Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0

Abstract

The limited per-pixel bandwidth of most microscopy methods requires compromises between field of view, sampling density and imaging speed. This limitation constrains studies involving complex motion or fast cellular signaling, and presents a major bottleneck for high-throughput structural imaging. Here, we combine high-speed intensified camera technology with a versatile, reconfigurable and dramatically improved Swept, Confocally Aligned Planar Excitation (SCAPE) microscope design that can achieve high-resolution volumetric imaging at over 300 volumes-persecond and over 1.2 GHz pixel rates. We demonstrate near-isotropic sampling in freely moving C. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Capturing volumetric dynamics at high speed in the brain by confocal light field microscopy

“…Besides confocal detection, selective plane illumination is another effective method to suppress background. In particular, oblique plane illumination and imaging through a single objective preserved the epi-illumination configuration and greatly enhanced its compatibility with biological studies 19,51 . Introducing this strategy to conventional LFM could also be attractive, but the large incident angle of the oblique illumination beam could make it difficult to maintain its beam shape in scattering tissue and reduce the efficiency of background suppression.…”

“…The high volumetric imaging speed and epi-illumination configuration together make LFM a well-suited technique to image freely moving animals, which are of great interests in neuroscience because of the extended behavioral paradigms to understand various complex brain functions 19,[36][37][38][39] . In particular, neural activity in groups of neurons during larval zebrafish's prey capture behavior, which is under extensive investigations [40][41][42][43] , has been recorded at relatively low spatial or temporal resolutions 31,44,45 .…”

3D neural network-based particle tracking reveals spatial heterogeneity of the cytosol

“…Using light sheet microscopy would reduce photobleaching and allow for longer videos. Currently, imaging speed for 3D microscopy with standard piezoelectric stepping is slower than the framerate of a typical camera, and many groups are working to eliminate this bottleneck (24,25). Higher acquisition speeds will generally further improve the quality of all tracking inferences, will enable tracking of smaller rapidly diffusing probes, and will allow for acquisition of thicker volumes.…”

Widespread Receptor Driven Modulation in Peripheral Olfactory Coding

“…High-speed volumetric imaging of intact epithelium was performed on a custom Swept Confocally-Aligned Planar Excitation (SCAPE) microscope extended from designs described in Bouchard et al 2015 and Hillman et al 2019 [15][16][17] . SCAPE is a form of light-sheet microscopy, providing low phototoxicity combined with very high-speed 3D imaging of intact samples through a single, stationary objective lens.…”

“…Suppression in blends has been known from psychophysical experiments for decades [10][11][12][13][14] , but the neural locus of those effects remains largely unknown (unlike vision where inhibition plays a large role, but only in higher circuitry). In the current study we utilized a new technology, Swept Confocally Aligned Planar Excitation (SCAPE) microscopy [15][16][17] . SCAPE enabled high-throughput, high-speed 3-dimensional, simultaneous monitoring of the intracellular calcium responses of large numbers of individual intact olfactory sensory neurons (OSNs) expressing GCaMP6f, imaged in-situ within a uniquely designed mouse hemi-head perfused preparation.…”