Light-sheet microscopy is a powerful method for imaging small translucent samples in vivo, owing to its unique combination of fast imaging speeds, large field of view, and low phototoxicity. This chapter briefly reviews state-of-the-art technology for variations of light-sheet microscopy. We review recent examples of optogenetics in combination with light-sheet microscopy and discuss some current bottlenecks and horizons of light sheet in all-optical physiology. We describe how 3-dimensional optogenetics can be added to an home-built light-sheet microscope, including technical notes about choices in microscope configuration to consider depending on the time and length scales of interest.

Optical aberrations affect the quality of light propagating through a turbid medium, where refractive index is spatially inhomogeneous. In multiphoton optical applications, such as two-photon excitation fluorescence imaging and optogenetics, aberrations non-linearly impair the efficiency of excitation. We demonstrate a sensorless adaptive optics technique to compensate aberrations in holograms projected into turbid media. We use a spatial light modulator to project custom three dimensional holographic patterns and to correct for local (anisoplanatic) distortions. The method is tested on both synthetic and biological samples to counteract aberrations arising respectively from misalignment of the optical system and from samples inhomogeneities. In both cases the anisoplanatic correction improves the intensity of the stimulation pattern at least two-fold.

We propose a sensor-less adaptive optics approach to correct local aberrations in holograms used for two-photon stimulation. Our method showed intensity enhancement of about 60 % in holograms projected into fixed zebrafish tissue.

Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammalian origin by use of photoswitches, visible light (typically), and genetic modification. Thus, synthetic optogenetics facilitates interrogation of native neuronal signaling mechanisms. However, the poor tissue penetration of visible wavelengths impedes the use of the technique in tissue, as two-photon excitation (2PE) is typically required to access the near-infrared window. Here, we describe an alternative technique that uses 2PE-compatible photoswitches (section 1) for photoactivation of genetically modified glutamate receptors (section 2). Furthermore, for fast, multi-region photoactivation, we describe the use of 2P-digital holography (2P-DH) (section 3). We detail how to combine 2P-DH and synthetic optogenetics with electrophysiology, or with red fluorescence Ca ²⁺ recordings, for all-optical neural interrogation. The time required to complete the methods, aside from obtaining the necessary reagents and illumination equipment, is ~3 weeks.