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Causes of Fluorescence Intensity Fluctuations

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How are Physical Parameters Extracted from FCS Data?

The real power of FCS comes when one relates the intensity fluctuations observed to the underlying physical processes to determine the fundamental physical constants of the system being studied. For instance, one might fit the data to isotropic three-dimensional diffusion and obtain the diffusion coefficients.

Below, you will find discussions on extracting physical parameters from these two properties of correlation functions:

Extracting Parameters from Correlation Timeback to top

The rate of decay of the correlation over time, the so-called correlation time, τD, describes the physical phenomenon, such as diffusion, that is causing the correlation. The longer the correlation persists, the slower the diffusion. Correlation persists longer for slowly diffusing particles and decays quickly for rapidly diffusing particles. In Figure 1, shifts in the correlation curve from the left to the right represent increases in correlation time (slower diffusing particles).

Figure 1
Figure 1: Schematic of a Set of Correlation Functions, G(Δt), Shown for Different Characteristic Correlation Times

Figure 2 displays correlation times for 3D diffusion for a wide range of small molecules, macromolecules, fluorescent beads, quantum dots, and pathogens. This is based on a detection system using a confocal beam radius of approximately 1 µm (See Confocal Optics).

Figure 2
Figure 2: Characteristic Diffusion Times for a 1 µm Beam

Extracting Parameters from the Interceptback to top

In Autocorrelation Due to 3D Diffusion, we calculated the value of g(Δt) at time t = 0 axis to be the reciprocal of the number of particles in the confocal volume.


where Nm is the number of particles in the confocal volume.

In General Intensity Autocorrelation, we showed that G(Δt) is simply related to g(Δt) through the following relationship:


As a result,


As illustrated in Figure 3, the intercept of the correlation function is inversely related to the number of fluorescent particles detected. As particle number decreases, the intercept increases.

Figure 3
Figure 3: Schematic of a Set of Correlation Functions, G(Δt), Showing the Inverse Relationship Between Intercept and Particle Number