From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 157

6

1
t D

R 0
r

K T ð r Þ¼ ½ 5 : 15

¼

for r R 0

K T r ðÞ¼ 1 = t D ½ 5 : 16

One obtains (from Eq. 5.14 )

ð 1

2 9000 ln 10

ðÞ

4 dl

R 0 ¼ F D k

F D ðÞ e A ðÞ l ½ 5 : 17

5 n 4 N A

128p

0

R 0 is given in Angstr ¨ m and may be simplified as

R 0 ¼ 10 5

2 J ðÞ

n 4 8 : 79 F D k ½ 5 : 18

This expression allows the F¨ rster distance to be calculated from the
spectral properties of the donor and the acceptor and from the donor quan-
tum yield F D. 13
FRET efficiency is dependent on the inverse sixth power of the inter-
molecular separation ( r ) (discussed in the next section).
Additionally, F ¨ rster distance is usually reported for an assumed value of

2

k of 2/3 characterizing free FRET pairs. The question of dipole-dipole ori-

entation is discussed later.
Directly, if the transfer rate is much faster than the decay rate, then
energy transfer will be efficient; otherwise, FRET will be inefficient.
A crucial step in the practical implementation of FRET is the knowledge
of several major parameters:
- the quantum yield F D of the fluorophore donor only;

- the overlap integral J (l) between the donor and acceptor fluorophores;

2 between two fluorophore dipoles.

- the orientation factor k

3.3. Overlap integral J( )

J (l) is the overlap integral between the donor emission and the acceptor
absorption spectra (expressed as M -1 cm -1 nm 4 ) and is defined as

l

ð 1

4 dl

F D ðÞ e A ðÞ l

0

ð 1

J ðÞ¼ ½ 5 : 19

F D ðÞ dl

0