Photon autocorrelation function analysis

The hydrodynamic radius of colloidal particles can be obtained from dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS). Here, the temporal fluctuations of scattered light intensity are measured to provide the autocorrelation function, analysis of which provides the translational diffusion coefficient. Then the Stokes-Einstein equation (Eq. 1.9) is used to determine a hydrodynamic radius. This method is described further in Section 1.9.2. 

In fluorescence correlation spectroscopy (FCS) a small volume element or a small area) of a sample is illuminated by a laser beam and the autocorrelation function of fluctuations in the fluorescence is determined by photon counting. From this autocorrelation function the mean number densities of the fluorophores and their diffusion coefficients can be extracted. Measurement and analysis of higher order correlation functions of the fluorescence has been shown to yield information concerning aggregation states of fluorophores ). 

Fig. 5A, B Proteolytic cleavage analysed by A cross-correlation analysis B FRET on single-molecule-scale. In A, during the course of a specific proteolytic reaction GFP and DsRed get separated leading to gradually decreasing cross-correlation amplitudes Gx(0) determined in 40-s intervals, in B, alternatively, autocorrelation functions and photon counts per molecule in kHz (inset) for rsGFP and DsRed were determined in parallel during a proteolytic digest. Changes in fluorescence intensity of FRET donor and acceptor were detected immediately after enzyme addition, whereas autocorrelation G(0) values and fluorescent particle numbers remained constant. The monitored increase of rsGFP fluorescence corresponds to approximately 35% FRET in the intact substrate...

Higher order autocorrelation analysis is analogous to the analysis of the higher order moments of the photon count distribution. Such a correlation function is given by. 

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