Abstract
The recent integration of light‐sheet microscopy and tissue‐clearing has facilitated an important alternative to conventional histological imaging approaches. However, the in toto cellular mapping of neural circuits throughout an intact mouse brain remains highly challenging, requiring complicated mechanical stitching, and suffering from anisotropic resolution insufficient for high‐quality reconstruction in 3D. Here, the use of a multiangle‐resolved subvoxel selective plane illumination microscope (Mars‐SPIM) is proposed to achieve high‐throughput imaging of whole mouse brain at isotropic cellular resolution. This light‐sheet imaging technique can computationally improve the spatial resolution over six times under a large field of view, eliminating the use of slow tile stitching. Furthermore, it can recover complete structural information of the sample from images subject to thick‐tissue scattering/attenuation. With Mars‐SPIM, a digital atlas of a cleared whole mouse brain (≈7 mm × 9.5 mm × 5 mm) can readily be obtained with an isotropic resolution of ≈2 µm (1 µm voxel) and a short acquisition time of 30 min. It provides an efficient way to implement system‐level cellular analysis, such as the mapping of different neuron populations and tracing of long‐distance neural projections over the entire brain. Mars‐SPIM is thus well suited for high‐throughput cell‐profiling phenotyping of brain and other mammalian organs.
Highlights
The recent integration of light-sheet microscopy and tissue-clearing has facilichallenges in neuroscience
The use of a multiangle-resolved subvoxel brain, 3D high-resolution (HR) imaging is required over a mesoscale sized volume.[1]. Creating such a large-scale brain dataset has posed a big challenge for current 3D optical microscopy methods, all selective plane illumination microscope (Mars-selective plane illumination microscopy (SPIM)) is proposed to achieve of which show relatively small optical high-throughput imaging of whole mouse brain at isotropic cellular resoluthroughputs.[2,3]
We developed a Mars-SPIM system, with a low-profile setup with wide-field of view (FOV) illumination and detection sufficient to cover the entire mouse brain
Summary
We developed a Mars-SPIM system, with a low-profile setup with wide-FOV illumination and detection sufficient to cover the entire mouse brain (see the Experimental Section and Figures S1 and S2, Supporting Information). The camera is synchronized to sequentially record a stack of plane images with a step-size significantly smaller than the laser-sheet thickness This nonaxial scanning mode of the Mars-SPIM encodes the LR raw image stack with subvoxel spatial shifts, which are used to reconstruct HR images through the application of an SVR algorithm (Figure S3, Supporting Information). The microbeads were scanned by a ≈12 μm thick (full width at half maximum value, FWHM) laser sheet with 280 nm step size and were detected by a 4×/0.16 objective This nonaxial scanning process (10° to the x–z and y–z planes) generated subresolution shifts of 48 and 272 nm in the lateral and axial directions respectively. In the following whole-brain applications, this underlying robustness allows the Mars-SPIM prototype to image the entire thick organ with high spatial-temporal performance while maintaining a simple setup
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