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Isotropic imaging across spatial scales with axially swept light-sheet microscopy


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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Fig. 1: ASLM.
Fig. 2: Multiscale imaging with ASLM.
Fig. 3: Principles of aberration-free remote focusing.
Fig. 4: Optical principle for axial sweeping of a light sheet.
Fig. 5: Numerical simulations for light sheets derived via ASLM and a scanned Bessel beam.
Fig. 6: Detailed optical schematics of generic variants of ASLM.
Fig. 7: Complete CAD rendering of ctASLMv1.
Fig. 8: Fine alignment of the ASLM scan.
Fig. 9: Adjusting the scan range and offset to the rolling shutter of the camera.
Fig. 10: ASLM imaging of fluorescent nanospheres embedded in agarose.
Fig. 11: Fine alignment of the light sheet.
Fig. 12: Rotational misalignment between beam waist and camera.
Fig. 13: Imaging of the beam waist in collagen.
Fig. 14: ASLM imaging of a fluorescent collagen sample.
Fig. 15: ASLM imaging of a cleared mouse brain sample.
Fig. 16: ASLM imaging in a BABB cleared mouse bone marrow sample.

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Data availability

Data have been uploaded in their original form on Zenodo:

Code availability

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 (


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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.

Author information

Authors and Affiliations



K.M.D, S.D. and R.F. wrote the manuscript. K.M.D., T.C., S.D., J.L., and R.F. contributed to the development of the protocol. G.G. wrote the microscope control software. O.M’S., H.T.M., M.S., E.T.S. and J.B. provided biological samples for evaluation of instrument performance. F.F.V. and F.H. provided data for the mesoSPIM variant of ASLM. All authors read and approved of the manuscript.

Corresponding authors

Correspondence to Kevin M. Dean or Reto Fiolka.

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Competing interests

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

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):

Dean, K. M. et al. Biophys. J. 108, 2807–2815 (2016):

Voigt, F. F. et al. Nat. Methods 16, 1105–1108 (2019):

Key data used in this protocol

Dean, K. M. et al. Zenodo10.5281/zenodo.5639726

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Supplementary Tables 1–9 and Supplementary Notes 1–6.

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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).

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