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

Abstract

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.

Data availability

Data have been uploaded in their original form on Zenodo: https://doi.org/10.5281/zenodo.5639726

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 (https://github.com/AdvancedImagingUTSW/manuscripts).

References

  1. Power, R. M. & Huisken, J. A guide to light-sheet fluorescence microscopy for multiscale imaging. Nat. Methods 14, 360–373 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Siedentopf, H. & Zsigmondy, R. Über Sichtbarmachung und Größenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser. Ann. Phys. 315, 1–39 (1902).

    Article  Google Scholar 

  3. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Voie, A. H., Burns, D. H. & Spelman, F. A. Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229–236 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Daetwyler, S. & Huisken, J. Fast fluorescence microscopy with light sheets. Biol. Bull. 231, 14–25 (2016).

    Article  PubMed  Google Scholar 

  6. Gao, L., Shao, L., Chen, B.-C. & Betzig, E. 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat. Protoc. 9, 1083–1101 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Born, M. et al. Principles of Optics (Cambridge Univ. Press, 2013).

  8. Fahrbach, F. O., Gurchenkov, V., Alessandri, K., Nassoy, P. & Rohrbach, A. Self-reconstructing sectioned Bessel beams offer submicron optical sectioning for large fields of view in light-sheet microscopy. Opt. Express 21, 11425–11440 (2013).

    Article  PubMed  Google Scholar 

  9. Vettenburg, T. et al. Light-sheet microscopy using an Airy beam. Nat. Methods 11, 541–544 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Chen, B.-C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Chang, B.-J. et al. Universal light-sheet generation with field synthesis. Nat. Methods 16, 235–238 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gao, L. Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet. Opt. Express 23, 6102–6111 (2015).

    Article  PubMed  Google Scholar 

  13. Chang, B.-J., Dean, K. M. & Fiolka, R. Systematic and quantitative comparison of lattice and Gaussian light-sheets. Opt. Express 28, 27052–27077 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Chakraborty, T. et al. Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution. Nat. Methods 16, 1109–1113 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dean, K. M. et al. Deconvolution-free subcellular imaging with axially swept light sheet microscopy. Biophys. J. 108, 2807–2815 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Preibisch, S. et al. Efficient Bayesian-based multiview deconvolution. Nat. Methods 11, 645–648 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Swoger, J., Verveer, P., Greger, K., Huisken, J. & Stelzer, E. H. Multi-view image fusion improves resolution in three-dimensional microscopy. Opt. Express 15, 8029–8042 (2007).

    Article  PubMed  Google Scholar 

  18. Wu, Y. et al. Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat. Biotechnol. 31, 1032–1038 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Chhetri, R. K. et al. Whole-animal functional and developmental imaging with isotropic spatial resolution. Nat. Methods 12, 1171–1178 (2015).

    Article  CAS  PubMed  Google Scholar 

  20. Botcherby, E. J., Juskaitis, R., Booth, M. J. & Wilson, T. Aberration-free optical refocusing in high numerical aperture microscopy. Opt. Lett. 32, 2007–2009 (2007).

    Article  PubMed  Google Scholar 

  21. Voigt, F. F. et al. The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue. Nat. Methods 16, 1105–1108 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Heintzmann, R. in Fluorescence Microscopy 393–401 (Wiley, 2013).

  23. Santi, P. A. et al. Thin-sheet laser imaging microscopy for optical sectioning of thick tissues. BioTechniques 46, 287–294 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Dean, K. M. & Fiolka, R. Uniform and scalable light-sheets generated by extended focusing. Opt. Express 22, 26141–26152 (2014).

    Article  PubMed  Google Scholar 

  25. Zurauskas, M., Barnstedt, O., Frade-Rodriguez, M., Waddell, S. & Booth, M. J. Rapid adaptive remote focusing microscope for sensing of volumetric neural activity. Biomed. Opt. Express 8, 4369–4379 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Botcherby, E. J. et al. Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates. Proc. Natl Acad. Sci. USA 109, 2919–2924 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hedde, P. N. & Gratton, E. Selective plane illumination microscopy with a light sheet of uniform thickness formed by an electrically tunable lens. Microsc. Res. Tech. 81, 924–928 (2018).

    Article  CAS  PubMed  Google Scholar 

  28. Chakraborty, T. et al. Converting lateral scanning into axial focusing to speed up three-dimensional microscopy. Light Sci. Appl. 9, 165 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sofroniew, N. J., Flickinger, D., King, J. & Svoboda, K. A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. eLife 5, e14472 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Remacha, E., Friedrich, L., Vermot, J. & Fahrbach, F. O. How to define and optimize axial resolution in light-sheet microscopy: a simulation-based approach. Biomed. Opt. Express 11, 8–26 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Planchon, T. A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Escobet-Montalbán, A. et al. Three-photon light-sheet fluorescence microscopy. Opt. Lett. 43, 5484–5487 (2018).

    Article  PubMed  Google Scholar 

  33. Welf, E. S. et al. Quantitative multiscale cell imaging in controlled 3D microenvironments. Dev. Cell 36, 462–475 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Landry, J., Hamann, S. & Solgaard, O. High-speed axially swept light sheet microscopy using a linear MEMS phased array for isotropic resolution. J. Biomed. Opt. 25, 106504 (2020).

    Article  PubMed Central  Google Scholar 

  35. Dodt, H. U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chen, F., Tillberg, P. W. & Boyden, E. S. Optical imaging. Expansion microscopy. Science 347, 543–548 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mohan, A. S. et al. Enhanced dendritic actin network formation in extended Lamellipodia drives proliferation in growth-challenged Rac1(P29S) melanoma cells. Dev. Cell 49, 444–460 e449 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Welf, E. S. et al. Actin-membrane release initiates cell protrusions. Dev. Cell 55, 723–736 e728 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Fahrbach, F. O., Voigt, F. F., Schmid, B., Helmchen, F. & Huisken, J. Rapid 3D light-sheet microscopy with a tunable lens. Opt. Express 21, 21010–21026 (2013).

    Article  PubMed  Google Scholar 

  41. Edelstein, A. D. et al. Advanced methods of microscope control using μManager software. J. Biol. Methods 1, e10 (2014).

    Article  PubMed  Google Scholar 

  42. Pinkard, H. et al. Pycro-Manager: open-source software for customized and reproducible microscope control. Nat. Methods 18, 226–228 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Marin, Z. et al. PYMEVisualize: an open-source tool for exploring 3D super-resolution data. Nat. Methods 18, 582–584 (2021).

    Article  CAS  PubMed  Google Scholar 

  44. Huisken, J. & Stainier, D. Y. R. Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt. Lett. 32, 2608–2610 (2007).

    Article  PubMed  Google Scholar 

  45. Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).

    Article  CAS  PubMed  Google Scholar 

  46. Jing, D. et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res. 28, 803–818 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. M’Saad, O. & Bewersdorf, J. Light microscopy of proteins in their ultrastructural context. Nat. Commun. 11, 3850 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Li, S., Van Den Diepstraten, C., D’Souza, S. J., Chan, B. M. C. & Pickering, J. G. Vascular smooth muscle cells orchestrate the assembly of type I collagen via α2β1 integrin, rhoa, and fibronectin polymerization. Am. J. Pathol. 163, 1045–1056 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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

Authors

Contributions

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.

Ethics declarations

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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Related links

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

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). https://doi.org/10.1038/s41596-022-00706-6

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