Biophotonics_Concepts_to_Applications

9.3 Fluorescence Correlation Spectroscopy

In addition to its use for identifying molecules and examining their external
behaviors,fluorescence spectroscopy has the ability to examine molecular diffusion
processes and to carry out noninvasive measurements inside of living cells. Toward
this goal, a technique calledfluorescence correlation spectroscopy(FCS) has been
devised for precisely investigating the basic physical and chemical (referred to as
physicochemical) processes in the smallest functional units in biological systems,
such as individual proteins and nucleic acids [ 18 – 21 ]. In general, FCS measure-
ments are made usingfluorescently labeled biomolecules that are diffusing in an
aqueous buffer solution. A laser spot in the focus of a confocal microscope or a
multiphotonfluorescence microscope defines the detection volume.
In contrast to other fluorescent spectroscopic methods that concentrate on
examining the spectral emission intensity, FCS is a statistical method that examines
thespontaneousfluorescent intensityfluctuationscaused by minute deviations of
the small biological system from thermal equilibrium. The intensityfluctuations can
originate from Brownian motion of dye labeled molecules, enzymatic activity,
rotational molecular motion, or protein folding. A key parameter of interest is the
lateral diffusionoffluorescent molecules in and out of the detection volume (in the
viewing plane of the microscope). The time duration in which molecules remain
within the laser spot depends on their size. For example, if a small, dye-tagged
molecule binds to a larger molecule, the tagged molecule will slow down and emit
photons for a longer time during its diffusion through the laser spot than if it were
attached to a smaller molecule. The intensityfluctuations due to the lateral diffusion
process range from milliseconds to seconds, whereas photochemical processes are
usually much faster. Thus, the study of the contributions to the intensityfluctuations
from the photochemical processes can be separated from the diffusion effects.
Experimental setups for FCS use nanomolar concentrations of molecules in sample

5 ns

1 ns

Double exponential

with lifetimes of

1 ns and 5 ns

Time (ns)

ln

I(t) [arbitrary units]

24 6 810

Fig. 9.8 Logarithmic plots of
single and double exponential
fluorescence decays

9.3 Fluorescence Correlation Spectroscopy 269