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Introduction |
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Fluorescence Correlation Spectroscopy (FCS) in its modern embodiment
measures and exploits temporal fluctuations in the fluorescence
emission of small numbers of molecules in an open excitation volume
(of ca 1 fl) defined by confocal optics. Fluctuations arise from
changes in the number of molecules observed as they diffuse in and
out of the volume element, as well as by the fluorescent intensity
or yield, particularly manifest via the autocorrelation function of
the time series. Since small ligands have an autocorrelation function
which falls off much more rapidly when they are free than when they
are bound to (say) proteins or nucleic acids, analysis of the shape
of the autocorrelation function allows one to discriminate bound
(macromolecular) and free ligands non-invasively and simultaneously.
The autocorrelation function is dominated by the diffusion coefficient,
is thus known as a 'mass-dependent' method, and is the simplest version
of this general principle of fluctuation analysis. However, the same
apparatus may be used to generate data which may be subjected to much
more powerful and more recently developed analyses such as
brightness-dependent fluorescence correlation spectroscopy as represented
by Fluorescence Histogram/Fluorescence Intensity Distribution Analysis
(FHA/FIDA) and related methods. As part of a program in the Analytical
Biotechnology of single molecules, we are developing and exploiting
variants of these strategies in a variety of areas.
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Last update: 19 February 2004
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