswitch; MAG (color coded as in (a); maleimide—green; azobenzene—red; glutamate—purple).