Photons are a fascinating reagent, flowing and reacting quite differently compared to more massive and less ephemeral particles of matter. The optogenetic palette comprises an ever growing set of light-responsive proteins, which open the possibility of using light to perturb and to measure biological processes with great precision in space and time. Yet there are limits on what light can achieve. Diffraction limits the smallest features, and scattering in tissue limits the largest. Photobleaching, diffusion of photogenerated products, and optical crosstalk between overlapping absorption spectra further muddy the optogenetic picture, particularly when one wants to use multiple optogenetic tools simultaneously.
But these obstacles are surmountable. Most light-responsive proteins and small molecules undergo more than one light-driven transition, often with different action spectra and kinetics. By overlapping multiple laser beams, carefully patterned in space, time, and wavelength, one can steer molecules into fluorescent or nonfluorescent, active or inactive conformations. By doing so, one can often circumvent the limitations of simple one-photon excitation and achieve new imaging and stimulation capabilities. These include subdiffraction spatial resolution, optical sectioning, robustness to light scattering, and multiplexing of more channels than can be achieved with simple one-photon excitation.
The microbial rhodopsins are a particularly rich substrate for this type of multiphoton optical control. The natural diversity of these proteins presents a huge range of starting materials. The spectroscopy and photocycles of microbial rhodopsins are relatively well understood, providing states with absorption maxima across the visible spectrum, which can be accessed on experimentally convenient time scales. A long history of mutational studies in microbial rhodopsins allows semirational protein engineering. Mutants of Archaerhodopsin 3 (Arch) come in all the colors of the rainbow. In a solution of purified Arch-eGFP, a focused green laser excites eGFP fluorescence throughout the laser path, while a focused red laser excites fluorescence of Arch only near the focus, indicative of multiphoton fluorescence. This nonlinearity occurs at a laser intensity ∼1010-fold lower than in conventional two-photon microscopy! The mutant Arch(D95H) shows photoswitchable optical bistability. In a lawn of E. coli expressing this mutant, illumination with patterned blue light converts the molecule into a state that is fluorescent. Illumination with red light excites this fluorescence, and gradually resets the molecules back to the non-fluorescent state. This review describes the new types of molecular logic that can be implemented with multi-photon control of microbial rhodopsins, from whole-brain activity mapping to measurements of absolute membrane voltage.
Part of our goal in this Account is to describe recent work in nonlinear optogenetics, but we also present a variety of interesting things one could do if only the right optogenetic molecules were available. This latter component is intended to inspire future spectroscopic, protein discovery, and protein engineering work.