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.

journal_name

Nat Biotechnol

journal_title

Nature biotechnology

authors

Hoebe RA,Van Oven CH,Gadella TW Jr,Dhonukshe PB,Van Noorden CJ,Manders EM

doi

10.1038/nbt1278

subject

Has Abstract

pub_date

2007-02-01 00:00:00

pages

249-53

issue

2

eissn

1087-0156

issn

1546-1696

pii

nbt1278

journal_volume

25

pub_type

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