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Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy
Author(s) -
HOEBE R.A.,
VAN DER VOORT H.T.M.,
STAP J.,
VAN NOORDEN C.J.F.,
MANDERS E.M.M.
Publication year - 2008
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2008.02009.x
Subject(s) - photobleaching , phototoxicity , fluorophore , microscopy , fluorescence microscope , fluorescence , singlet oxygen , microscope , confocal microscopy , chemistry , materials science , photochemistry , biophysics , optics , physics , oxygen , biology , biochemistry , in vitro , organic chemistry
Summary Phototoxicity and photobleaching are major limitations in live‐cell fluorescence microscopy. They are caused by fluorophores in an excited singlet or triplet state that generate singlet oxygen and other reactive oxygen species. The principle of controlled light exposure microscopy (CLEM) is based on non‐uniform illumination of the field of view to reduce the number of excited fluorophore molecules. This approach reduces phototoxicity and photobleaching 2‐ to 10‐fold without deteriorating image quality. Reduction of phototoxicity and photobleaching depends on the fluorophore distribution in the studied object, the optical properties of the microscope and settings of CLEM electronics. Here, we introduce the CLEM factor as a quantitative measure of reduction in phototoxicity and photobleaching. Finally, we give a guideline to optimize the effect of CLEM without compromising image quality.