Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 326

B

A D

1

440 nm 530 nm

FRET

440 nm 480 nm

0.5

J

505 nm

0

400 500 600

AB

Wavelength (nm)

C

440 nm 530 nm 440 nm

FRET

FRET

q T

480 nm

q D

A

D

r

q A

2 = (cos 2

q T -3cos q A cos q D ) 2

k

E

AB

R 0 :Försterdistance

Cooled CCD Camera

PC

1

F d ¢

E = 1-

F d Chamber box

R 0 6
R 0 6

E =

6

+ r

0.5

Xenon lamp

F d

Emission
filter wheel

F d ¢

R 0

Excitation
filter wheel

0

Donor
Acceptor

Distance

Figure 8.3 The principle behind FRET. (A) When CFP exists alone, CFP excitation results
in CFP emission. When CFP exists close to YFP, the excited energy of CFP is transferred to
YFP; thus, YFP emission can be observed. In the bottom graph, the relative FRET effi-
ciency is plotted against the distance between the chromophores (assuming that
k

2

¼2/3). (B) Spectral overlap (J) of donor emission and acceptor excitation spectra.
Dashed and solid lines indicate the excitation and emission spectra, respectively, while
gray and black lines indicate the spectra of CFP and YFP. (C) The relative orientations of
chromophore dipoles and orientation coefficients. (D) Schematic representation of the
photobleaching method. In the presence of FRET, disruption of YFP by photobleaching
results in recovery of CFP emission. In the bottom graph, a typical photobleaching ex-
periment is shown. From the donor emission intensities before (F d 0 ) and after (F d ) pho-
tobleaching, the FRET efficiency can be calculated using the equation indicated. (E) An
epifluorescence microscopic system for FRET analysis.

Here, r is the distance between the centers of the donor and acceptor
chromophores, k

2

is an orientation factor for dipole-dipole interaction

( Fig. 8.3C ), D is a rate constant for the donor fluorescence emission, and

J is the spectral overlap integral between the donor emission and the acceptor
adsorption ( Fig. 8.3B ). The refractive index n of the medium between the
chromophores is generally taken as 1.33 in water; thus, k T is proportional to
1/ r. 6,84 When rotational diffusion prior to energy transfer randomizes the

l