A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view (FOV). We report a subvoxel light-sheet microscopy (SLSM) method enabling high-throughput volumetric imaging of mesoscale specimens at cellular resolution. A nonaxial, continuous scanning strategy is developed to rapidly acquire a stack of large-FOV images with three-dimensional (3-D) nanoscale shifts encoded. Then, by adopting a subvoxel-resolving procedure, the SLSM method models these low-resolution, cross-correlated images in the spatial domain and can iteratively recover a 3-D image with improved resolution throughout the sample. This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times, with high acquisition speeds of gigavoxels per minute. By fast reconstruction of 3-D cultured cells, intact organs, and live embryos, SLSM method presents a convenient way to circumvent the trade-off between mapping large-scale tissue (>100 mm3) and observing single cell (∼1-μm resolution). It also eliminates the need of complicated mechanical stitching or modulated illumination, using a simple light-sheet setup and fast graphics processing unit-based computation to achieve high-throughput, high-resolution 3-D microscopy, which could be tailored for a wide range of biomedical applications in pathology, histology, neuroscience, etc.
Light-sheet fluorescence microscopy (LSFM) uses an additional laser-sheet to illuminate selective planes of the sample, thereby enabling three-dimensional imaging at high spatial-temporal resolution. These advantages make LSFM a promising tool for high-quality brain visualization. However, even by the use of LSFM, the spatial resolution remains insufficient to resolve the neural structures across a mesoscale whole mouse brain in three dimensions. At the same time, the thick-tissue scattering prevents a clear observation from the deep of brain. Here we use multi-view LSFM strategy to solve this challenge, surpassing the resolution limit of standard light-sheet microscope under a large field-of-view (FOV). As demonstrated by the imaging of optically-cleared mouse brain labelled with thy1-GFP, we achieve a brain-wide, isotropic cellular resolution of ~3μm. Besides the resolution enhancement, multi-view braining imaging can also recover complete signals from deep tissue scattering and attenuation. The identification of long distance neural projections across encephalic regions can be identified and annotated as a result.