Ülo Mets

Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed.

Triplet-state monitoring by fluorescence correlation spectroscopy

The effects of high excitation intensities in fluorescence correlation spectroscopy (FCS) in terms of saturation and triplet-state build-up have been studied for the case of Rh6G in aqueous solution. It was found that FCS provides a powerful means for the determination of intersystem crossing and triplet-state depopulation rates of fluorophores in solution.

Submillisecond detection of single rhodamine molecules in water

Using a modified confocal fluorescence microscope and a CW argon laser, we have measured fluorescence bursts from diffusing single Rh6G molecules that clearly exceed the background intensity. The exact average number of molecules in the observable volume elements was measured directly via the fluorescence intensity autocorrelation function. This allowed us to estimate the probability of finding several molecules simultaneously in the volume element. A tradeoff between the number of detected fluorescence photons and the signal-to-background ratio was observed. In a volume element of 0.24 fl, 4 photoelectrons on average were detected from a molecule of Rh6G with a fluorescence-to-background ratio of 1000, while the volume element of 60 fl yielded on average 100 photoelectrons with a background of 25 counts. In fast single-molecule detection the intersystem crossing into the triplet state plays an important role, affecting the maximum emission rate from the molecule.

Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy

Abstract GFPs are upon excitation influenced by many different photophysical and photochemical processes effective over a very broad time scale. Much effort has been spent to investigate these processes. However, in the microsecond to millisecond time-range many processes still remain to be further characterized. This time-range can be conveniently covered by FCS, and is used here to study the photodynamical behaviour of wild-type (WT) and a F64L S65T mutant (BioST) of GFP. In addition to intersystem crossing to the triplet state, additional photophysical processes are seen, showing identical fluctuations in fluorescence to those found for a reversible photo-induced isomerization process, as well as fluctuations, not influenced by the electronic state of the chromophore unit. In the nanosecond time-range a contribution to the fluorescence correlation function is observed which can be attributed to rotational diffusion, suggesting a convenient way to measure rotational diffusion of proteins expressed with GFP on a microscopic scale.

Interactions and Kinetics of Single Molecules as Observed by Fluorescence Correlation Spectroscopy

Random fluctuations of the intensity of individual molecules excited to fluorescence by a stationary light source provide information on important molecular properties such as rotational motion [1–5], translational diffusion [6, 7], chemical kinetics [7–9] as well as the lifetime of the excited state [1, 2, 10].

Application of the antibunching in dye fluorescence: measuringthe excitation rates in solution

Abstract Experiments on antibunching in fluorescence of Rhodamine 6G in aqueous solution have been carried out under optimized conditions. The dependence of the antibunching correlation times on the excitation intensity has been studied, taking into account the saturation effects in the Gaussian intensity profile the exciting laser beam. Information on the decay and excitation rates can be obtained and, with the knowledge of the excitation intensity, the absorption cross-section can be estimated.

13C-1H NMR Relaxation and Fluorescence Anisotropy Decay Study of Tyrosine Dynamics in Motilin

Tyrosine ring dynamics of the gastrointestinal hormone motilin was studied using two independent physical methods: fluorescence polarization anisotropy decay and NMR relaxation. Motilin, a 22-residue peptide, was selectively (13)C labeled in the ring epsilon-carbons of the single tyrosine residue. To eliminate effects of differences in peptide concentration, the same motilin sample was used in both experiments. NMR relaxation rates of the tyrosine ring C(epsilon)-H(epsilon) vectors, measured at four magnetic field strengths (9.4, 11.7, 14.1, and 18.8 Tesla) were used to map the spectral density function. When the data were analyzed using dynamic models with the same number of components, the dynamic parameters from NMR and fluorescence are in excellent agreement. However, the estimated rotational correlation times depend on the choice of dynamic model. The correlation times estimated from the two-component model-free approach and the three-component models were significantly different (1.7 ns and 2.2 ns, respectively). Various earlier studies of protein dynamics by NMR and fluorescence were compared. The rotational correlation times estimated by NMR for samples with high protein concentration were on average 18% longer for folded monomeric proteins than the corresponding times estimated by fluorescence polarization anisotropy decay, after correction for differences in viscosity due to temperature and D(2)O/H(2)O ratio.