Photobleaching of asymmetric cyanines used for fluorescence imaging of single DNA molecules

The photobleaching of the cyanine dyes YO and YOYO has been investigated fo r both free and DNA-bound dyes, using absorption and fluorescence spectrosc opy coupled with fluorescence microscopy. For the free dyes, the nature of the reactive species involved in the photodegradation process is different for the monomer and the dimer, as shown by scavenger studies. For DNA-bound dyes, photoinduced fading of the visible absorption band occurs by differe nt pathways depending on the drug binding mode and can be attenuated by app ropriate scavengers. However, none of these scavengers were found to have a ny significant effect on the photobleaching of dye fluorescence. It appears that the reduction of fluorescence intensity comes from a quenching of the dye fluorescence by modified DNA bases, possibly 8-oxo-7,8-dihydro-2 ' -de oxyguanosine.