ZEISS
Center Screen Area / Image Containers - Display and Image Analysis
ELYRA 7
408
000000-2262-999
03/2019 V_02
6.20
Polarization Imaging
Linear (plane) polarized light, which is light whose wave goes only one direction, exciting a fluorescent
molecule with a preferred dipole orientation results in polarized emitted light. It provides a contrast-
enhancing method that is especially useful in the study of molecules that are fixed in their orientation or
are greatly restricted in their rotational diffusion. Anisotropy is directly related to polarization and is
defined as the ratio of the polarized light component intensity to the total light intensity.
In polarization microscopy using laser WF systems the sample is irradiated with vertical polarized light (in
respect to the optical table) from a laser source. The emitted fluorescence is passed sequentially through
emission polarizers (analyzers) that are housed in the
Notch filter cascade
and that transmit either the
vertical (I
VV
) or horizontal (I
VH
) polarized emitted light onto the Quasar detector (L format fluorescence
polarization). Since the vertical component of the emission light is parallel polarized to the vertical
polarized excitation light, it is often also referred to as the parallel component (I
p
, I
||
). Likewise, the
horizontal polarized emission light is also designated as the perpendicular ("senkrecht" in German)
component (I
S
, I
|
). In the Software the "p" and "s" designations are used.
Polarisation
P
and anisotropy
r
are defined as:
s
p
s
p
I
I
I
I
P
+
−
=
and
s
p
s
p
I
I
I
I
r
⋅
+
−
=
2
with 0
≤
r
≤
1.
Hence they can be interconverted to each other in the following way:
r
r
P
+
⋅
=
2
3
and
P
P
r
+
⋅
=
3
2
.
In a completely polarized sample (I
S
=0) the anisotropy r=1. In a completely non-polarized sample (I
s
=I
p
)
anisotropy r=0.
The formulas for polarization
P
and anisotropy
r
as given above are strictly true only, if the optical
transmission for both emission polarizers are identical. Any differences must be corrected by introducing
a correction factor
G
that is multiplied with
I
p
. Hence the anisotropy
r
in such a case would be calculated
according to:
s
p
s
p
I
G
I
I
G
I
r
⋅
⋅
+
⋅
−
=
2
G
can be measured using horizontally polarized excitation light and is defined as
HH
HV
I
I
G
=
.
However, since in laser WF systems the polarization of the excitation light can not be changed easily from
vertical to horizontal, G has to be determined with an isotropic fluorescent dye solution as the ratio
between the mean intensities I
p
and I
s
, e.g. obtained from the histogram view at the image container.
Please note that the G factor is not the mean intensity of the ratio (
R
) channel, where every pixel is
computed seperately. It has to be calculated from the ratio of the mean intrensities of the I
p
and I
s
images.
As the formulas implies:
Anisotropy
r
is the preferential display as anisotropy of single species will be simply additive. Note that
the ZEN Software provides for a formula to display the anisotropy directly in ratio imaging. Images could
also be computed in the
Processing
tool`s
Calculator
.
For Laser WF polarization imaging, a dual camera setup with a polarizing beam splitter is required.