Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy

Nature Biotechnology volume 39, pages 609–618 (2021)Cite this article

Subjects

Abstract

Elucidating the volumetric architecture of organelles and molecules inside cells requires microscopy methods with a sufficiently high spatial resolution in all three dimensions. Current methods are limited by insufficient resolving power along the optical axis, long recording times and photobleaching when applied to live cell imaging. Here, we present a 3D, parallelized, reversible, saturable/switchable optical fluorescence transition (3D pRESOLFT) microscope capable of delivering sub-80-nm 3D resolution in whole living cells. We achieved rapid (1–2 Hz) acquisition of large fields of view (~40 × 40 µm2) by highly parallelized image acquisition with an interference pattern that creates an array of 3D-confined and equally spaced intensity minima. This allowed us to reversibly turn switchable fluorescent proteins to dark states, leading to a targeted 3D confinement of fluorescence. We visualized the 3D organization and dynamics of organelles in living cells and volumetric structural alterations of synapses during plasticity in cultured hippocampal neurons.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Interference based OFF-switching pattern in 3D pRESOLFT.
Fig. 2: 3D pRESOLFT imaging scheme and parallelization.
Fig. 3: Resolution assessment in 3D pRESOLFT.
Fig. 4: Four-dimensional imaging and rendering in full-length human cells.
Fig. 5: Structural alteration of mitochondria following oxidative stress.
Fig. 6: Intrasynaptic neuronal imaging with isotropic 3D pRESOLFT.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The software used to acquire the super-resolved data (hardware control, image reconstruction and deconvolution pipeline) were developed by our laboratory and available upon request.

References

  1. Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).

    Article  CAS  Google Scholar 

  2. Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. USA 106, 3125–3130 (2009).

    Article  CAS  Google Scholar 

  3. Aquino, D. et al. Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat. Methods 8, 353–359 (2011).

    Article  CAS  Google Scholar 

  4. Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    Article  CAS  Google Scholar 

  5. Juette, M. F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

    Article  CAS  Google Scholar 

  6. Pavani, S. R. et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl Acad. Sci. USA 106, 2995–2999 (2009).

    Article  CAS  Google Scholar 

  7. Willig, K. I., Harke, B., Medda, R. & Hell, S. W. STED microscopy with continuous wave beams. Nat. Methods 4, 915–918 (2007).

    Article  CAS  Google Scholar 

  8. Huang, F. et al. Ultra-high resolution 3D imaging of whole cells. Cell 166, 1028–1040 (2016).

    Article  CAS  Google Scholar 

  9. Gustafsson, M. G. L. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).

  10. Shao, L., Kner, P., Rego, E. H. & Gustafsson, M. G. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat. Methods 8, 1044–1046 (2011).

    Article  CAS  Google Scholar 

  11. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    CAS  PubMed  Google Scholar 

  12. Klar, T. A., Engel, E. & Hell, S. W. Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes. Phys. Rev. E 64, 066613 (2001).

    Article  CAS  Google Scholar 

  13. Hell, S. W., Jakobs, S. & Kastrup, L. Imaging and writing at the nanoscale with focused visible light through saturable optical transitions. Appl. Phys. A Mater. Sci. Process. 77, 859–860 (2003).

    Article  CAS  Google Scholar 

  14. Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S. W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl Acad. Sci. USA 102, 17565–17569 (2005).

    Article  CAS  Google Scholar 

  15. Grotjohann, T. et al. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature 478, 204–208 (2011).

    Article  CAS  Google Scholar 

  16. Testa, I. et al. Nanoscopy of living brain slices with low light levels. Neuron 75, 992–1000 (2012).

    Article  CAS  Google Scholar 

  17. Bohm, U., Hell, S. W. & Schmidt, R. 4Pi-RESOLFT nanoscopy. Nat. Commun. 7, 10504 (2016).

    Article  Google Scholar 

  18. Grotjohann, T. et al. rsEGFP2 enables fast RESOLFT nanoscopy of living cells. eLife 1, e00248 (2012).

    Article  Google Scholar 

  19. Pennacchietti, F. et al. Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy. Nat. Methods 15, 601–604 (2018).

    Article  CAS  Google Scholar 

  20. Chmyrov, A. et al. Nanoscopy with more than 100,000 ‘doughnuts’. Nat. Methods 10, 737–740 (2013).

    Article  CAS  Google Scholar 

  21. Masullo, L. A. et al. Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems. Nat. Commun. 9, 3281 (2018).

  22. Rego, E. H. et al. Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution. Proc. Natl Acad. Sci. USA 109, E135–E143 (2012).

    Article  CAS  Google Scholar 

  23. Li, D. et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).

    Article  Google Scholar 

  24. Xue, Y. & So, P. T. C. Three-dimensional super-resolution high-throughput imaging by structured illumination STED microscopy. Opt. Express 26, 20920–20928 (2018).

    Article  CAS  Google Scholar 

  25. Mahecic, D., Testa, I., Griffie, J. & Manley, S. Strategies for increasing the throughput of super-resolution microscopies. Curr. Opin. Chem. Biol. 51, 84–91 (2019).

