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

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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Figure 1: The concept and implementation of CLEM.
Figure 2: The quality of CLEM images.
Figure 3: CLEM reduces photobleaching, formation of reactive oxygen species (ROS) and phototoxicity.

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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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to E M M Manders.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Reduction of photobleaching and phototoxicity by CLEM depends of fluorophore distribution. (PDF 6740 kb)

Supplementary Video 1

CLEM strongly reduces photobleaching. Left: non-CLEM; Right: CLEM (MOV 1200 kb)

Supplementary Video 2

CLEM strongly reduces phototoxicity. Left: non-CLEM; Right: CLEM (MOV 9713 kb)

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Hoebe, R., Van Oven, C., Gadella, T. et al. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging. Nat Biotechnol 25, 249–253 (2007). https://doi.org/10.1038/nbt1278

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