Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale

Abstract

Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions, e.g. in a solvent of high viscosity, a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion, fluorescence quenching interactions, and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.

Multi-Fractional Analysis of Molecular Diffusion in Polymer Multilayers by FRAP: A New Simulation-Based Approach

“…Therefore, the evaluation procedures require adaptation to the specifics of the experiment, but they typically employ some general concept of FRAP data evaluation in the core. Evaluation of FCS data by the autocorrelation function may seem more straightforward, but its results are dependent on knowledge about involved phenomena and its precise parameters (e.g., a geometry of the detection volume, bleaching and photophysical dark states of a fluorophore, viscosity of local environment). , …”

“…Evaluation of FCS data by the autocorrelation function may seem more straightforward, but its results are dependent on knowledge about involved phenomena and its precise parameters (e.g., a geometry of the detection volume, bleaching and photophysical dark states of a fluorophore, viscosity of local environment). 5,6 In this work, we used the FRAP technique because it leads to a rather intuitive data evaluation and because a standard confocal microscope is sufficient for this type of experiment. The most original approach to the analysis of FRAP data was described by Axelrod et al 1 In this approach, a circular area within a sample is bleached and the recovery of fluorescence is evaluated from the temporal development of the average fluorescence intensity within the bleached area.…”

On the Possibility of Exploring Tip-Molecule Interactions with STM Experiments

“…RTD (11), because the respective a n in Eq. ( 8) are zero for these odd solutions due to the symmetry of the potential and of the initial condition (2).…”

“…Thus, this method is particularly well suited for molecules with high mobility since the tunneling current can be measured at a high time resolution. It can also be applied in other fields as, for example, fluorescence correlation spectroscopy [7][8][9][10][11].…”

Diffusion Coefficients from Signal Fluctuations: Influence of Molecular Shape and Rotational Diffusion

“…Both of them are suitable to determine absolute values of D . Though the methods have been developed for surface diffusion, the concepts can also be useful in other fields such as, e.g., single-molecule fluorescence correlation spectroscopy. − …”