A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes

Abstract

The exquisite selectivity, sensitivity, and spatial resolution obtained with fluorescence spectroscopy and imaging have led to an ever-increasing number of applications. With the development of detectors approaching 100 % quantum efficiencies and sophisticated collection optics, the bottleneck of current fluorescence microscopy is the fluorophores used, which pose severe limitations owing to photobleaching and blinking. Most of the basic dye structures that are currently used in fluorescence microscopy have been known since their use in the development of dye lasers.[1] Increasing demands posed by fluorescence microscopy and single-molecule and high-resolution applications [2,3] have spurred the development of new kinds of emitters such as semiconductor nanocrystals, silver nanoclusters, and new derivatives of fluorescent proteins.[4] In comparison, the advancement of classical organic dyes such as rhodamine or cyanine derivatives has been incremental despite some progress with regard to labeling chemistry, solubility in water, and the availability of bright and photostable near-IR dyes. Approaches for their improvement comprise increasing brightness by multichromophore systems, intramolecular triplet quenching, and decreasing the sensibility for reactions with singlet oxygen. [5] For different reasons, none of these approaches has been implemented with great success in fluorescence microscopy.Here we present a new approach to minimize photobleaching and blinking by recovering reactive intermediates. The method is based on the removal of oxygen and quenching of triplet as well as charge-separated states by electrontransfer reactions. For this reason, a structure that contains reducing as well as oxidizing agents, that is, a reducing and oxidizing system (ROXS) is used. The success of the approach is demonstrated by single-molecule fluorescence spectroscopy of oligonucleotides labeled with different fluorophores, that is, cyanines, (carbo-)rhodamines, and oxazines, in aqueous solvents; individual fluorophores can be observed for minutes under moderate excitation with increased fluorescence brightness. Thermodynamic considerations of the underlying redox reactions support the model, yielding a comprehensive picture of blinking and photobleaching of organic fluorophores.Typically, the photophysics of fluorophores is described by a three-state model including the ground and first excited singlet states, S 0 and S 1 , respectively, and the lowest triplet state T 1 . Owing to its longer lifetime, T 1 is considered to be the photochemically most active state. Quenching of T 1 by molecular oxygen, for example, can generate reactive singlet oxygen, and therefore oxygen is removed in demanding applications, for example, with the aid of an enzymatic oxygen-scavenging system.[6] The disadvantage of oxygen removal, however, is the increase of the triplet state lifetime with negative effects for the brightness of the fluorophore and increased probability for other follow-up reactions from the triplet state. Alternatively, redu...

Accelerated single photon emission from dye molecule-driven nanoantennas assembled on DNA

“…Biotin-functionalized single particles and dimers are grafted on the surface of home-made Neutravidin-coated microfluidic chambers using methods described elsewhere 14 . An oxygen scavenger (10 mM protocatechuic acid and 200 nM protocatechuate-3,4,dioxygenase) 20 and reducing and oxidizing agents (1 mM ascorbic acid and methyl viologen) 21,22 are added in the final 50 mM NaCl/10 mM Tris buffer (pH = 8) before sealing the microchamber with hot wax. The sample is illuminated in an inverted microscope (Olympus IX71) using a 1.3 NA objective with an unpolarized supercontinuum laser (SC450-pp, Fianium), spectrally filtered around 635 nm, which delivers 50-ps pulses at a 10-MHz repetition rate.…”

“…To minimize photobleaching and blinking of SMs, we add an oxygen scavenger 20 and reducing and oxidizing agents 21,22 in the microchamber. The sample is illuminated with a pulsed supercontinuum source spectrally filtered at 635 nm.…”

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

“…This suggests a combination of additives to be used. 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 .…”

Watching two conjugated polymer chains breaking each other when colliding in solution

“…Fluorescence blinking and bleaching of an individual chromophore are examples of photo-induced electron-transfer reactions. [18][19][20][21] To observe the dynamics of the same single molecule on the scale of seconds, the molecule must be immobilized on a surface or embedded into a transparent polymer matrix. The chemical reactions of single molecules have also been directly observed using scanning probe microscopy, 22 a technique that is limited to the investigation of surfaces and molecules attached to surfaces.…”