challenges in neuroscience. To unravel the various neuronal profiles of different physiological functions in the whole brain, 3D high-resolution (HR) imaging is required over a mesoscale sized volume. [1] However, creating such a large-scale brain dataset has posed a big challenge for current 3D optical microscopy methods, all of which show relatively small optical throughputs. [2,3] Furthermore, light scattering and attenuation are outstanding issues for the turbid tissues that limit the extraction of signals from deep brain. To address these issues, 3D tile stitching combined with brain sectioning has been a popular strategy for obtaining mammalian brain atlases, which can be a meaningful platform for mapping neuronal populations, activities, or connections over the entire brain. [4] For example, sequential two-photon tomography (STPT) can 3D image the brain at subcellular high resolution, [5,6] but at the cost of long acquisition times of up to several days and a highmaintenance system setup. The advent of light-sheet fluorescence microscopy [7] (LSFM) in conjunction with tissue-clearing [8] eliminates the need for complicated mechanical slicing of samples by instead applying nondestructive optical sectioning. Although LSFM still needs repetitiveThe 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 highquality 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 cellprofiling phenotyping of brain and other mammalian organs.

Propagating-path uniformly scanned light sheet excitation microscopy for isotropic volumetric imaging of large specimens," J.Abstract. We demonstrate a propagating-path uniformly scanned light sheet excitation (PULSE) microscopy based on the oscillation of voice coil motor that can rapidly drive a thin light sheet along its propagation direction. By synchronizing the rolling shutter of a camera with the motion of laser sheet, we can obtain a uniform planeilluminated image far beyond the confocal range of Gaussian beam. A stable 1.7-μm optical sectioning under a 3.3 mm × 3.3 mm wide field of view (FOV) has been achieved for up to 20 Hz volumetric imaging of large biological specimens. PULSE method transforms the extent of plane illumination from one intrinsically limited by the short confocal range (μm scale) to one defined by the motor oscillation range (mm scale). Compared to the conventional Gaussian light sheet imaging, our method greatly mitigates the compromise of axial resolution and successfully extends the FOV over 100 times. We demonstrate the applications of PULSE method by rapidly imaging cleared mouse spinal cord and live zebrafish larva at isotropic subcellular resolution. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Volumetric imaging of dynamic signals in a large, moving, and light-scattering specimen is extremely challenging, owing to the requirement on high spatiotemporal resolution and difficulty in obtaining high-contrast signals. Here...