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.

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In deep tissue imaging, pulsed near-infrared lasers commonly provide high peak powers needed for nonlinear absorption, but average power and linear absorption can be limiting factors for tissue damage through heat. We implemented intra-cavity dumping within a mode-locked Ti:Sapphire laser used for two-photon computer generated holography stimulation. This system enables photostimulation in which pulse energy, average power, and repetition rate can each be varied and harnessed as degrees of freedom. We demonstrate how this system provides a new dimension of temporal control in photostimulation experiments to alter the ratio of nonlinear to linear light-tissue interactions, namely by tuning the laser repetition rate between single-shot and ≈ 3 MHz. Repetition rates below 1 MHz, yielding pulse energies over 60 nJ, facilitated holographic projections with more regions of interest than the highest repetition rate of 3 MHz. Even lower repetition rates ( ≈ 10 kHz) diminished thermal load on the sample, as characterized by quantification of heat shock protein expression in zebrafish tissue.@en

Optogenetics is a powerful addition to the spectrum of techniques available in neuroscience to investigate neurophysiology and unravel how neural circuit structure is related to circuit function. This technique relies on introducing lightsensitive proteins or molecules as actuators to transduce an optical signal into a physiological perturbation of a living cell in vitro or in a living animal. To date, optogenetics has allowed remote control of neural activity in living and awake animals at different scales from single cells to complex networks of neurons to the investigation of animal behaviours. This wide range of experimental scales has been accomplished through joint progress on engineering the biological sensors and the optical design of instruments capable of manipulating with cellular spatial precision and millisecond temporal resolution.@en

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.

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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.

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The use of spatial light modulators to project computer generated holograms is a common strategy for optogenetic stimulation of multiple structures of interest within a three-dimensional volume. A common requirement when addressing multiple targets sparsely distributed in three dimensions is the generation of a points cloud, focusing excitation light in multiple diffraction-limited locations throughout the sample. Calculation of this type of holograms is most commonly performed with either the high-speed, low-performance random superposition algorithm, or the low-speed, high performance Gerchberg–Saxton algorithm. This paper presents a variation of the Gerchberg–Saxton algorithm that, by only performing iterations on a subset of the data, according to compressive sensing principles, is rendered significantly faster while maintaining high quality outputs. The algorithm is presented in high-efficiency and high-uniformity variants. All source code for the method implementation is available as Supplementary Materials and as open-source software. The method was tested computationally against existing algorithms, and the results were confirmed experimentally on a custom setup for in-vivo multiphoton optogenetics. The results clearly show that the proposed method can achieve computational speed performances close to the random superposition algorithm, while retaining the high performance of the Gerchberg–Saxton algorithm, with a minimal hologram quality loss@en

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.

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