    Article  CAS  Google Scholar 

  26. Stiel, A. C. et al. 1.8 Angstrom bright-state structure of the reversibly switchable fluorescent protein dronpa guides the generation of fast switching variants. Biochem. J. 402, 35–42 (2007).

    Article  CAS  Google Scholar 

  27. Bodén, A., Casas Moreno, X., Cooper, B. K., York, A. G. & Testa, I. Predicting resolution and image quality in RESOLFT and other point scanning microscopes [Invited]. Biomed. Opt. Express 11, 2313–2327 (2020).

    Article  Google Scholar 

  28. van Niel, G., D’Angelo, G. & Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19, 213–228 (2018).

    Article  Google Scholar 

  29. Chan, D. C. Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241–1252 (2006).

    Article  CAS  Google Scholar 

  30. Ding, W. X. et al. Electron microscopic analysis of a spherical mitochondrial structure. J. Biol. Chem. 287, 42373–42378 (2012).

    Article  CAS  Google Scholar 

  31. Miyazono, Y. et al. Uncoupled mitochondria quickly shorten along their long axis to form indented spheroids, instead of rings, in a fission-independent manner. Sci. Rep. 8, 350 (2018).

    Article  Google Scholar 

  32. Meyer, D., Bonhoeffer, T. & Scheuss, V. Balance and stability of synaptic structures during synaptic plasticity. Neuron 82, 430–443 (2014).

    Article  CAS  Google Scholar 

  33. Masch, J. M. et al. Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling. Proc. Natl Acad. Sci. USA 115, E8047–E8056 (2018).

    Article  CAS  Google Scholar 

  34. Hruska, M., Henderson, N., Le Marchand, S. J., Jafri, H. & Dalva, M. B. Synaptic nanomodules underlie the organization and plasticity of spine synapses. Nat. Neurosci. 21, 671–682 (2018).

    Article  CAS  Google Scholar 

  35. Tao-Cheng, J. H., Thein, S., Yang, Y., Reese, T. S. & Gallant, P. E. Homer is concentrated at the postsynaptic density and does not redistribute after acute synaptic stimulation. Neuroscience 266, 80–90 (2014).

    Article  CAS  Google Scholar 

  36. Spahn, C., Grimm, J. B., Lavis, L. D., Lampe, M. & Heilemann, M. Whole-cell, 3D, and multicolor STED imaging with exchangeable fluorophores. Nano Lett. 19, 500–505 (2019).

    Article  CAS  Google Scholar 

  37. Booth, M. J. Adaptive optical microscopy: the ongoing quest for a perfect image. Light Sci. Appl. 3, e165 (2014).

    Article  Google Scholar 

  38. Ratz, M., Testa, I., Hell, S. W. & Jakobs, S. CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells. Sci. Rep. 5, 9592 (2015).

    Article  CAS  Google Scholar 

  39. Hosokawa, T., Rusakov, D. A., Bliss, T. V. & Fine, A. Repeated confocal imaging of individual dendritic spines in the living hippocampal slice: evidence for changes in length and orientation associated with chemically induced LTP. J. Neurosci. 15, 5560–5573 (1995).

    Article  CAS  Google Scholar 

  40. Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

I.T. thanks the ERC (ERC_StG 638314, MoNaLISA) and the Swedish Foundation for Strategic Research (FFL15-0031) for supporting the project.

Author information

Authors and Affiliations

  1. Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden

    Andreas Bodén, Francesca Pennacchietti, Giovanna Coceano, Martina Damenti & Ilaria Testa

  2. Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden

    Michael Ratz

Authors
  1. Andreas Bodén
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesca Pennacchietti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Giovanna Coceano
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martina Damenti
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Ratz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ilaria Testa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

I.T. designed and supervised the project. A.B. engineered and built the microscope with associated software. A.B. performed the experiments and data analysis. F.P. carried out RSFP switching experiments and data analysis. G.C. and M.D. performed the bioimaging treatments and imaging. M.R. cloned the constructs and provided biological guidance. I.T. and A.B. wrote the manuscript with assistance from all the authors.

Corresponding author

Correspondence to Ilaria Testa.

Ethics declarations

Competing interests

I.T. and A.B. have filed a provisional patent on the 3D pRESOLFT technology (no. N.1930406-2, Sweden, December 2019).

Additional information

Peer review information Nature Biotechnology thanks Reto Fiolka, Valentin Nagerl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Notes 1–9, Figs. 1–15 and Tables 1–5.

Reporting Summary

Supplementary Video 1

Mitochondrial 3D dynamics.

Supplementary Video 2

Entire mitochondrial network of a U2OS cell.

Supplementary Video 3

Entire actin cytoskeleton of a U2OS cell.

Supplementary Video 4

Actin architecture in hippocampal neuron.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bodén, A., Pennacchietti, F., Coceano, G. et al. Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy. Nat Biotechnol 39, 609–618 (2021). https://doi.org/10.1038/s41587-020-00779-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-020-00779-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing