Skip to main content

Thank you for visiting 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.

Assessing phototoxicity in live fluorescence imaging


Are the answers to biological questions obtained via live fluorescence microscopy substantially affected by phototoxicity? Although a single set of standards for assessing phototoxicity cannot exist owing to the breadth of samples and experimental questions associated with biological imaging, we need quantitative, practical assessments and reporting standards to ensure that imaging has a minimal impact on observed biological processes and sample health. Here we discuss the problem of phototoxicity in biology and suggest guidelines to improve its reporting and assessment.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: The effect of low levels of blue light on cell proliferation.
Figure 2: The four main considerations for live imaging.
Figure 3: Identifying species-specific criteria for the assessment of phototoxicity.


  1. Magidson, V. & Khodjakov, A. Methods Cell Biol. 114, 545–560 (2013).

    Article  Google Scholar 

  2. Stelzer, E.H.K. Nat. Methods 12, 23–26 (2015).

    CAS  Article  Google Scholar 

  3. Jemielita, M., Taormina, M.J., Delaurier, A., Kimmel, C.B. & Parthasarathy, R. J. Biophotonics 6, 920–928 (2013).

    CAS  Article  Google Scholar 

  4. Tinevez, J.-Y. et al. Methods Enzymol. 506, 291–309 (2012).

    CAS  Article  Google Scholar 

  5. Waters, J.C. & Wittmann, T. (eds.) Quantitative Imaging in Cell Biology. (Academic Press, 2014).

    Google Scholar 

  6. Goldman, R.D., Swedlow, J.R. & Spector, D.L. (eds.) Live Cell Imaging: A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press, 2009).

    Google Scholar 

  7. Daddysman, M.K., Tycon, M.A. & Fecko, C.J. Methods Mol. Biol. 1148, 1–17 (2014).

    CAS  Article  Google Scholar 

  8. Cole, R. Cell Adh. Migr. 8, 452–459 (2014).

    Article  Google Scholar 

  9. Cranfill, P.J. et al. Nat. Methods 13, 557–562 (2016).

    CAS  Article  Google Scholar 

  10. Donnert, G., Eggeling, C. & Hell, S.W. Nat. Methods 4, 81–86 (2007).

    CAS  Article  Google Scholar 

  11. Murray, J.M., Appleton, P.L., Swedlow, J.R. & Waters, J.C. J. Microsc. 228, 390–405 (2007).

    Article  Google Scholar 

  12. Murray, J.M. An icosahedral virus as a fluorescent calibration standard: methods for counting protein molecules in cells by fluorescence microscopy. Preprint at (2016).

  13. Spielmann, H. et al. Altern. Lab. Anim. 28, 777–814 (2000).

    CAS  PubMed  Google Scholar 

  14. Bianchi, J.I., Stockert, J.C., Buzzi, L.I., Blázquez-Castro, A. & Simonetta, S.H. PLoS One 10, e0128898 (2015).

    Article  Google Scholar 

  15. AlGhamdi, K.M., Kumar, A. & Moussa, N.A. Lasers Med. Sci. 27, 237–249 (2012).

    Article  Google Scholar 

  16. Harnett, M.T., Makara, J.K., Spruston, N., Kath, W.L. & Magee, J.C. Nature 491, 599–602 (2012).

    CAS  Article  Google Scholar 

  17. Galli, R. et al. PLoS One 9, e110295 (2014).

    Article  Google Scholar 

  18. Logg, K., Bodvard, K., Blomberg, A. & Käll, M. FEMS Yeast Res. 9, 875–884 (2009).

    CAS  Article  Google Scholar 

  19. Knoll, S.G., Ahmed, W.W. & Saif, T.A. Sci. Rep. 5, 18513 (2015).

    CAS  Article  Google Scholar 

  20. Roehlecke, C., Schaller, A., Knels, L. & Funk, R.H.W. Mol. Vis. 15, 1929–1938 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. McDonald, A., Harris, J., Macmillan, D., Dempster, J. & McConnell, G. Biomed. Opt. Express 3, 1266–1273 (2012).

    CAS  Article  Google Scholar 

  22. Wäldchen, S., Lehmann, J., Klein, T., van de Linde, S. & Sauer, M. Sci. Rep. 5, 15348 (2015).

    Article  Google Scholar 

  23. Tomer, R., Khairy, K., Amat, F. & Keller, P.J. Nat. Methods 9, 755–763 (2012).

    CAS  Article  Google Scholar 

  24. Wu, Y. et al. Proc. Natl. Acad. Sci. USA 108, 17708–17713 (2011).

    CAS  Article  Google Scholar 

  25. Icha, J., Kunath, C., Rocha-Martins, M. & Norden, C. J. Cell Biol. 215, 259–275 (2016).

    CAS  Article  Google Scholar 

  26. Strnad, P. et al. Nat. Methods 13, 139–142 (2016).

    CAS  Article  Google Scholar 

  27. Strobl, F. & Stelzer, E.H.K. Development 141, 2331–2338 (2014).

    CAS  Article  Google Scholar 

  28. Carlton, P.M. et al. Proc. Natl. Acad. Sci. USA 107, 16016–16022 (2010).

    CAS  Article  Google Scholar 

  29. König, K. in Methods in Cellular Imaging (ed. Periasamy, A.) 236–251 (Oxford University Press, 2001).

    Book  Google Scholar 

  30. Hoebe, R.A. et al. Nat. Biotechnol. 25, 249–253 (2007).

    CAS  Article  Google Scholar 

  31. Kuznetsova, D.S. et al. J. Biophotonics 8, 952–960 (2015).

    CAS  Article  Google Scholar 

  32. Icha, J. et al. J. Vis. Exp. 110, e53966 (2016).

    Google Scholar 

  33. Dobrucki, J.W., Feret, D. & Noatynska, A. Biophys. J. 93, 1778–1786 (2007).

    CAS  Article  Google Scholar 

  34. Wu, Y. et al. Sci. Rep. 5, 14766 (2015).

    CAS  Article  Google Scholar 

  35. Boudreau, C. et al. Sci. Rep. 6, 30892 (2016).

    CAS  Article  Google Scholar 

  36. Chen, B.-C. et al. Science 346, 1257998 (2014).

    Article  Google Scholar 

  37. Ji, N., Magee, J.C. & Betzig, E. Nat. Methods 5, 197–202 (2008).

    CAS  Article  Google Scholar 

  38. US Environmental Protection Agency. Guidelines for Exposure Assessment. Report No. EPA/600/Z-92/001 (US EPA, 1992).

Download references


The authors thank all the delegates of LSFM2016 who attended and contributed to an exciting roundtable discussion. LSFM2016 was supported by the Company of Biologists (EA1170). P.P.L. thanks R. Heintzmann, S. Fraser, J. Huisken, and J.A. Laissue for discussions on phototoxicity. This work was supported by the Royal Society (IF150018 to P.P.L.) and by King Abdulaziz University, Rabigh Campus, Jeddah, Saudi Arabia (scholarship to R.A.A.).

Author information

Authors and Affiliations


Corresponding author

Correspondence to P Philippe Laissue.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Note 1 (PDF 615 kb)

Supplementary Table 1

Full methodology and data for Fig. 1 in main article (XLS 52 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Laissue, P., Alghamdi, R., Tomancak, P. et al. Assessing phototoxicity in live fluorescence imaging. Nat Methods 14, 657–661 (2017).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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