Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the Ensemble and Single-Molecule Levels

Abstract

Photobleaching is a severely limiting factor in the optical study of single biomolecules. We investigate the photobleaching of rhodamine 6G (R6G) ensembles in poly(vinyl alcohol) (PVA) as a function of illumination time, excitation intensity, the presence of oxygen, and temperature. We observe nonexponential kinetics related to primary photobleaching through two dark statesthe triplet state and a radical anionand to secondary photobleaching after the optical excitation of those dark states. Reactions of the metastable states with oxygen can lead either to photoproducts or to a recovery of the ground state. Oxygen can therefore enhance or reduce photobleaching, depending on the experimental conditions. At low temperature, photobleaching is reduced although not completely suppressed. Despite the presence of the long-lived radical anion, we are able to observe single R6G molecules in PVA. At room temperature, only relatively bleaching-resistant molecules are resolved as individuals. At low temperature, the observation times become considerably longer. Our study shows that metastable states other than the triplet drastically affect photobleaching.

Power-Law-Distributed Dark States are the Main Pathway for Photobleaching of Single Organic Molecules

“…It has been noted by Zondervan et al that dark states other than the triplet, like the radical state, can drastically affect photobleaching [16], as now strongly and directly evidenced by the results above. Using single-molecule fluorescence measurements, we cannot distinguish whether electron tunneling takes place from the singlet excited state or from the triplet state [14].…”

“…In fact, Zondervan et al have recently found that the presence of O 2 not only enhances photo- bleaching but also assists in repopulation of the ground state from the dark state [16]. Interestingly, Ha and Xu reported the existence of multiple photobleaching intermediates, each with single-exponential kinetics, in a liquid environment, based on the observation of a complex O 2 -dependent behavior of the distribution of dark states [17].…”

“…A complex pathway involving multiple intermediate states was suggested by Ha et al [17]. Recently, it was also shown that radical dark states might be involved in the photobleaching pathway [16]. Singlemolecule fluorescence studies, however, cannot unambiguously distinguish between a (temporary) excursion to a LD state longer than the observation time and a (permanent) photobleaching event.…”

“…In particular the triplet and higher excited states have been considered as important intermediate states in photobleaching [16]. Although little is known about the nature of this reaction and the pathway involved, it appears that oxygen plays an important role [15,17], either being the primary oxidation agent [7,16], or assisting in the population and depopulation of other excited states than the singlet [16].…”

Iodide as a Fluorescence Quencher and Promoter—Mechanisms and Possible Implications

“…Also the excitation conditions can have a substantial influence [11][12][13][14] . High excitation rates, as often used in SMD or FCS experiments, or in other applications where high read-out rates or high sensitivities are required, can strongly increase the generation of several long-lived, photo-induced states of the dyes, such as triplet states, photo-isomerised states, photo-oxidized states or other states generated by photo-induced charge transfer 8,[12][13][14][15] . The multitude of states generated, and that the effect of the added photo-stabilizers and quenchers may lead to the formation of additional intermediate states increases the complexity.…”