Specifically, the FCS measurements in this research were performed on a
custom apparatus, which has been developed from an Olympus IX70 confocal
microscope. The excitation light of a 488 nm line of an Ar laser is focused onto the
sample through an Olympus 60x water-immersion objective (numerical aperture 1.2).
The fluorescence is collected by the same objective, separated from the excitation light
by a dichroic mirror, then sent onto an avalanche photodiode (APD) (SPCM-AQR-14,
Perkin Elmer) by the means of an optical fiber with 50 pm-inner diameter after passing
through a 515-555 nm bandpass filter. The sample is illuminated with an excitation
power of 2 mW at the back aperture of the objective. The measurements are performed
in a volume element of 0.44 fL with halfaxes -xy = 0.22 pJm and wz = 1.56 pJm.
Autocorrelation is processed by a hardware correlator (ALV 5000/EPP, ALV-GmBH,
Langen, Germany).
FCS for Molecular Diffusion Studies in Solution
FCS is a highly sensitive tool for molecular diffusion studies in solution. There are
two direct read-outs from the autocorrelation curves, the absolute number of fluorescent
molecules in the focus volume, N, and the mean diffusion time TD.
Quantitative Studies of Molecular Numbers in the Focus Volume
Figure 2-5A shows the autocorrelation function measured for dye Rhodamine
123 at different concentrations (0.01nM-10nM). The increase in the number of
molecules N is proportional to the increase in concentration, and the autocorrelation
decreases as N increases, as given by Equation 2-3.