Photophysical Aspects of Single‐Molecule Detection by Two‐Photon Excitation with Consideration of Sequential Pulsed Illumination

Niesner, Raluca; Roth, Wolfgang R.; Gericke, Karl‐Heinz

doi:10.1002/cphc.200300881

Cited by 15 publications

(30 citation statements)

References 29 publications

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“…Because higher electronic-excited states couple quite efficiently with ionic states in polar solvents such as water, two-and multistep absorption processes open additional channels for photobleaching. [58][59][60][61][62] This two-step photolysis is readily obtained with cw or pulsed lasers, as shown previously in the case of coumarins, [63][64][65] rhodamines, [43,51,54,66,67] fluorescein, [51] fluorescent proteins, [67,68] and other fluorophores. [64] The photobleaching following pulsed excitation depends on the peak irradiance as well as on the repetition rate.…”

Section: Introductionmentioning

confidence: 91%

“…[64] The photobleaching following pulsed excitation depends on the peak irradiance as well as on the repetition rate. [54,63,65] 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.…”

Section: Introductionmentioning

confidence: 99%

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Molecular Photobleaching Kinetics of Rhodamine 6G by One‐ and Two‐Photon Induced Confocal Fluorescence Microscopy

2005

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Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.

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Section: Introductionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Molecular Photobleaching Kinetics of Rhodamine 6G by One‐ and Two‐Photon Induced Confocal Fluorescence Microscopy

2005

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“…There is an extensive body of publications that deal with the impact of high-intensity excitation on confocal fluorescence microscopy, both in one-and multiphoton excitation regimes (see e.g., refs. [16][17][18]). The topic usually discussed in relation to high excitation intensities is photobleaching, which shifts the autocorrelation to shorter times.…”

Section: Introductionmentioning

confidence: 99%

Optical Saturation in Fluorescence Correlation Spectroscopy under Continuous‐Wave and Pulsed Excitation

Gregor¹,

Patra²,

Enderlein³

2005

ChemPhysChem

110

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A detailed theoretical and experimental study of the dependence of fluorescence correlation measurements on optical excitation power due to optical saturation effects is presented. It is shown that the sensitivity of a fluorescence correlation measurement on excitation power becomes increasingly stronger for decreasing excitation power. This makes exact measurements or diffusion coefficients with fluorescence correlation spectroscopy rather difficult. A strong difference of this behavior for continuous-wave and pulsed excitation is found.

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“…(2). Triplet and saturation effects have been studied by several groups [3,33,46,47,48,49,50,51,52,53,54,55,56,62,63] and several methods for accounting for saturation in the ACF fitting function have been proposed in the literature [48,52,64,65].…”

Section: Effects Of Triplet Photophysics and Saturationmentioning

confidence: 99%

“…Monte Carlo simulations [3,34,39,44,45,46,47,48,i] and numerical calculations [49,50,51,52,53,54,55,56] (which are also referred to as 'simulations' by some authors) have provided invaluable tools for quantitatively studying distortion and errors in the experimental ACF. Ref.…”

Section: Introductionmentioning

confidence: 99%

Accounting for Triplet and Saturation Effects in FCS Measurements

Davis¹,

Shen²

2006

CPB

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Abstract:Fluorescence correlation spectroscopy (FCS) is an increasingly important tool for determining low concentrations and dynamics of molecules in solution. Oftentimes triplet transitions give rise to fast blinking effects, which are accounted for by including an exponential term in the fitting of the autocorrelation function (ACF). In such cases, concomitant saturation effects also modify the amplitude and shape of the remaining parts of the ACF. We review studies of triplet and saturation effects in FCS and present a simple procedure to obtain more accurate results of particle concentrations and diffusional dynamics in experiments where triplet kinetics are evident, or where moderate laser powers approaching saturation levels are used, for example, to acquire sufficient photon numbers when observation times are limited. The procedure involves use of a modified function for curve-fitting the ACF, but there are no additional fitting parameters. Instead, a simple calibration of the total fluorescence count rate as a function of relative laser power is fit to a polynomial, and the non-linear components of this fit, together with the relative laser power used for the FCS measurement, are used to specify the magnitude of additional terms in the fitting function. Monte Carlo simulations and experiments using Alexa dyes and quantum dots, with continuous and pulsed laser excitation, demonstrate the application of the modified fitting procedure with first order correction terms, in the regime where distortions in the ACF due to photobleaching and detector dead time are small compared to those of fluorescence saturation and triplet photophysics.

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Photophysical Aspects of Single‐Molecule Detection by Two‐Photon Excitation with Consideration of Sequential Pulsed Illumination

Cited by 15 publications

References 29 publications

Molecular Photobleaching Kinetics of Rhodamine 6G by One‐ and Two‐Photon Induced Confocal Fluorescence Microscopy

Molecular Photobleaching Kinetics of Rhodamine 6G by One‐ and Two‐Photon Induced Confocal Fluorescence Microscopy

Optical Saturation in Fluorescence Correlation Spectroscopy under Continuous‐Wave and Pulsed Excitation

Accounting for Triplet and Saturation Effects in FCS Measurements

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