Relationship between fluorescence anisotropy, r and rotational correlation time ρ
is shown in Perrin-Weber equation,
1
r ¼
1
r 0 1þ
3τ
ρ
ð Þ
where r 0 , and ρ are r values of
fluorescence life time and rotational correlation time of molecule at 0 K, respectively. If fluorescence lifetime is obtained by such as pulse excitation method,
rotational correlation time ρ can be calculated using the above equation. By use of
Einstein’s relationship, ρ ¼
4πr
3
kT η where ρ, r, k, T and η are rotational correlation
time, radius of fluorescence probe, Boltzmann constant, absolute temperature and
local viscosity, respectively. And local viscosity is obtained by introducing rotational correlation time. Stokes-Einstein equation among diffusion constant, D,
viscosity of solvent, η and radius of fluoresce probe, R is shown as D ¼
kT
6πηR
where ρ, k, η, T and are rotational correlation time, Boltzmann constant, local
viscosity of solvent, absolute temperature and radius of fluorescence probe, respectively. Therefore, diffusion constant of fluorescence probe is obtained in above
procedure.
Fluorescence probe DPH (1,6-diphenyl-1,3,5-hexatriene) is widely used for
measurement of fluorescence polarization anisotropy (Fig. 3.17).
However, it is not easy to estimate radius of fluorescence probe because its shape
affects the molecular structure. Therefore, fluorescence photo-bleaching recovery
(FPR) is used for measurement of lateral diffusion of a molecule in membranes.
I VH
I VV
Excitation light
Sample
Monochrometer
Polarizer(vertical(V))
Monochrometer
Polarizer
(vertical(V)/horizontal(H))
Measured fluorescence intensity
G
I
I
G
I
I
r
VH
VV
VH
VV
⋅
+
⋅
−
=
2
HH
VV
I
I
G =
Fig. 3.16 Arrangement of optical system for measurement of fluorescence polarization
anisotropy, r
Polarization light ((I VV ) and fluorescence intensity of horizontal polarized light (I VH ) are measured.
And r is calculated by above equation. G is instrument constant for correction of sensitivity
difference between vertical polarized light and horizontal polarized light
46
3 Methods for Physical Properties of Biomembranes and Cells
is shown in Perrin-Weber equation,
1
r ¼
1
r 0 1þ
3τ
ρ
ð Þ
where r 0 , and ρ are r values of
fluorescence life time and rotational correlation time of molecule at 0 K, respectively. If fluorescence lifetime is obtained by such as pulse excitation method,
rotational correlation time ρ can be calculated using the above equation. By use of
Einstein’s relationship, ρ ¼
4πr
3
kT η where ρ, r, k, T and η are rotational correlation
time, radius of fluorescence probe, Boltzmann constant, absolute temperature and
local viscosity, respectively. And local viscosity is obtained by introducing rotational correlation time. Stokes-Einstein equation among diffusion constant, D,
viscosity of solvent, η and radius of fluoresce probe, R is shown as D ¼
kT
6πηR
where ρ, k, η, T and are rotational correlation time, Boltzmann constant, local
viscosity of solvent, absolute temperature and radius of fluorescence probe, respectively. Therefore, diffusion constant of fluorescence probe is obtained in above
procedure.
Fluorescence probe DPH (1,6-diphenyl-1,3,5-hexatriene) is widely used for
measurement of fluorescence polarization anisotropy (Fig. 3.17).
However, it is not easy to estimate radius of fluorescence probe because its shape
affects the molecular structure. Therefore, fluorescence photo-bleaching recovery
(FPR) is used for measurement of lateral diffusion of a molecule in membranes.
I VH
I VV
Excitation light
Sample
Monochrometer
Polarizer(vertical(V))
Monochrometer
Polarizer
(vertical(V)/horizontal(H))
Measured fluorescence intensity
G
I
I
G
I
I
r
VH
VV
VH
VV
⋅
+
⋅
−
=
2
HH
VV
I
I
G =
Fig. 3.16 Arrangement of optical system for measurement of fluorescence polarization
anisotropy, r
Polarization light ((I VV ) and fluorescence intensity of horizontal polarized light (I VH ) are measured.
And r is calculated by above equation. G is instrument constant for correction of sensitivity
difference between vertical polarized light and horizontal polarized light
46
3 Methods for Physical Properties of Biomembranes and Cells