Letter | Published:

Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging

Nature Biotechnology volume 25, pages 249253 (2007) | Download Citation

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Abstract

Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy (CLEM), a simple imaging approach that reduces photobleaching and phototoxicity two- to tenfold, depending on the fluorophore distribution in the object. By spatially controlling the light-exposure time, CLEM reduces the excitation-light dose without compromising image quality. We show that CLEM reduces photobleaching sevenfold in tobacco plant cells expressing microtubule-associated GFP-MAP4 and reduces production of reactive oxygen species eightfold and prolongs cell survival sixfold in HeLa cells expressing chromatin-associated H2B-GFP. In addition, CLEM increases the dynamic range of the fluorescence intensity at least twofold.

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Acknowledgements

E.M.M.M., R.A.H., C.H.V.O., C.J.F.V.N. and T.W.J.G. were supported by the Amsterdam Genomics Centre (AmGC) of the University of Amsterdam, E.M.M.M. by the Dutch Technology Foundation (STW, ATF-7394); E.M.M.M., R.A.H. and T.W.J.G., by the EU integrated project “Molecular Imaging” (LSHG-CT-2003-503259); and P.B.D. and T.W.J.G., by the Netherlands Organization for Scientific Research (NWO, grant 805-47.012). We thank Merel Adjobo-Hermans, Joachim Goedhart and Bernd Rieger for technical assistance, G.J. Brakenhoff for scientific advice and Nikon Europe for providing microscope equipment.

Author information

Author notes

    • P B Dhonukshe

    Present address: Developmental Genetics, ZMBP, University of Tübingen, Auf der Morgenstelle 3, 72076 Tübingen, Germany.

Affiliations

  1. Center for Advanced Microscopy, Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.

    • R A Hoebe
    • , T W J Gadella Jr
    • , P B Dhonukshe
    •  & E M M Manders
  2. Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

    • R A Hoebe
    • , C H Van Oven
    •  & C J F Van Noorden

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Contributions

R.A.H., experimental data, engineering, computer simulations; C.H.V.O., electronic design and development; T.W.J.G., biofluorescence expertise; P.B.D., plant cell experiments; C.J.F.V.N., scientific and editorial management; E.M.M.M., inventor of CLEM and general team leader.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to E M M Manders.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Reduction of photobleaching and phototoxicity by CLEM depends of fluorophore distribution.

Videos

  1. 1.

    Supplementary Video 1

    CLEM strongly reduces photobleaching. Left: non-CLEM; Right: CLEM

  2. 2.

    Supplementary Video 2

    CLEM strongly reduces phototoxicity. Left: non-CLEM; Right: CLEM

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DOI

https://doi.org/10.1038/nbt1278

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