Light-sheet fluorescence microscopy is a rapidly growing technique that has gained tremendous popularity in the life sciences owing to its high-spatiotemporal resolution and gentle, non-phototoxic illumination. In this protocol, we provide detailed directions for the assembly and operation of a versatile light-sheet fluorescence microscopy variant, referred to as axially swept light-sheet microscopy (ASLM), that delivers an unparalleled combination of field of view, optical resolution and optical sectioning. To democratize ASLM, we provide an overview of its working principle and applications to biological imaging, as well as pragmatic tips for the assembly, alignment and control of its optical systems. Furthermore, we provide detailed part lists and schematics for several variants of ASLM that together can resolve molecular detail in chemically expanded samples, subcellular organization in living cells or the anatomical composition of chemically cleared intact organisms. We also provide software for instrument control and discuss how users can tune imaging parameters to accommodate diverse sample types. Thus, this protocol will serve not only as a guide for both introductory and advanced users adopting ASLM, but as a useful resource for any individual interested in deploying custom imaging technology. We expect that building an ASLM will take ~1–2 months, depending on the experience of the instrument builder and the version of the instrument.
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Data have been uploaded in their original form on Zenodo: https://doi.org/10.5281/zenodo.5639726
All software and CAD documents are publicly available on Zenodo: DOI:10.5281/zenodo.6048284. Software redistribution, in source or binary forms, with or without modification, is permitted for academic and research use only according to the license described on the associated GitHub repository (https://github.com/AdvancedImagingUTSW/manuscripts).
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H.T.M. is associated with the Integrated Graduate Program in Physical and Engineering Biology and supported by NIBIB T32EB019941. R.F. is supported by NCI R33CA235254 and NIGMS R35GM133522. K.M.D. is supported by NIDDK R01DK127589, NIMH R01MH120131 and NICHD R21HD105189. S.D. is supported by the Schweizerischer Nationalfonds zur Fӧrderung der Wissenschaftlichen Forschung (P2SKP3_191347). The authors thank J. Manton for help with the numerical simulations of light sheets and point-spread functions.
K.M.D. and R.F. have a patent covering ASLM (US10989661) and consultancy agreements with 3i, Inc (Denver, CO, USA). K.M.D. has an ownership interest in Discovery Imaging Systems, LLC.
Peer review information
Nature Protocols thanks Dayong Jin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Chakraborty, T. et al. Nat. Methods 16, 1109-1113 (2019): https://doi.org/10.1038/s41592-019-0615-4
Dean, K. M. et al. Biophys. J. 108, 2807–2815 (2016): https://doi.org/10.1016/j.bpj.2015.05.013
Voigt, F. F. et al. Nat. Methods 16, 1105–1108 (2019): https://doi.org/10.1038/s41592-019-0554-0
Key data used in this protocol
Dean, K. M. et al. Zenodo10.5281/zenodo.5639726
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Dean, K.M., Chakraborty, T., Daetwyler, S. et al. Isotropic imaging across spatial scales with axially swept light-sheet microscopy. Nat Protoc 17, 2025–2053 (2022). https://doi.org/10.1038/s41596-022-00706-6