The observation of fluctuations in FCS requires that we observe only a few particles at any point in time. If too many particles are present, the ACF amplitude becomes small and cannot be detected anymore against the noise of the ACF. The number of particles we detect will depend on the volume we observe and the concentration of the probe. Assuming a concentration range of nM - µM, which is quite typical for many biological applications, we need a volume on the order of femtoliters (10-15 liters) to reach sufficiently low particle numbers. E.g., using a high NA objective (NA 1.2) and laser light around 500 nm we can achieve a confocal observation volumes (Vobs) on the order of 0.3 fl. At a concentration range of nM - µM, this would result in about 0.2 - 200 particles, N, in the observation volume (N = c Vobs NA, with c being the concentration and NA the Avogadro constant). This is about the range where FCS can be used, although lower and higher concentrations have been reported (~0.1 nM - 40 µM), sometimes with special setup modifications.
When a fluorescent particle passes through the observation volume (represented as the blue "peanut" on the left) it will be excited by the laser light and will emit photons that are recoreded by a detector (middle). The length of the photon bursts (or fluorescence fluctuations) provide information how long the particle took to pass through the observation volume. As we have seen under correlations we can measure transition tiome of the particles by the ACF as only photons stemming from the smae particles will be correlated. By observing many particles one can obtain the average transition time which is proportiona to the diffusion coefficient. In FCS we extract this avergae transition time from the width of the autocorrelation function of the signal (graph on the right).
How the the amplitude and width of the ACF change with number of particles in the observation volume and diffusion coefficient of the particles can be found in LINK TO ...
The volume of the confocal obervation volume Vobs and the number of particles N is given by:
The correlation function for 3D diffusion is given by:
For more information on different models for the correlation fucntion, please see "FCS".