title: 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.
authors.affiliation: Center for Advanced Microscopy, Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
authors.forename: R A
authors.initials: RA
authors.lastname: Hoebe
authors.forename: C H
authors.initials: CH
authors.lastname: Van Oven
authors.forename: T W J
authors.initials: TW
authors.lastname: Gadella
authors.forename: P B
authors.initials: PB
authors.lastname: Dhonukshe
authors.forename: C J F
authors.initials: CJ
authors.lastname: Van Noorden
authors.forename: E M M
authors.initials: EM
authors.lastname: Manders
mesh_terms: D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D004307:Dose-Response Relationship, Radiation; D006367:HeLa Cells; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching / Q000528:radiation effects; D011829:Radiation Dosage; D014026:Nicotiana / Q000166:cytology* / Q000502:physiology / Q000528:radiation effects*