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The Fluorescence Correlation Spectroscopy (FCS) Method

FCS is a fluorescence-based measurement method. Fluorescent molecules passing through a strongly focused, fixed laser beam are excited for fluorescence emission. After passing a confocal pinhole, the emitted photons are registered using very sensitive detectors.

FCS measurements are performed with the Leica TCS SP8 SMD system.

Quantitative parameters derived from autocorrelation curve

The moving particles give rise to intensity fluctuations. An autocorrelation of this reveals information on the molecular scale: the amplitude of the correlation curve shows the particle number. The time at the inflection point contains information about particle mobility. Finally, the steepness and shape of the curve reflect the type of diffusion. Particle concentration, particle mass, viscosity, and bound fraction can be derived from these parameters.

Principle of FCS data acquisition and analysis

  1. Laser illumination at a fixed point of interest (beam parking) excites fluorescent particles in the excitation volume. The particle movement in and out of the confocal volume causes intensity fluctuations.
  2. Registration of intensity fluctuations using detectors in single photon counting mode.
  3. Calculation of the correlation function.
  4. Fitting of the corresponding biophysical model to the correlation curve. Obtain parameters of interest:
    • particle concentration
    • diffusion coefficient
    • viscosity
    • molecular mass
    • binding constant
    • photo-physical properties