Evaluation
The reciprocal of the number of particles determines the correlation amplitude, whereas the kinetics of the triplet state (relaxation time, τF) and particle mobility (diffusion time, τD) determine the characteristic decays in the curve. The decay of the correlation curve in the case of the diffusion process is a result of a decrease in the overlap of the two signals with increasing lag times. How does FCS work?

Measurement
Calculation
Evaluation
Result
Confocor Sanduhr
Computed fluorescence autocorrelation curve for 3-dimensional diffusion including triplet state
A biophysical model describes the intensity fluctuations due to diffusion of different sizes molecules through a confocal volume.
The model may contribute for electronically properties of dyes like triplet states.
Models describing other sources of intensity fluctuations have been described in literature.
Find Biophysical Model
If δI (t) results from molecules diffusing through a light spot with gausian profile, one gets this form for the normalized auto correlation function.
Were N stand for the number of molecules and s describes the ratio between the elongation of the spot in xy and z direction.
τD describes the time the molecule needs to diffuse through the light spot.
The diffusion coefficient of the molecule is related to its hydrodynamical radius and thus its mass.
Fitting Mathematical Model to Correlation Curve
Measuring fluctuating intensities
Calculating auto correlation function
Comparing (fitting) auto correlation function with biophysical model by adjusting free parameters
Revealing biophysical parameters
Size Dependence of Correlation
  • The mean time interval of the correlation gives an information of the size and the molecular weight of the observed molecules:
  • 25-50 µs are typical for pure dye molecules (MW: 550 g/mol)
  • An E.coli cell has a diffusion time of 150 ms
    • Typical Binding Assay
  • FCS detects the change in mobility of a chromophore.
  • The chromophore is attached to a small, fast moving molecule.
  • The small molecule binds to a larger molecule.
  • Now, the chromophore is attached to a slow moving compound.
    • Typical times
    Order of
    magnitude    		TimeExample
    Sample
    10-6 s (MHz)50 µsDiffusion time of dye molecule
    200 µsDiffusion time of 40 bp DNA
    400 µsDiffusion time of antibody
    10-3 s150 msDiffusion time of E. coli
    Electronic Properties of Dye
    10-9 s1 nsFluorescence life time
    10-6 s (MHz)3 µsFluorescence triplet time
    10 µsMean time between two photons (100 kcpm)
    Instrumentation
    10-9 s35 nsDead time of photo detector (APD)
    10-0 s30 sMeasurement time
    Amplitude of Correlation and Sample Concentration
  • The amplitude of the correlation increases if the concentration decreases.
  • The start of the correlation curves gives the mean number of molecules inside the confocal volume.
  • A number of 1.5 particles in the detection volume of the ConfoCor 3 belongs to a sample concentration of 10 nM.
    • Typical Binding Assay
  • FCS detects the number of chromophores.
  • The chromophores are attached to free moving molecules.
  • The free molecules bind to immobile molecules.
  • Now, the chromophores do no longer contribute to the fluctuation, thus the correlation increases.
    • Different correlation curves add up
    If several species of molecules are present, their correlation curves add up.
    The different molecules or compounds can be separated by there different diffusion times.
    curve mainly determined by particle 1
    curve mainly determined by particle 2
    Typical Binding Assay
    FCS detects the change in concentration of a chromophore with a specific mobility.
    The fraction of molecules of the fast moving species corresponds to free molecules.
    The fraction of molecules of the slow moving species corresponds to bound molecules.


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