Biomedical Engineering Reference
In-Depth Information
excitation and emission wavelengths. For best results, the filter cube should be removed for
PolScope observations to avoid the polarization distortions caused by the dichromatic mirror.
For observing fluorescence, on the other hand, the PolScope analyzer should be removed
because it attenuates the fluorescence emission by at least 50%. To meet both requirements,
the PolScope analyzer can be mounted in an otherwise empty filter cube holder, which is
moved into the optical path as the fluorescence cube is moved out and vice versa.
If, however, the option of removing the fluorescence cube is unavailable, the cube can
remain in the optical path for observations with polarized light and the LC-PolScope.
In this case, the following points should be considered:
For the light source of the PolScope, choose a wavelength that is compatible with the
emission wavelength filter in the fluorescence cube. For example, fluorescein
isothiocyanate (FITC) requires excitation with blue light (485 nm) and fluoresces in
green. The FITC dichromatic beam splitter and a barrier filter passing green fluorescence
light with wavelength longer than 510 nm would be compatible with 546 nm light for
polarized light observations using a mercury arc burner for the transmission light path.
The light source for one imaging mode must be blocked while observing with the other
imaging mode.
Removing the polarization analyzer, while observing fluorescence, more than doubles
the fluorescence intensity.
The LC-PolScope must be calibrated with the fluorescence filter cube in place.
The polarization distortions caused by the dichromatic mirror are partially counteracted
by the calibrated settings of the universal compensator.
15.5 Polarized Light Imaging in Three Dimensions
In this last section, we briefly address the question of how the three-dimensional (3D) nature
of a specimen affects the images and measurements recorded with a polarizing microscope.
I consider two issues here: (a) the effect of optical anisotropy located in sections of the
specimen that are not in focus, and (b) the fact that anisotropy itself is a 3D property.
A polarizing microscope generates 2D images of 3D objects. Each image contains
information from all specimen planes simultaneously, from the in-focus plane and the
planes that are out of focus. The out-of-focus blur superimposes on the sharp details of
structures that are located in the in-focus plane. This characteristic also applies to the
measurement of birefringence of a particular structure in the image. In
Figure 15.9
, the
retardance image of a meiotic spindle clearly delineates the birefringence of the kinetochore
fibers (k-fibers) that extend between the chromosomes and the spindle poles. However, the
retardance of the k-fibers is superimposed on the retardance of the whole spindle, which
more than doubles the retardance value that is attributable to a k-fiber alone. Nevertheless,
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