Laser photolysis of laser dyes and the influence of triplet quenchers

Asimov, M. M.; Gavrilenko, V. N.; Рубинов, А. Н.

doi:10.1016/0022-2313(90)90020-c

Cited by 19 publications

(6 citation statements)

References 18 publications

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“…Similarly, the intersystem crossing rate, k ISC , remained the same at different COT concentrations, while the triplet decay rate increased linearly with the COT concentration (see inset Figure c). At concentrations above approximately 3 mM, the triplet quenching effect of COT was diminished probably because of micelle formation of the COT molecules . Below this limit, the addition of COT enhanced the fluorescence output from the Rh6G molecules.…”

Section: Resultsmentioning

confidence: 96%

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Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

et al. 2006

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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.

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Section: Resultsmentioning

confidence: 96%

“…Here, we study the effects of mercaptoethylamine (MEA) in water and cyclo-octatetraene (COT) in ethanol. In the past, the addition of triplet state quenchers has proven to be a useful strategy to improve both outcome of fluorescence spectroscopic studies 5,36,37 as well as performance of dye lasers. − …”

Section: Introductionmentioning

confidence: 99%

Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

et al. 2006

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“…Unfortunately, the triplet state lifetimes of standard fluorophores such as rhodamine, oxazine, and carbocyanine dyes are short and vary between less than 1 µs up to a few hundred µs in air-saturated solution, with intersystem crossing rates k isc from 4.2 × 10 5 to 2.8 × 10 7 s −1 [26][27][28][29][30][31] corresponding to relatively low quantum yields, Φ isc < 0.1%. In order to use the triplet state for photoswitching microscopy, relatively high excitation intensities are required to efficiently populate the triplet state.…”

Section: Resultsmentioning

confidence: 99%

Photoswitching microscopy with standard fluorophores

et al. 2008

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We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as ß-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of ∼20 nm. The method requires widefield illumination with only a single laser beam for readout and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.

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“…In addition, it should be mentioned that our measurements have been performed under ambient conditions (room temperature, air) where direct observation of intersystem crossing is extremely difficult due to short triplet lifetimes (collisional quenching by oxygen) as well as rapid photobleaching. Therefore most single-molecule triplet studies have been performed at cryogenic temperatures ,, or under vacuum conditions. , In air-saturated ensemble solutions, the triplet state lifetimes τ T of rhodamine, oxazine, and carbocyanine dyes vary between less than 1 μs up to several μs with intersystem crossing rates k ISC from 4.2 × 10 5 to 2.8 × 10 7 s -1 . ,− In vacuum, Weston et al found triplet lifetimes of 4−100 ms for single DiIC 12 molecules (a carbocyanine derivative) with a triplet yield of 4 × 10 -4 . In accordance with these results, English et al .…”

Section: Resultsmentioning

confidence: 99%

Photophysical Dynamics of Single Molecules Studied by Spectrally-Resolved Fluorescence Lifetime Imaging Microscopy (SFLIM)

2001

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A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds to milliseconds time resolution. Discrete jumps in fluorescence intensity from single molecules which lacked spectral diffusion and changes in radiative lifetime have been observed with correlation times (triplet lifetimes) spanning several orders of magnitude (from 2 µs for the rhodamine derivative up to several seconds for the oxazine dye) and amplitude. For the carbocyanine derivative Cy5, fast spectral fluctuations to red-shifted dim-states which appear partly as offstates with a lifetime in the millisecond range were determined. In addition, these dim-states exhibit the same radiative decay rate of ∼2 ns as the normal on-state. Our results imply that a direct correlation between the radiative decay time and spectral fluctuations is not necessarily given in each of the three chromophores. Both parameters seem to be independent characteristic of each individual molecule. About 5-15% of all molecules independent of the dye structure, respectively, exhibited a constant emission spectrum but strong fluctuations in fluorescence lifetime directly correlated to the intensity. The results indicate that a combined analysis of emission spectrum, intensity and radiative decay rate is a valuable approach for classification and quantification of the underlying photophysical dynamics.

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Laser photolysis of laser dyes and the influence of triplet quenchers

Cited by 19 publications

References 18 publications

Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

Photoswitching microscopy with standard fluorophores

Photophysical Dynamics of Single Molecules Studied by Spectrally-Resolved Fluorescence Lifetime Imaging Microscopy (SFLIM)

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