Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

Abstract

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.

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

“…However, when dissociated in aqueous solution the iodide ions are known to act as mild reducing agents 10 respectively. The determined k ox1 and k oxn rates for RhGr are well in agreement with the corresponding rates previously determined for Rh6G 8 . With regard to the uncertainty of some of the parameters used for the determination of these rates, in particular the excitation cross-sections of the higher excited singlet and triplet states as well as the lifetimes of these states, the k ox1 and k oxn rates can only be determined within a factor of 2 to 3.…”

“…With increasing KI concentrations, the additional relaxation term in the FCS curves due to photooxidation was gradually diminished, and totally vanished at KI concentrations in the range of 20 -30 µM. This behavior is similar to that found for the well known antioxidants n-propyl gallate (nPG) and ascorbic acid (AA) in a previous study 8 . Interestingly, KI is not primarily known as an antioxidant and antifading compound, as it appears to be for the dyes of the second group, but rather the opposite -as a fluorescence quencher.…”

“…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.…”

“…Along this strategy, a combination of oxygen removal and addition of both a reducing and an oxidizing agent recently proved to be useful to minimize photobleaching and blinking for a range of organic dyes 16 . In addition, for several additives there is a balance between their deactivation of triplet states and photo-induced radical 4 states, and their quenching of fluorophores in their fluorescently viable states 8 . The optimal concentration of an effective additive can vary considerably depending on the combination of fluorophore/additive and the excitation conditions.…”

“…The optimal concentration of an effective additive can vary considerably depending on the combination of fluorophore/additive and the excitation conditions. Under conditions relevant for biomolecular studies by SMD or FCS, the FCS technique itself has proven useful for characterizing population dynamics of a range of photo-induced states of dyes, including triplet states 17 , photo-isomerized states 18 , and states generated by photo-induced charge transfer 19 , including photo-oxidation 8 . In this work, FCS was used to investigate the effect of potassium iodide (KI) on a set of organic dyes.…”