Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 13
Figure 1.15. Effects of polarized excitation and rotational diffusion on
the polarization or anisotropy of the emission.
tion times and are expected to yield lower anisotropies. The
anisotropies of larger proteins can also be low if they are
labeled with long-lifetime fluorophores. The essential point
is that the rotational correlation times for most proteins are
comparable to typical fluorescence lifetimes. As a result,
measurements of fluorescence anisotropy will be sensitive
to any factor that affects the rate of rotational diffusion. The
rotational rates of fluorophores in cell membranes also
occur on the nanoscale timescale, and the anisotropy values
are thus sensitive to membrane composition. For these reasons,
measurements of fluorescence polarization are widely
used to study the interactions of biological macromolecules.
1.6. RESONANCE ENERGY TRANSFER
Another important process that occurs in the excited state is
resonance energy transfer (RET). This process occurs
whenever the emission spectrum of a fluorophore, called
the donor, overlaps with the absorption spectrum of another
molecule, called the acceptor. 19 Such overlap is illustrated
in Figure 1.16. The acceptor does not need to be fluorescent.
It is important to understand that RET does not
involve emission of light by the donor. RET is not the result
of emission from the donor being absorbed by the acceptor.
Such reabsorption processes are dependent on the overall
concentration of the acceptor, and on non-molecular factors
such as sample size, and thus are of less interest. There is
no intermediate photon in RET. The donor and acceptor
are coupled by a dipole–dipole interaction. For these reasons
the term RET is preferred over the term fluorescence
resonance energy transfer (FRET), which is also in
common use.
The extent of energy transfer is determined by the distance
between the donor and acceptor, and the extent of
spectral overlap. For convenience the spectral overlap (Figure
1.16) is described in terms of the Förster distance (R 0 ).
The rate of energy transfer k T (r) is given by
k T (r) 1
τ D
( R0 6 ) r
(1.11)
where r is the distance between the donor (D) and acceptor
(A) and τ D is the lifetime of the donor in the absence of
energy transfer. The efficiency of energy transfer for a single
donor–acceptor pair at a fixed distance is
E R6 0
R 6 0 r 6
(1.12)
Hence the extent of transfer depends on distance (r).
Fortunately, the Förster distances are comparable in size to
biological macromolecules: 30 to 60 Å. For this reason
energy transfer has been used as a "spectroscopic ruler" for
measurements of distance between sites on proteins. 20 The
value of R 0 for energy transfer should not be confused with
the fundamental anisotropies (r 0 ).
The field of RET is large and complex. The theory is
different for donors and acceptors that are covalently
linked, free in solution, or contained in the restricted
geometries of membranes or DNA. Additionally, depending
on donor lifetime, diffusion can increase the extent of energy
transfer beyond that predicted by eq. 1.12.
Figure 1.16. Spectral overlap for fluorescence resonance energy
transfer (RET).