Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS
10/2014 V_01 000000-2071-464 527
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 the LSM 710 and LSM 780 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 the LSM 710 and LSM 780 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 .