Biomedical Engineering Reference
In-Depth Information
Fig. 3.5
Basic concepts of FRET. FRET is the nonradiative energy transfer from an excited-state
donor (D) to an acceptor (A) at the ground state, in close proximity (1-10 nm), via a long-range
dipole-dipole coupling mechanism. The energy transfer efficiency (E) from the D to the A of a
FRET pair is dependent on the inverse of the sixth power of the distance between them, subject
to the Forster distance of the FRET pair (see Eqs.
3.1
and
3.2
in Sect.
3.3.4
). (
a
) Plotting E as
a function of the D-A distance for a FRET pair with a Forster distance of 5 nm indicates that
measuring E can provide sensitive indication on the change of the D-A distance within 2-8 nm.
Other than a close distance between the D and the A, FRET also requires (
b
) a significant overlap
between the spectra of the D emission and the A excitation (covered by the
gray area
)and
(
c
) a favorable dipole moment -
2
3 cos
D
cos
A
/,where
T
is the angle between the
transition dipoles of the D emission and the A absorption;
D
and
A
are the angles between these
dipoles and the vector joining the D and the A; no FRET occurs for
2
D .cos
T
D 0, and the likelihood of
FRET increases with a larger
2
(Adapted from [
43
])
estimate the E by measuring the intensity change of the donor in the absence and
the presence of the acceptor [
77
-
79
]. FLIM-FRET methods quantify the E from
measuring the change in the donor lifetimes in the absence and the presence of
the acceptor (described in Sect.
3.4
). The most commonly used FRET microscopy
methods are based on the detection of the sensitized emission from the acceptor -
the FRET signal. For example, ratiometric FRET microscopy provides a simple
way to demonstrate the change in FRET significances due to different treatments
for a biological system with a fixed stoichiometry of the donor and the acceptor
(e.g., FRET-based biosensors) [
80
-
82
]. However, quantitative determination of
the E in sensitized emission measurements usually requires removing the spectral
bleedthrough (SBT) components from the donor and the acceptor that contaminate
the FRET signal, and many algorithms have been developed for this purpose
[
83
-
100
].
We developed the processed FRET (PFRET) method that utilizes both donor-
alone and acceptor-alone control specimens and algorithm-based software to
achieve accurate SBT corrections for different donor and acceptor fluorescence
levels [
93
,
100
,
101
] (also see Chapter
7
in [
74
]). Two examples of applying PFRET
in biological studies are presented here: (1) detect dimerization of C=EBP˛ in
live mouse pituitary cell nuclei using a widefield microscope (see Fig.
3.2
)and
(2) study receptor-ligand binding and internalization in polarized live MDCK cells
using a confocal microscope (see Fig.
3.3
). We also developed the spectral FRET
(sFRET) microscopy method [
97
]. Spectral imaging microscopy produces -stacks
consisting of x-, y- (spatial), and - (spectral) dimensions, measuring the emission
signals in a series of spectral intervals equally sampled over a spectral range at each
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