Fluorescence Correlation
Spectroscopy (FCS)
Olga Pekar
Helin Räägel
EMBO Practical Course 2009
Heidelberg, DE
Why do FCS?
Study the kinetics of molecules in relation to:
- their surrounding environment (temperature,
viscosity, etc.)
- molecule size
- modified forms of the molecule.
Theory behind FCS
Dye dilution in water
1/N = 0,2
number of molecules = 5
concentration of dye
4.15 x 10-16 L
focal point volume
a=1.5 µm
a
b
b=0.25 µm
concentration
1/N
N
τD
Diffusion coefficient
20 nM
0.2
5
0.021 msec
7.44 x 10-10 m2/sec
200 nM
0.0164
61
0.028 msec
5.58 x 10-10 m2/sec
FCS in live cells
before
monoGFP
tetraGFP
after
1/N
N (molecules)
τD1 (msec)
Diffusion coefficient 1
τD2 (msec)
nucleus
0.047
21
0.23
6.82 x 10-8 m2/sec
199.4
cytoplasm
0,058
17
0.17
9.01 x 10-8 m2/sec
34.7
nucleus
0.035
28
0.81
1.93 x 10-8 m2/sec
229.5
cytoplasm
0.068
15
0.48
3.25 x 10-8 m2/sec
810.5
*theoretically diffusion time should increase by a factor of 41/3 = 1.58
FCS technique
Advantages
 Measure the diffusion
of molecules
 Transfected cells can
be used
 Very small sample
volumes
 Single molecule
sensitivity
 Provides direct
measure of
stoichiometry
Disadvantages
 Only cells with low
expression of the
fluorescently tagged
protein can be used
 movement of cells
during measurement
 Need for special
equipment
Thank You:
Nicolas
Jörg
Oleg