Sub-voxel light-sheet microscopy for high-resolution, high-throughput volumetric imaging of large biomedical specimens

A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view (FOV). Here, we report a sub-voxel light-sheet microscopy (SLSM) method enabling high-throughput volumetric imaging of mesoscale specimens at cellular-resolution. A non-axial, continuous scanning strategy is used to rapidly acquire a stack of large-FOV images with three-dimensional (3-D) nanoscale shifts encoded. Then by adopting a sub-voxel-resolving procedure, the SLSM method models these low-resolution, cross-correlated images in the spatial domain and iteratively recovers 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. As demonstrated by quick reconstruction (minutes to hours) of various samples, e.g., 3-D cultured cells, an intact mouse heart, mouse brain, and live zebrafish embryo, the SLSM method presents a high-throughput way to circumvent the tradeoff between intoto mapping of large-scale tissue (>100 mm3) and isotropic imaging of single-cell (~1-m resolution). It also eliminates the need of complicated mechanical stitching or precisely modulated illumination, using a simple light-sheet setup and fast graphics-processing-unit (GPU)-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.