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Assessing photodamage in live-cell STED microscopy

Nature Methods volume 15, pages 755–756 (2018)Cite this article

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To the Editor — The recent breakthroughs in the development of optical nanoscopy have provided unprecedented views of the inner workings of cells. Stimulated emission depletion (STED) microscopy, in particular, allows real-time observation of living cells at resolutions of 50 nm or less1,2. However, the high irradiation intensities used in STED nanoscopy have raised concerns about the validity of live-cell observations obtained with this and similar approaches3,4. We report here that, under the right conditions, living cells can be imaged by STED nanoscopy without substantial photodamage.

We observed, however, that use of a slower scanner (1 kHz) led to a more pronounced FluoForte response, which suggests that concentrating the irradiation of an area in time, rather than distributing it more evenly, increases photodamage (Supplementary Note 5). The stress response also depended on which cellular compartment—ER, mitochondria (outer membrane protein 25 (OMP25)), Golgi (α-mannosidase II), or histones (H2B)—was labeled (Supplementary Methods, Supplementary Note 6), and it increased with the amount of SiR dye present in each cell (Supplementary Note 7). These last observations suggest that stress was mediated through light absorption of the SiR dye.

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Fig. 1: Short- and long-term effects of live-cell STED imaging on COS7 and HeLa cells.

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Acknowledgements

We thank S. Baguley, F. Bottanelli, and L.K. Schroeder for generating and providing plasmids; A.E.S. Barentine for determining the STED microscope resolution; E.B. Kromann for support with the custom-built STED microscope; M. Germino and Y. Zhang for helpful discussions regarding data analysis; A. Kumar (Enzo Life Sciences, Inc.) for support regarding the use of FluoForte; and B.G. Rash for comments on the manuscript. This work was supported by grants from the Wellcome Trust (095927/A/11/Z and 203285/B/16/Z to D.T., J.E.R., and J.B.) and the G. Harold & Leila Y. Mathers Foundation (to J.B.). The used Leica SP8 STED microscope was supported by NIH (S10OD020142). M.D.L. and J.B. acknowledge support by the NIH-funded Yale Center of Excellence in Hematology (U54DK106857). D.T. and J.B. were supported by the Yale Diabetes Research Center (NIH P30DK045735).

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Author notes

  1. These authors contributed equally: Nicole Kilian and Alexander Goryaynov.

Authors and Affiliations

  1. Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA

    Nicole Kilian, Alexander Goryaynov, Mark D. Lessard, Derek Toomre, James E. Rothman & Joerg Bewersdorf

  2. Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, NY, USA

    Giles Hooker

  3. Department of Statistical Science, Cornell University, Ithaca, NY, USA

    Giles Hooker

  4. Nanobiology Institute, Yale University, West Haven, CT, USA

    Derek Toomre, James E. Rothman & Joerg Bewersdorf

  5. Department of Biomedical Engineering, Yale University, New Haven, CT, USA

    Joerg Bewersdorf

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  1. Nicole Kilian
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Contributions

N.K., A.G., and J.B. conceived and designed the experiments. N.K., A.G., and M.D.L. performed the experiments. N.K., A.G., M.D.L., G.H., D.T., J.E.R., and J.B. analyzed and discussed the data. G.H. performed the statistical analysis. N.K., A.G., and J.B. wrote the manuscript.

Corresponding author

Correspondence to Joerg Bewersdorf.

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Competing interests

J.B. has financial interests in Bruker Corp. and Hamamatsu Photonics. The other authors declare no competing interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Notes

Reporting Summary

Supplementary Video 1

STED time-lapse video of a representative COS7 cell

Supplementary Video 2

Video of a COS7 cell surviving for at least 24 h after STED irradiation

Supplementary Video 3

Video of a COS7 cell dying a few hours after STED irradiation

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Kilian, N., Goryaynov, A., Lessard, M.D. et al. Assessing photodamage in live-cell STED microscopy. Nat Methods 15, 755–756 (2018). https://doi.org/10.1038/s41592-018-0145-5

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