Question

1) Describe the basic principles of Fluorescence Correlation Spectroscopy (FCS). What do you measure in an FCS experiments and how do you perform the measurements.

Answer 1

Fluorescence correlation spectroscopy (FCS) monitors the relative fluorescence fluctuations in a small confocal volume element (light green in figure) which is typically less than 1 femtoliter. The fluorescence photons emitted from molecules in this volume element pass through a pinhole and are detected by a highly sensitive detector. The signal-to-noise ratio achieved by this method is very high, since signal interference from scattered laser light, background fluorescence and Raman emission can be largely eliminated. This allows measurements at the single molecule level. Diffusion of molecules, entering and leaving the volume element (see figure) is one of the most studied sources of fluctuation but also other processes like triplet state dynamics, isomerization, quenching or protonation of the chromophore have been studied. In the case of diffusion, the average time required for the passage of a single fluorescent molecule through the volume element is determined by its diffusion coefficient, which, in turn, is also related to the size of the molecule. Therefore FCS can be used to study the molecular interactions by observing a faster diffusing ligand and the slower diffusing complex.

Fluorescence correlation spectroscopy (FCS) is a correlation analysis of fluctuation of the fluorescence intensity. FCS is such a sensitive analytical tool because it observes a small number of molecules (nanomolar to picomolar concentrations) in a small volume (~1μm³). The analysis provides parameters of the physics under the fluctuations. One of the interesting applications of this is an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles (molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles.

In FCS, the autocorrelation of this fluorescence variation is used to evaluate the temporal progression of a system around its equilibrium state. The autocorrelation is the cross-correlation of a signal with itself and is obtained by comparing a measured value at a time t with that at a later time (delayed by ). In this sense, one would expect two signals taken anearly the same time to have a high correlation value and those taken farther apart to result in a lower correlation value. The amplitude of the autocorrelation function is influenced by the number of molecules in the detection volume. The relative effect of one particular molecule on the total measured fluorescence decreases as the number of molecules increases, and the normalized amplitude of the autocorrelation function declines accordingly.2 It is for this reason that extremely dilute concentrations are used for FCS studies, such that approximately five molecules are desired in the detection volume at one time.