Biomedical Engineering Reference
In-Depth Information
colocalization of two photons within the absorption cross section of the fluorophore.
The rate of excitation is proportional to the square of the instantaneous intensity.
This extremely high local instantaneous intensity is produced by the combination
of diffraction-limited focusing of a single laser beam in the specimen plane and
the temporal concentration of a femtosecond mode-locked laser (typically of the
order of 10
50
to 10
49
cm
4
s/photon/molecule) [
57
]. Multiphoton (i.e., 3-photon
or 4-photon) excitation microscopy is the extension of TPE microscopy [
58
].
Compared to SPE confocal microscopy (see Sect.
3.3.2
), TPE microscopy offers
several advantages listed in Table
3.1
. TPE microscopy has been widely used
in many areas of the biomedical sciences including cell biology, microbiology,
molecular biology, developmental biology, neuroscience and neurology, tissue
engineering, etc. [
48
,
59
-
66
]. In Sect.
3.4
, we describe an example of employing
TPE to implement a FLIM system used for studying protein-protein interactions in
living cells.
3.3.4
FRET Microscopy
FRET is the nonradiative energy transfer from an excited molecule (the donor) to
another nearby molecule (the acceptor) via a long-range dipole-dipole coupling
mechanism. The most basic concepts of FRET are described by Eqs.
3.1
-
3.2
[
67
-
70
] (also see Chapter 13 in [
13
]) and Fig.
3.5
. More details about FRET theory
and history can be read in the literature [
71
].
D
Ro
1
E
1
1
6
Ro
6
.Ro
6
E
D
or
r
(3.1)
C
r
6
/
R
0
f
D
./f
A
./
4
d
R
0
1
6
;
Ro
D
0:211
f
2
n
4
QY
D
J
g
J
D2
A
(3.2)
f
D
./d
AsshowninEq.
3.1
and Fig.
3.5
A, the efficiency of energy transfer (E) from
the donor to the acceptor is dependent on the inverse of the sixth power of the
distance (r ) separating them, subject to Ro - a characteristic distance at which half
of the excited-stated energy of the donor is transferred to the acceptor (E
D
50 %).
The correct calculation form of Ro (named Forster distance) was first described by
Theodor Forster in the mid-1940s [
67
,
68
]. Determination of the Ro (in angstrom)
for a FRET pair is given by Eq.
3.2
,where
2
is the dipole moment factor described
in Fig.
3.5
C; n is the medium refractive index; QY
D
is the donor quantum yield; J
is the degree of the overlap between the donor emission spectrum and the acceptor
absorption spectrum (see Fig.
3.5
B); "
A
is the extinction coefficient of the acceptor
at its peak absorption wavelength; is the wavelength; f
D
./ and f
A
./ are the
normalized donor emission and acceptor absorption spectra, respectively. Since
FRET is usually limited to distances less than about 10 nm, FRET microscopy
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