Fluorescence Correlation Spectroscopy (FCS) is a method that allows to detect the dynamics of fluorescing particles (e.g. molecules, quantum dots, small beads). It may be applied to motion in solution - also inside living cells - in gels and on surfaces. We use a standard FCS setup consisting of an inverted confocal microscope with a high-NA objective, which produces a narrow laser beam focus inside the sample. Molecules that diffuse in the focus will be excited and therefore show fluorescence which may subsequently be detected by means of the same microscope. So there is only a small volume (often less than 1 femto liter or 1 Ám3) from which fluorescence is detected. The next image shows a schematic overview of the setup:
The fluorescence photons are separated from the excitation light by a dichroic mirror and may then be detected on a single-photon detector (we use an avalanche photodiode, APD). This APD generates a pulse train where each pulse represents one detected photon. After binnig this gives a rather random intensity signal I(t):
Then we do a noise analysis, i.e. we calculate the time correlation function g(τ) of this signal which is proportional to the propability to find a photon a time t+τ if there was one at time t.
If you have only random noise in your signal this will result in no correlations at all, but in the described system a fluorescing particle enters and leaves the focal volume, which will generate a slower decay of the correlation function. The decay time estimated from this may then be interpreted as the average time a particle stays in the detection volume which is clearly connected to the motional properties of the particle. From theoretical modelling one knows the form of the correlation function for different types of motion, so we can fit the models to the experimental data and from this learn something about the motion inside the sample. The next image shows an example correlation curve (blue) together with a fit (red/orange):
We use a home-built microscope called FFM for FCS measurements. The abbreviation FFM stands for Fluorescence Fluctuation Microscope and describes a combination of a confocal laser scanning microscope (CLSM) with a FCS module developed in our group. The FCS module is coupled to two APDs and thus also allows cross-correlation measurements. The correlation function is calculated by an ALV-5000 multi-tau correlator card with a time resolution of up to 12.5 ns. The FFM uses a 2D galvanometric scanner to position the laser focus within the field of view.
Our FCS data evaluation software QuickFit 3.0 is available for free.