Biophotonics_Concepts_to_Applications

Example 9.2Suppose the distance between the donor and acceptor is

increased by a factor of three from R = R 1 =R 0 to R = R 2 =3R 0. What is

the decrease in energy transfer efficiency?

Solution: Letting the parameter x = R/R 0 , then Eq. (9.1) can be written as

E = 1/(1 + x^6 ). Thus, letting x 1 =R 1 /R 0 and x 2 =R 2 /R 0 , the ratio of the

energy efficiencies is

ERðÞ 1

ERðÞ 2

¼

1 þx^62

1 þx^61

¼

1 þ 36

1 þ 16

¼

1 þ 729

1 þ 1

¼ 365

Thus the energy transfer efficiency decreases by a factor of 365 when the

distance between thefluorophores increases from R 1 =R 0 to R 2 =3R 0.

Table9.2lists some commonly used FRET pairs, the corresponding Förster
distance, the donor excitation wavelength, and the acceptor emission wavelength.

9.2.2 Fluorescence Lifetime Imaging Microscopy.

In a FRET process, the decay times offluorescent emissions from an acceptor
molecule can be used at the cellular level to study protein interactions, confor-
mational changes, and parameters such as viscosity, temperature, pH, refractive
index, and ion and oxygen concentrations. An imaging procedure that makes use of
FRET isfluorescence lifetime imaging microscopy(FLIM), which is an advanced
version of thefluorescence microscopy technique described in Sect.8.4. By gen-
eratingfluorescence images based on the differences in the exponential decay times

Table 9.2 Commonly used FRET pairs and their spectral parameters

Donor (excitation wavelength) Acceptor (emission wavelength) Förster distance

(nm)

Fluorescein (512 nm) QSY-7 dye (560 nm) 6.1

Fluorescein (512 nm) Tetramethylrhodamine (TRITC:

550 nm)

5.5

Cyanfluorescent protein (CFP:

477 nm)

Yellowfluorescent protein (YFP:

514 nm)

5.0

IAEDANS (336 nm) Fluorescein (494 nm) 4.6

Bluefluorescent protein (BFP:

380 nm)

Greenfluorescent protein (GFP:

510 nm)

3.5

EDANS (340 nm) DABCYL (393 nm) 3.3

266 9 Spectroscopic Methodologies