Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

Widengren, Jerker; Chmyrov, Andriy; Eggeling, Christian; Löfdahl, Per‐Åke; Seidel, Claus A. M.

doi:10.1021/jp0646325

Cited by 217 publications

(307 citation statements)

References 58 publications

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“…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.…”

Section: Selective Quenching Of the Fluorophore Triplet States By Kisupporting

confidence: 89%

“…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.…”

Section: Selective Quenching Of the Fluorophore Triplet States By Kisupporting

confidence: 88%

“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

2010

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In this work, Fluorescence Correlation Spectroscopy (FCS) was used to investigate the effects of potassium iodide (KI) on the electronic state population kinetics of a range of organic dyes in the visible wavelength range. Apart from a heavy atom effect promoting intersystem crossing to the triplet states in all dyes, KI was also found to enhance the triplet state decay rate by a chargecoupled deactivation. This deactivation was only found for dyes with excitation maximum in the blue range, not for those with excitation maxima at wavelengths in the green range or longer.Consequently, under excitation conditions sufficient for triplet state formation, KI can promote the triplet state build-up of one dye and reduce it for another, red-shifted dye. This anti-correlated, spectrally separable response of two different dyes to the presence of one and the same agent may provide a useful readout for biomolecular interaction and micro-environmental monitoring studies. In contrast to the typical notion of KI as a fluorescence quencher, the FCS measurements also revealed that when added in micromolar concentrations KI can act as an anti-oxidant, promoting the recovery of photo-oxidized fluorophores. However, in millimolar concentrations 2 KI also reduces intact, fluorescently viable fluorophores to a considerable extent. In aqueous solutions, an optimal concentration of KI of approximately 5 mM can be defined at which the fluorescence signal is maximized. This concentration is not high enough to allow full triplet state quenching. Therefore, as a fluorescence enhancement agent, it is primarily the anti-oxidative properties of KI that play a role.

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Section: Selective Quenching Of the Fluorophore Triplet States By Kisupporting

confidence: 89%

Section: Selective Quenching Of the Fluorophore Triplet States By Kisupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

2010

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Single‐Molecule Studies of Biomolecules

Blanchard

Altman

Geggier

et al. 2008

Wiley Encyclopedia of Chemical Biology

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Imaging biological processes at the single‐molecule scale enables the direct observation of the temporal and spatial dynamics of cellular processes. This relatively new branch of biophysical investigation, which was founded on a platform of physics and chemistry research, is pushing forward the frontiers of medical science by providing unprecedented new illuminations into the molecular basis of life. Although it is far from comprehensive, this article provides the advanced reader with a review of single‐molecule methods, as well as a discussion of the aims, considerations, achievements, and future directions of this field in the context of ongoing investigations of structure–function relationships inherent to biomolecules.

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Fluorophore Selection for Single‐Molecule Fluorescence Spectroscopy (SMFS) and Photobleaching Pathways

Sauer

Hofkens

Enderlein

2011

Handbook of Fluorescence Spectroscopy and Imaging

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To emphasize the advantage of single-molecule over ensemble experiments, imagine that you are at a large station observing hundreds of passengers arriving on an unknown number of trains. From such an observation, you cannot answer questions about the individual routes that the trains took, how many passengers boarded the train at which station, and at what times, or how many stops, each train made. You observe simply an average and can only conclude that trains typically transport hundreds of passengers at a time. If single-molecule spectroscopy is used to monitor reactions, individual properties can be measured, whereas in standard experiments, only the overall average response is observed. The technique provides the basis for a direct comparison of models, which are usually derived by envisaging individual molecules, with solid experimental results. Furthermore, we can determine whether each molecule exhibits a different but temporally constant reaction rate (static inhomogeneity) or changes its rate with time (dynamic inhomogeneity, which can be caused by perturbations that are analogous to elevations on a trains route). Thus, single-molecule fluorescence spectroscopy (SMFS) is used in a range of scientific areas in physics, chemistry, and biology, and has been explained in detail in various excellent reviews [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16].The ongoing success of SMFS has been facilitated not only through the development of optical single-molecule techniques, but in addition through new refined organic synthesis methods and the large repertoire of molecular biology techniques. The ability to specifically label many different sites on macromolecules, for example, provides a great toolbox for the application of several spectroscopic techniques, such as fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET). The dream of being able to detect individual molecules optically arose in conjunction with the confirmation of their existence. Nevertheless, it took more than half a century before the group working with Hirschfeld made the first successful attempts at detecting the fluorescence signal of single antibody molecules statistically labeled with 80-100 fluorophores [17]. Subsequently, at the beginning of the 1990s, Kellers group was finally able to detect a single fluorophore in a biologically relevant environment, that is, in an aqueous solvent [18]. Simultaneously, but independently, Moerner, Orrit and coworkers successfully detected single molecules at cryogenic temperatures using optical means [19,20].

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Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

Cited by 217 publications

References 58 publications

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

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

Single‐Molecule Studies of Biomolecules

Fluorophore Selection for Single‐Molecule Fluorescence Spectroscopy (SMFS) and Photobleaching Pathways

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