All-optical experiments promise neuroscientists an unprecedented possibility to manipulate and measure neuronal circuits with single-cell resolution. They rely on highly fine-tuned microscopes with complex optical designs. Of similar importance are genetically encoded optical actuators and indicators that also have to be optimized for such experiments. A particular challenge in these experiments is the detection of natural firing patterns via genetically encoded indicators while avoiding optical cross-activation of neurons that are photon-sensitized to allow optical replay of these patterns. Most optogenetic tools are sensitive in a broad spectral range within the visible spectrum, which impedes artifact-free read-and-write access to neuronal circuits. Nonetheless, carefully matching biophysical properties of actuators and indicators can permit unambiguous excitation with a single wavelength in a so-called single-beam all-optical experiment.
In this chapter, we evaluate the current understanding of these biological probes and describe the possibilities and limitations of those tools in the context of the all-optical single-beam experiment. Furthermore, we review new insights into the photophysical properties of actuators, and propose a new strategy for a single-beam two-photon excitation experiment to monitor activity minimizing cross-activation with the actuators. Finally, we will highlight aspects for future developments of these tools.
Copyright 2023, The Author(s).