Multiparamter Fluorescence Detection and Fluorescence Correlation Spectroscopy (PIE-MFD/FCS)
Single-molecule measurements can be performed on freely diffusing species using a confocal microscope. Single-molecule conditions are ensured by using picomolar sample concentrations, so that single molecules diffusing through the confocal volume cause bursts in the detected fluorescence intensity. Our setup combines the techniques of pulsed interleaved excitation (PIE) and multiparameter fluorescence detection (MFD) in order to gain as much information as possible about every single molecule. MFD uses time-correlated single photon counting (TCSPC) and pulsed laser excitation to determine several characteristic parameters, like lifetime, anisotropy or FRET-efficiency, in one measurement. In PIE, several fluorophores are excited alternatingly by different lasers, so all the parameters can be determined for each dye on a molecule and additional characteristics, like the labeling stoichiometry, become available.
Fluorescence correlation spectroscopy (FCS) measurements can also be performed with this setup. Diffusion and kinetics of fluorescently labeled molecules cause fluctuations of the measured fluorescence intensity. Thus, information on diffusion behavior and kinetic rate constants can be gained from auto- or cross-correlation of the signals.
C. Eggeling, S. Berger, L. Brand, J. R. Fries, J. Schaffer, A. Volkmer, and C. A. M. Seidel
Journal of biotechnology (2001) 86, 163-180
J. Widengren, V. Kudryavtsev, M. Antonik, S. Berger, M. Gerken and C. A. M. Seidel
Analytical Chemistry (2006), 78, 2039-2050
B. K. Müller, E. Zaychikov, C. Bräuchle and D. C. Lamb
Biophysical Journal (2005), 89, 3508-3522
Bayesian-Inference-Based Flourescence Correlation Spectroscopy and Single-Moledule Burst Analysis Reveal the Influence of Dye Selection on DNA Hairpin Dynamics
Fluorescence correlation spectroscopy (FCS) is a powerful tool to gain information about dynamics of biomolecules. However, the key problem is to extract the rates hidden in the FCS data by fitting the data to a meaningful model. A number of different fitting approaches have been described in recent years but the extraction of relevant information to date has still been limited by numerous experimental problems and the fact that the set of starting parameter values chosen could often predefine the result. We establish a new way to globally analyze FCS data based on Bayesian inference to overcome these issues. Moreover, the influence of other remaining experimental error sources, for example, photophysics, is excluded by additional means. Using this approach in combination with the results from single-molecule burst analysis, we investigate the kinetics of DNA hairpins labeled with a variety of different fluorescent probes as a function of the salt concentration. We find that the rates of hairpin opening and closing as well as the equilibrium constant of the transition depend on the characteristics of the dye molecules used to label the hairpin. Thus, great caution has to be used when utilizing dye molecules as reporters for the kinetics of dynamic macromolecular structures.