Chemistry Reference
In-Depth Information
absorption spectrum of an acceptor (A)
uorophore. In other words, the
uorescence
of the donor and acceptor must be in resonance for the energy transfer to occur.
Hence, we will continue to use the appellation
uorescence.
When two
fluorophores are close enousgh together, the excitation of one
uor-
ophore may be transferred to the other. Figure 9.1B shows how the FRET process
appears in a Jablonski diagram. Fluorophore D, with higher energy excitation levels,
is brought close to
uorophore A, with lower energy levels. If there are a large number
of emissions pathways from S
1
to the manifold of vibronic energy levels of S
0
that
match energy of excitation pathways fromS
0
to the vibronic energy levels of S
1
, then
ef
cient FRET can occur. FRET adds another de-excitation pathway for S
1
with a
distance dependent rate,
6
1
t
R
0
R
k
FRET
¼
ð
9
:
4
Þ
0
where
0
is the donor
fluorescence lifetime in the absence of an acceptor, R is the
distance betweenD and A, and the Forster radius R
0
is the distance at which 50%of D
excitations are transferred A.
t
Figure 9.2 Effects of diffraction on localization
accuracy of single fluorescent molecules.
(A) Even though fluorophores are small
(
of light, the center of each spot may be
determined with very good accuracy, as long as
each emitting molecule is well separated as in A.
(B) If emission from two molecules is spectrally
separable, the relative centers of the two spots
may be determined to very high accuracy, even if
they overlap. Emission from two fluorophores
may also be separated even if they are of same
color, but turned on or off at different times.
0.5 nm), diffraction causes emission from the
fluorophores to be imaged as larger spots with
sizes comparable to the wavelength of light. The
diffraction-limited spots are shown as larger
yellow or red circles around the fluorophores.
Even though the spot size is near the wavelength
<
