UV-Fluorescence Correlation Spectroscopy of 2-Aminopurine

Wennmalm, Stefan; Blom, Hans; Wallerman, Lennart; Rigler, Rudolf

doi:10.1515/bc.2001.048

Cited by 21 publications

(26 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Single-molecule and FCS experiments should therefore circumvent the use of femtosecond pulsed-excitation in the UV region, which has already been stated previously. [63,75,98] …”

Section: One-photon Femtosecond-pulsed Excitation At 350 Nm Into Highmentioning

confidence: 99%

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

2005

View full text Add to dashboard Cite

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.

show abstract

“…Single-molecule and FCS experiments should therefore circumvent the use of femtosecond pulsed-excitation in the UV region, which has already been stated previously. [63,75,98] …”

Section: One-photon Femtosecond-pulsed Excitation At 350 Nm Into Highmentioning

confidence: 99%

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

2005

View full text Add to dashboard Cite

show abstract

“…15). Although the most general single molecule application relies on the use of fluorescent dyes, which are attached to the biomolecule of interest, the intrinsic fluorescence emission of biomolecules has also been utilized in some cases (16)(17)(18).…”

mentioning

confidence: 99%

Two-photon excitation microscopy of tryptophan-containing proteins

Lippitz

Erker²,

Decker³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.

show abstract

“…, N̄ . To correct for bias in the amplitude, the “true” number of molecules, N 0 , may be extracted using the following expression: N̄ 0 = N̄ (1 + B / F ) −2 , where B is the background signal that can be measured for a sample of pure water, and F is the detected fluorescence signal [24,40]. Incorporating this correction, with a B / F ratio that varied from 0.07 to 0.02 as the ionic strength increased from 0.1 mM to 150 mM, leads to a modest change of the surface potential from −29.7 mV to −32.4 mV at 0.1 mM (cf.…”

Section: Resultsmentioning

confidence: 99%

Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy

Blom

Hassler

Chmyrov

et al. 2010

IJMS

Self Cite

View full text Add to dashboard Cite

Electrostatic interactions between dielectric surfaces and different fluorophores used in ultrasensitive fluorescence microscopy are investigated using objective-based Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacial dynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein, zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengths at physiological pH. As analyzed by means of the amplitude and time-evolution of the autocorrelation function, the fluorescent molecules experience electrostatic attraction or repulsion at the glass surface depending on their charges. Influences of the electrostatic interactions are also monitored through the triplet-state population and triplet relaxation time, including the amount of detected fluorescence or the count-rate-per-molecule parameter. These TIR-FCS results provide an increased understanding of how fluorophores are influenced by the microenvironment of a glass surface, and show a promising approach for characterizing electrostatic interactions at interfaces.

show abstract

UV-Fluorescence Correlation Spectroscopy of 2-Aminopurine

Cited by 21 publications

References 22 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

Two-photon excitation microscopy of tryptophan-containing proteins

Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy

Contact Info

Product

Resources

About