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.
Low-cost and high-performance 3-D light-sheet fluorescence imaging on pre-owned conventional microscopes
Tingting Zhu, Xinlin Xie, Yao Yao, Dan Zhu2,* and Peng Fei1, 2, *
1. School of Optics and Electronic Information, Huazhong University of Science and Technology, Wuhan 430074, China
2. Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China
Abstract: light-sheet fluorescence microscopy (LSFM) is a promising imaging technique that enable imaging of samples in three dimensions at high speed and low phototoxicity1.LSFM is fundamentally characterized by its separate laser-sheet illumination path that provides optical sectioning of the samples and otherwise shows the same wide-field fluorescence detection with the conventional microscopes. Most of LSFM modalities are currently independent from either conventional microscopes, containing highly complicated setting with large form factors and high maintenance. Given the number of epifluorescence microscopes in service, a much easier way to access advanced LSFM imaging can be realized by creating a laser-sheet illumination and make use of existing conventional microscope for fluorescence readout. Here we present light-sheet imaging plugin (LIP) method that readily enables multi-dimensional, high spatiotemporal resolution imaging from single cells to whole organisms on an inverted microscope2.Furthermore, LIP can be also combined with microfluidics techniques to achieve high-throughput, high-resolution 3-D imaging/screening at a speeds up to 30 samples per seconds3.The ability to achieve high-speed, multi-dimensional imaging on a conventional microscope renders LIP device a valuable tool for many biomedical applications such as embryo development, tissue pathology and neuroscience. Furthermore, its compact add-on format allows the full use of pre-owned equipment and renovation at an affordable expense, which could substantially benefit smaller and less-funded departments / laboratories for not limiting the access to greater biological research in the absence of commercial light-sheet systems.
Keywords: Light-sheet microscopy, light-sheet imaging plugin, microfluidics, 3-D biomedical imaging
1. Power R M, Huisken J. A guide to light-sheet fluorescence microscopy for multiscale imaging.[J]. Nature Methods, 2017, 14(4):360.
2. Guan Z, Lee J, Jiang H, et al. Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope[J]. Biomedical Optics Express, 2015, 7(1):194.
3. Jiang H, Zhu T, Zhang H, et al. Droplet-based light-sheet fluorescence microscopy for high-throughput sample preparation, 3-D imaging and quantitative analysis on a chip[J]. Lab on A Chip, 2017.
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.