Rapid 3D imaging of whole organs at cellular resolution is vital to pathological research, but this task remains challenging in light sheet microscopy due to the compromise between axial resolution and field of view. In the meanwhile, needed magnification varies when doing different analysis. Here, we report on a high-throughput multi-scale light sheet microscopy which combines adjustable beam shaping system providing continuous imaging magnification from 1.26X~12.6X, sample holding device for fast sample switching, modulation mask to generate thin optical sectioning and large illumination field, capable of imaging a lung lobe with cellular resolution (~2.8μm), centimeter-scale field of view (1.1cm×0.8cm) within 1 minute. We demonstrate the imaging of hundreds of lung lobes of mice and other large-scale tissue. Using our ultra-high throughput light sheet microscopy with multiple magnification, we are able to quickly get the 3D view of the whole tissue and the statistical data of the volume of tumors, the number tumor cells and so on for further biological analysis.
High-throughput 3D imaging of multiple organs and organisms at cellular resolution is a recurring challenge in statistical experiments. Here we report on a computational light-sheet microscopy achieving high-throughput high-resolution mapping of multiple macro-scale organs. Through combining a dual-side confocally-scanned Bessel light-sheet illumination with a content-aware compressed sensing (CACS) computation, our approach yields 3D images with high, isotropic spatial resolution and rapid acquisition over two-order-of-magnitude faster than conventional 3D microscopy implementations. And we designed a holder suitable for multi-sample imaging to avoid wasting time during switching samples when imaging a batch of biological samples. This multi-sample holding module improves the switching time to 1s per sample, providing notably higher throughput for batch samples imaging. In addition, we can analyze the imaging results in different application requirements, such as accurately region segmentation, nuclei counting, etc., which have played an important role in the research of biomedical issues.
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 plane-illuminated 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.
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.
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