Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy.
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.
Hoebe
R A
RA
Centre for Microscopy Research (CMO), Department of Cell Biology & Histology, Academic Medical Centre, Amsterdam, The Netherlands. r.a.hoebe@amc.uva.nl
Van der Voort
H T M
HT
Stap
J
J
Van Noorden
C J F
CJ
Manders
E M M
EM
D045744:Cell Line, Tumor; D051936:Centromere Protein B / Q000235:genetics / Q000378:metabolism*; D017484:Dermatitis, Phototoxic; D004307:Dose-Response Relationship, Radiation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008027:Light; D008853:Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence; D038761:Photobleaching / Q000528:radiation effects*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D051936:Centromere Protein B; D006657:Histones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins