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


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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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




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.

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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).

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