Light-Sheet Microscopy in Neuroscience

Abstract

Light-sheet microscopy is an imaging approach that offers unique advantages for a diverse range of neuroscience applications. Unlike point-scanning techniques such as confocal and two-photon microscopy, light-sheet microscopes illuminate an entire plane of tissue, while imaging this plane onto a camera. Although early implementations of light sheet were optimized for longitudinal imaging of embryonic development in small specimens, emerging implementations are capable of capturing light-sheet images in freely moving, unconstrained specimens and even the intact in vivo mammalian brain. Meanwhile, the unique photobleaching and signal-to-noise benefits afforded by light-sheet microscopy's parallelized detection deliver the ability to perform volumetric imaging at much higher speeds than can be achieved using point scanning. This review describes the basic principles and evolution of light-sheet microscopy, followed by perspectives on emerging applications and opportunities for both imaging large, cleared, and expanded neural tissues and high-speed, functional imaging in vivo.

Keywords: GCaMP; functional imaging; light sheet; microscopy; tissue clearing.

Figure 1

Microscopy approaches for optical sectioning: (a) point-scanning confocal, (b) two-photon point scanning, (c) conventional light-sheet microscopy, and (d) oblique light-sheet (e.g. swept confocally aligned planar excitation) microscopy. In all panels, blue (or red) shading indicates excitation light, and green indicates fluorescence emission. Arrows indicate the direction of scanning to form a 3D image. (e) Depiction of the useful versus unused light generated in point scanning. (f) A confocal line-scanning approach to lateral parallelization of illumination. (g) Conventional and oblique light-sheet geometries that more efficiently form a sheet with photons have multiple chances to generate useful interactions with fluorophores along their path, while generating less extraneous fluorescence and thus photobleaching. (h) Depiction of wide-field, temporal-focusing, two-photon microscopy.

Figure 2

Examples of light-sheet approaches for neuroscience applications. (a) Demonstration of ultramicroscopy for light-sheet imaging of cleared samples (left), including green fluorescent protein (GFP)-expressing neurons in the mouse brain (right). Panel a adapted with permission from Dodt et al. (2007). (b) Whole-brain imaging of calcium activity (GCaMP) in a zebrafish larva at around 1 volume per second (right). (Left) The system used scanned light sheets incident from both sides of the sample, with the detection lens moved by a piezo actuator, to maintain focus on the sheet. The fish was positioned in an agarose tube between the three objective lenses. Panel b adapted with permission from Ahrens et al. (2013). (c) Swept confocally aligned planar excitation (SCAPE) imaging of GCaMP activity in apical dendrites of layer 5 neurons in awake, behaving mouse cortex at 10 volumes per second. (Right, top and middle) Time-color-encoded firing events over a 6-s period shown as maximum-intensity projections over z and x. (Right, bottom) Raw time courses of fluorescence change (ΔF/F) extracted from the locations indicated in the right, top and middle panels, demonstrating minimal photobleaching and a high signal-to-noise ratio. Panel c adapted with permission from Hillman et al. (2018).

Figure 3

Determinants of spatial resolution in light-sheet microscopy. (a) A typical light-sheet geometry combines the light sheet’s Gaussian beam properties with the PSF of the detection optics (see also Equation 1), with resolution depending on the NA of both. (b) A lookup table of Gaussian beam properties for different NA foci shows how the beam’s minimum waist diameter scales with its R (the distance along the sheet at which the waist is $\sqrt{2}$× the minimum beam waist), modeled for 488 nm in water, refractive index n = 1.33. (c) Plots show simulated PSFs based on Equation 1 using a single-beam PSF model (PSF_Generator) by Kirshner et al.=(2013) (left). Plots show intensity cross sections through the combined PSF over z and x directions (right). Abbreviations: em, emission; ex, excitation; NA, numerical aperture; PSF, point spread function; R, Rayleigh range.