overlap, quantum yield of the donor, and relative orientation of the dipole
moments,κ^2 :
r^60 ∝κ^2 (refractive index)−^4 ×(overlap) ×(yield) (19.4)
In steady-state measurements of fluorescence intensity, the relative donor
and acceptor emission ratios can be examined. Alternatively, the donor
and acceptor emissions can be detected independently in the FRET
measurement.
By combining the power of fluorescence microscopy with the optical
properties of GFP, it is possible to probe intracellular processes and reac-
tions in cells. FRET is very sensitive and can be used to determine whether
two proteins come close together or whether two ends of a protein (or
DNA) come close together. For example, consider the case of two proteins,
identified as A and B, that under some circumstances can form a complex.
Protein A is labeled with GFP that serves as the donor and protein B is labeled
with an acceptor. When the two proteins are far apart then the excitation
of GFP results in emission from GFP. However, when the two proteins form
a complex, excitation of GFP is followed by energy transfer to the acceptor
and emission from the acceptor. Thus, the relative amount of emission
from GFP compared to the acceptor is directly related to the ratio of the
proteins as monomers compared to those forming the complex.
Imaging of GFP in cells
Fluorescence imaging techniques can be performed not only under
steady-state conditions but also as transient measurements. Among the
possible approaches are photobleaching studies (Figure 19.8). In the pre-
sence of a strong light, pigments will undergo changes that result in loss
of an absorption band and correspondingly loss of fluorescence. After the
illumination beam is turned off, the molecules will recover, restoring the
absorption and fluorescence if the bleaching is reversible. This photo-
bleaching property provides an opportunity to specifically monitor a
selected pool of GFP that is distinguished from other GFPs at different
locations in the cell. In one case, an area of the cell is photobleached with
a high-intensity laser pulse. The movement of the unbleached GFP from
neighboring areas into the bleached area is monitored by recording the
recovery of fluorescence in the photobleached area. The recovery of
the fluorescence in the selected area will increase until all of the GFP has
recovered. Alternatively, photobleaching can be performed repeatedly
so that the GFP in the selected area does not have time to recover and
is always bleached. With time, the bleached GFP will exchange with the
GFP from the outside areas, diminishing the amount of fluorescence in
the outside areas. Any GFP that enters the selected area is quickly photo-
bleached so the fluorescence in the selected area remains minimal. As the
412 PART 3 UNDERSTANDING BIOLOGICAL SYSTEMS USING PHYSICAL CHEMISTRY
