Abstract
Imaging large cleared tissues requires scaling the throughput of imaging techniques. Light sheet microscopy is a promising technique for high-throughput imaging; however, its reliance on conventional microscope objectives limits the optimization of the trade-off between spatial resolution and field of view. Here we introduce curved light sheet microscope to perform optical sectioning with curved light sheets. This concept addresses the long-standing field curvature problem and lowers the barriers in designing high-throughput objectives. Leveraging a customized objective, the curved light sheet microscope achieves diffraction-limited resolution of 1.0 μm laterally and 2.5 μm axially, with uniform contrast over a field of view of more than 1 × 1 cm2. Our technique is also compatible with various tissue clearing techniques. We demonstrate that imaging an entire intact cleared mouse brain at a voxel size of 0.625 × 0.625 × 1.25 μm3 can be completed in less than 3 h, without the need for image tiling. We share a full optical description of the objective and report imaging of neuronal and vascular networks, as well as tracing of brain-wide long-distance axonal projections in intact mouse brains.
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Data availability
Design data of the objective and curved light sheet generation module—along with the associated imaging parameters—are provided in Supplementary Information. Due to their large size, imaging datasets acquired with the curved light sheet microscope are not available in a public repository and are available from the corresponding author upon reasonable request.
Code availability
The imaging datasets were acquired using vendor-provided software for stage scanning (Kinesis, Thorlabs) and TDI camera control (CamExpert, Teledyne). The code for the PSF analysis is available from the corresponding author on reasonable request.
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Acknowledgements
This work was supported by the CAMS Innovation Fund for Medical Sciences (2024-I2M-3-024 to J. Wu), the start-up funds from CIBR (to J. Wu), and the Beijing Municipal Science & Technology Commission (Z220009 to J. Wu). We thank the CIBR LARC staff for animal care and the CIBR Imaging Core, Instrumentation Core, Computing and Data Science Core for their support.
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Contributions
J. Wu conceived the project. J. Wu and H.Z. supervised the research. J. Wu and L.T. designed the microscope. L.T. constructed the microscope, and collected and analysed the data. J. Wang, J.D., J.S., X.-j.C., R.S., P.C., R.G., W.-p.G., W.S. and H.Z. prepared the samples. Q.S. and F.X. traced the neurons. J. Wu and L.T. wrote the paper with inputs from all authors.
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J. Wu and L.T. are inventors on a patent application related to this work filed by CIBR. The other authors declare no competing interests.
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Nature Photonics thanks Tonmoy Chakraborty, Kevin Dean and Adam Glaser for their contribution to the peer review of this work.
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Supplementary Information
Supplementary Figs. 1–19, Table 1 and Notes 1–4.
Supplementary Video 1
Whole-brain imaging of a PEGASOS-cleared Thy1-eGFP mouse brain.
Supplementary Video 2
Whole-brain vascular network imaging of a PEGASOS-cleared mouse brain (Tie2-Cre::Ai47).
Supplementary Video 3
Whole-brain vascular network imaging of a PEGASOS-cleared mouse brain (Pitx2-Cre::Ai47).
Supplementary Video 4
Imaging of a propidium-iodide-labelled mouse brain cleared by iDISCO.
Supplementary Video 5
Imaging of a propidium-iodide-labelled mouse kidney cleared by iDISCO.
Supplementary Video 6
Imaging of a propidium-iodide-labelled mouse brain cleared by a commercial hydrophilic tissue clearing kit.
Supplementary Video 7
Imaging of a mouse kidney (Tie2-Cre::Ai47) cleared by CUBIC.
Supplementary Video 8
Imaging of sparsely labelled neurons in the motor cortex of a PEGASOS-cleared mouse brain (AAV2/9-hsyn-Cre and AAV2/9-EF1 α-DIO-mScarlet injection).
Supplementary Video 9
Two-colour imaging of a PEGASOS-cleared intact mouse brain with sparse labelling of neurons co-expressing eGFP and mScarlet in the primary motor cortex.
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Tang, L., Wang, J., Ding, J. et al. Curved light sheet microscopy for centimetre-scale cleared tissue imaging. Nat. Photon. 19, 577–584 (2025). https://doi.org/10.1038/s41566-025-01659-4
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DOI: https://doi.org/10.1038/s41566-025-01659-4
