Geminate recombination as a photoprotection mechanism for fluorescent dyes.
Despite common presumption due to fast photodestruction pathways through higher excited states, we show that further improvement of photostability is still achievable with diffusion-limited photoprotection formulas. Single-molecule fluorescence spectroscopy reveals that thiolate ions effectively quench triplet states of dyes by photoinduced electron transfer. Interestingly, this reaction rarely yields a radical anion of the dye, but direct return to the ground state is promoted by an almost instantaneous back electron transfer (geminate recombination). This type of mechanism is not detected for commonly used reductants such as ascorbic acid and trolox. The mechanism avoids the formation of radical cations and improves the photostability of single fluorophores. We find that a combination of β-mercaptoethanol and classical reducing and oxidizing systems yields the best results for several dyes including Atto532 and Alexa568.
Technische Universität Braunschweig, Institut für physikalische und theoretische Chemie, Hans-Sommer-Strasse 10, 38106 Braunschweig (Germany).
Phil
P
Holzmeister
Andreas
A
Gietl
Philip
P
Tinnefeld
D001205:Ascorbic Acid / Q000737:chemistry; D002839:Chromans / Q000737:chemistry; D004579:Electron Transport; D005456:Fluorescent Dyes / Q000737:chemistry*; D008623:Mercaptoethanol / Q000737:chemistry; D010084:Oxidation-Reduction; D038761:Photobleaching; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014466:Ultraviolet Rays
D002839:Chromans; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; D001205:Ascorbic Acid; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
fluorescence; geminate recombination; photophysics; photoprotection; single-molecule studies