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Low-cost and scalable projected light-sheet microscopy for the high-resolution imaging of cleared tissue and living samples

Nature Biomedical Engineering volume 8, pages 1109–1123 (2024)Cite this article

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Abstract

Light-sheet fluorescence microscopy (LSFM) is a widely used technique for imaging cleared tissue and living samples. However, high-performance LSFM systems are typically expensive and not easily scalable. Here we introduce a low-cost, scalable and versatile LSFM framework, which we named ‘projected light-sheet microscopy’ (pLSM), with high imaging performance and small device and computational footprints. We characterized the capabilities of pLSM, which repurposes readily available consumer-grade components, optimized optics, over-network control architecture and software-driven light-sheet modulation, by performing high-resolution mapping of cleared mouse brains and of post-mortem pathological human brain samples, and via the molecular phenotyping of brain and blood-vessel organoids derived from human induced pluripotent stem cells. We also report a method that leverages pLSM for the live imaging of the dynamics of sparsely labelled multi-layered bacterial pellicle biofilms at an air–liquid interface. pLSM can make high-resolution LSFM for biomedical applications more accessible, affordable and scalable.

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Fig. 1: Implementation of projected light-sheet microscopy.
Fig. 2: Characterization of projected light-sheet microscopy.
Fig. 3: High-resolution pLSM imaging and quantitative analysis of intact mouse brain samples cleared with iDISCO.
Fig. 4: High-resolution pLSM imaging of CLARITY-cleared mouse Thy1-eYFP brain.
Fig. 5: Rapid, high-resolution pLSM imaging of large post-mortem human brain samples.
Fig. 6: Phenotyping of multiple hiPS cell-derived brain and vessel organoids.
Fig. 7: Mapping the dynamic layered architecture of sparsely labelled (2.5%) bacterial pellicle biofilms.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary information. All the raw data are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

All the code is made available publicly as a GitHub repository at https://github.com/tomerlab/pLSM-Control.

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Acknowledgements

We are grateful to R. Etchenique for initial discussions related to laser projectors and A. Teich and S. Small for providing brain samples and associated information. We thank the families for donating the brain tissue used in this study. R.T. discloses support for the research described in this study from NIH (grant numbers DP2MH119423 and UH3TR002151), and Columbia University Arts and Sciences startup grant. K.W.L. discloses support from NIH (grant numbers UH3TR002151 and UH3NS115598). L.E.P.D. discloses support from NIH (grant number R01AI103369). M.B. discloses support from NIH (grant numbers AI164769, AG076949, MH133561 and AG080790). We thank the Columbia University Alzheimer’s Disease Research Center, funded by NIH (grant number P30AG066462) to S. Small (principal investigator).

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Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, NY, USA

    Yannan Chen, Shradha Chauhan, Cheng Gong, Hannah Dayton, Estanislao Daniel De La Cruz, Yu-Young Wesley Tsai, Malika S. Datta, Lars E. P. Dietrich & Raju Tomer

  2. Department of Biomedical Engineering, Columbia University, New York, NY, USA

    Yannan Chen, Cheng Gong, Cong Xu, Kam W. Leong & Raju Tomer

  3. Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, NY, USA

    Yu-Young Wesley Tsai, Malika S. Datta & Raju Tomer

  4. Department of Psychiatry, Columbia University Irving Medical Center, New York State Psychiatric Institute, New York, NY, USA

    Gorazd B. Rosoklija, Andrew J. Dwork, J. John Mann & Maura Boldrini

  5. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    Andrew J. Dwork

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  1. Yannan Chen
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Contributions

Y.C. and R.T. conceptualized and designed the pLSM framework. Y.C. implemented and characterized pLSM, with inputs from C.G. on optical simulation and CAD design, and from S.C. on initial prototyping. The imaging experiments were conducted as follows: Y.C., Y.-Y.T., S.C., E.D.d.l.C., C.G., M.S.D. and R.T. prepared mouse and stained human brain samples, and performed pLSM imaging. G.B.R., A.J.D., J.J.M. and M.B. collected and phenotype the neurotypical human brain tissue utilized in this study. C.X., S.C., R.T. and K.W.L. generated and imaged the brain and vessel organoids. S.C., H.D., L.E.P.D. and R.T. developed the live-imaging assay for pellicle biofilms and performed the live-imaging experiments. R.T. and Y.C. analysed all the data and wrote the paper, with input from all the authors. R.T. supervised the project.

Corresponding author

Correspondence to Raju Tomer.

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Competing interests

Columbia University has filed a provisional patent application related to pLSM.

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Nature Biomedical Engineering thanks Ali Erturk, Anne Rios and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figures, Tables, Notes and Video captions.

Reporting Summary

Supplementary Video 1

Complete brain-wide visualization of TH+ neurons mapped with pLSM.

Supplementary Video 2

Whole-brain segmentation of TH+ neurons in the same sample imaged with pLSM as well as the COLM system.

Supplementary Video 3

Whole-brain rendering of CLARITY-cleared Thy1-eYFP transgenic mouse brain imaged using pLSM.

Supplementary Video 4

Small-volume rendering of CLARITY-cleared Thy1-eYFP transgenic mouse brain imaged using pLSM.

Supplementary Video 5

Multi-channel pLSM imaging of vasculature in neurotypical post-mortem human brain sample.

Supplementary Video 6

High-resolution rendering of vasculature image volumes at different depths of an intact human brain sample.

Supplementary Video 7

High-resolution imaging of APP in Alzheimer’s disease and neurotypical cortical samples.

Supplementary Video 8

Volumetric rendering of the pLSM imaging of an ensemble of hiPS cell-derived brain organoids.

Supplementary Video 9

Volumetric rendering of hiPS cell-derived vessel organoids imaged with pLSM.

Supplementary Video 10

Live imaging of sparsely labelled (2.5%) bacterial biofilm pellicle at the air–liquid interface.

Supplementary Video 11

Live imaging of sparsely labelled (2.5%) bacterial biofilm pellicle at the air–liquid interface.

Supplementary Video 12

Live imaging of a highly motile sparsely labelled (2.5%) bacterial biofilm pellicle at the air–liquid interface.

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Chen, Y., Chauhan, S., Gong, C. et al. Low-cost and scalable projected light-sheet microscopy for the high-resolution imaging of cleared tissue and living samples. Nat. Biomed. Eng 8, 1109–1123 (2024). https://doi.org/10.1038/s41551-024-01249-9

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