horiba FluoroMax-3, Operation Manual

Page: 137 / 220

FluoroMax-3 v. 3.1 (6 Mar 2006) Automated Polarizers
10-3
r
P
P
=
−
2
3
(7)
For single-photon excitation, the allowed values for the emission anisotropy are gov-
erned by:
r P
=
0 4
2
. (cos )
α
(8)
where P x
x
2
2
3 1
2
( )
=
−
is the second Legendre polynomial, and α is the angle between
the molecule’s absorption and emission dipoles. The angle α may vary from 0 to 90°.
Thus the allowed values for < r > and P are:
Parameter
α = 0° α = 90°
P +0.5 0.333
< r > +0.4 0.2
Values outside of this range indicate scattered light is present in the measurement of
<
r >. If the sample is excited with depolarized light—a less common technique—the
measured value of P ranges from –1/7 to +1/3 (and < r > from –1/11 to +1/4). The indi-
vidual intensity components ( I
HH
, I
HV
, I
VH
, I
VV
) are also referred to as raw polarization .
Experimentalists often multiply polarization units by 1000 to yield
millipolarization
units, m P , for very small changes in the polarization.
Polarization geometries
Polarization measurements are taken in two basic geometries:
•
L-format uses two polarizers, which are both rotated between horizontal and verti-
cal positions for measurements. If the
G factor is determined beforehand, only two
measurements are required: the VV and VH components, found by rotating only the
emission polarizer. The FluoroMax
®
-3 uses the L-format geometry.
•
T-format uses one excitation and two emission polarizers. The excitation polarizer
is rotated between horizontal and vertical for measurements, while the emission po-
larizers are fixed—one horizontal and the other vertical. If the
G factor is deter-
mined beforehand, it is possible to obtain the anisotropy or polarization in one
measurement cycle, for the VV and VH components are available simultaneously
on the two emission detectors. The
G factor is measured differently in the T-format
technique.
A schematic diagram of the L-format geometry is shown on the next page: