Principles of Fluorescence Spectroscopy

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In the previous chapter we described fluorescence imaging
and spectroscopy on single molecules. Individual fluorophores
can be studied if the observed volume is restricted
and the fluorophores are immobilized on a surface. With
present technology it is difficult to track freely diffusing
single molecules. Single-molecule detection (SMD) on surfaces
is a powerful technique because it avoids ensemble
averaging and allows single events to be observed. If a
dynamic process such as a chemical reaction is being studied,
there is no need to synchronize the population because
the individual kinetic events can be observed. However,
SMD has its limitations. The most stable fluorophores typically
emit 10 5 to 10 6 photons prior to irreversible photodestruction.
Because of the modest detection efficiency of
optical systems, and the need for high emissive rates for
detection of the emission over background, single molecules
can only be observed for a brief period of time—1
second or less—which may be too short to observe many
biochemical processes. When the fluorophore is bleached
the experiment must be started again with a different molecule.
Additionally, SMD requires immobilization on a surface,
which can affect the functioning of the molecule and
slow its access to substrates and/or ligands because of
unstirred boundary layers near the surface.
Fluorescence correlation spectroscopy (FCS) is also a
method based on observation of a single molecule or several
molecules. In contrast to SMD, FCS does not require surface
immobilization and can be performed on molecules in
solution. The observed molecules are continuously replenished
by diffusion into a small observed volume. FCS thus
allows continuous observation for longer periods of time
and does not require selection of specific molecules for
observation. FCS is based on the analysis of time-dependent
intensity fluctuations that are the result of some dynamic
process, typically translation diffusion into and out of a
small volume defined by a focused laser beam and a confocal
aperture. When the fluorophore diffuses into a focused
Fluorescence
Correlation
Spectroscopy
light beam, there is a burst of emitted photons due to multiple
excitation-emission cycles from the same fluorophore.
If the fluorophore diffuses rapidly out of the volume the
photon burst is short lived. If the fluorophore diffuses more
slowly the photon burst displays a longer duration. Under
typical conditions the fluorophore does not undergo photobleaching
during the time it remains in the illuminated volume,
but transitions to the triplet state frequently occur. By
correlation analysis of the time-dependent emission, one
can determine the diffusion coefficient of the fluorophore.
In this case "time-dependent" refers to the actual time and
not to a time delay or time-dependent decay following
pulsed excitation.
FCS has many applications because a wide variety of
processes can result in intensity fluctuations. In addition
to translation diffusion, intensity fluctuations can occur due
to ligand–macromolecule binding, rotational diffusion,
internal macromolecule dynamics, intersystem crossing,
and excited-state reactions. The data are interpreted in
terms of correlation functions. Different equations are needed
to describe each process, and usually two or more
processes affect the data at the same time. It is also necessary
to account for the size and shape of the observed volume.
As a result the theory and equations for FCS are rather
complex.
FCS was first described in the early 1970s in a series of
classic papers. 1–4 An extensive description of FCS and its
applications can be found in a recent monograph. 5 These
papers describe the theory for FCS and recognize its potential
for measurement of diffusion and reaction kinetics. The
theory showed that FCS would allow measurement of kinetic
constants even when the system was in equilibrium, if the
reversible process caused spectral changes. The FCS data
will contain information on the reaction rates because a
reaction at equilibrium still proceeds in both directions, so
that the spectral properties will continue to fluctuate. However,
the early FCS measurements were plagued by low sig-
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