Fluorescence Correlation Spectroscopy

The contents of this section are derived from the following website: www.probes.com/handbook/boxes/1571.html. This website provides a very lucid description of the method of fluorescence correlation spectroscopy. Another suggested reference is a review by Thompson (1991).

Flow Cytometry

Time Time

Figure 4.15. (a) Fluorescence intensity fluctuations caused by diffusion of small molecules; (b) fluorescence intensity fluctuations caused by diffusion of less mobile biopolymers. (Reproduced with permission from the highlighted website.)

Time Time

Figure 4.15. (a) Fluorescence intensity fluctuations caused by diffusion of small molecules; (b) fluorescence intensity fluctuations caused by diffusion of less mobile biopolymers. (Reproduced with permission from the highlighted website.)

In fluorescence correlation spectroscopy, often abbreviated as FCS, spontaneous fluorescence intensity fluctuations in a microscopic volume consisting of only a small number of molecules is monitored as a function of time. The volume typically sampled is about 10-15L (or a femtoliter) compared to that of 0.1-1.0mL or even larger, typically sampled by conventional fluorescence spectroscopy. The fluorescence intensity fluctuations measured by FCS relates to dynamical processes occurring in the interrogation volume. These dynamical processes can be due to changes in the number of fluorescing molecules due to their diffusion in and out of the microscopic volume sampled. They can also represent a change in the fluorescence quantum yield due to processes occurring in the interrogation volume.

Fluctuations caused by diffusion of molecules depend on their size. Rapidly diffusing small molecules produce rapid intensity fluctuations as shown in Figure 4.15a. In contrast, large molecules and biopolymers such as proteins or protein bound ligands exhibit slowly fluctuating patterns of bursts of fluorescence, as shown in Figure 4.15b. Quantitatively, the fluorescence intensity fluctuation is characterized by a function G(t), called the autocorrelation function, which correlates the fluctuation SF(t) in fluorescence intensity at time t with that [SF(t +1)] at time (t + t), where t is a variable time interval., averaged over all data points in the time series. Thus, G(t) is defined as

The brackets in this expression represent the average over all data points at different times t.

Figure 4.16. Simulated FCS autocorrelation functions representing a free ligand and a corresponding bound ligand. The intermediate curve represents a mixture. (Reproduced with permission from the above highlighted website.)

Figure 4.16. Simulated FCS autocorrelation functions representing a free ligand and a corresponding bound ligand. The intermediate curve represents a mixture. (Reproduced with permission from the above highlighted website.)

A typical autocorrelation function G(t) plotted as a function of time interval t is represented in Figure 4.16 for a free ligand (low molecular weight) which can diffuse faster and a bound ligand which diffuse slower. The initial amplitude of the autocorrelation function is inversely proportional to the number of molecules in the sampled volume. The decay of the autocorrelation function is fast for a free ligand and relatively slow for a bound ligand. Thus, the decay behavior of G(t) provides information on the diffusion rates of the fluorescing species.

FCS is an excellent probe for monitoring biomolecular association and dissociation processes. With the recent progress of increase of detection sensitivity for fluorescence to the limit of single molecule detection, FCS has emerged as a valuable tool to investigate a variety of biological processes such as protein-protein interactions, binding equilibria for drugs, and clustering of membrane bound receptors. Another extension of FCS is dual color cross-correlation, which measures the cross-correlation of the time-dependent fluorescence intensities of two different dyes fluorescing at different wavelengths (Schwiller et al., 1997). This method has the advantage that cross-correlated fluorescence is only generated by molecules or biopolymers fluorescently labelled (chemically attached) with both dyes, allowing quantitation of interacting dyes.

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