Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 315

￿ If cells have to be kept in plastic Petri dishes during observations, only use dishes with

glass coverslip bottoms. The strong and random birefringence of a plastic bottom ruins
the polarization of the light.

￿ Prepare specimens such that cells or structures of interest are as close to the coverslip as

possible. When using high NA ( . 0.5) oil immersion or dry optics, a layer of more than
10 m of aqueous medium introduces enough spherical aberration to noticeably reduce

the resolution of cell images. The use of water-immersion optics alleviates this problem.

μ

￿ For observations of structures near the cell surface, it might be necessary to increase the

medium's refractive index to match the refractive index of the cytoplasm and reduce
the effect of edge birefringence [28] . Adding polyethyleneglycol, polyvinylpyrrolidone,
or some other harmless polymer or protein substitute to the medium will increase its
refractive index to match that of the cytoplasm inside the cell. The concentration of
substitute required might vary from a few percent to over 10%, depending on the
average refractive index of the cytoplasm of the cells of interest. It is best to test
various media containing different polymer concentrations by suspending cells in them
and examining the refractive index mismatch in a slide and coverslip preparation with
DIC or polarized light. At the optimal polymer concentration, no distinct cell boundary
is visible, giving the impression that the organelles and cytoskeleton are freely
suspended. After determining the optimal concentration for imaging, the medium should
be tested for compatibility with growing cells. Many cells grow and develop normally
in media with inert additives. The molecular weight of the polymer needs to be around
40 kD or more to prevent its uptake into cells.

￿ For observations with the LC-PolScope, prepare the specimen so that when mounted in

the microscope, at least one clear area without cells or birefringent material can be
identified and moved into the viewing field when required. A clear area is needed for
calibrating the PolScope and for recording background images. The background images
are used to remove spurious background retardance from specimen images, allowing
precise measurement and imaging of the cells and structures of interest. Many cell
cultures and cell-free systems can be prepared without special attention to this
requirement. For example, a free area can often be found around sparsely plated cells.
Cultures of free-swimming cells might have to be diluted before mounting a small drop
of the suspension between slide and coverslip to observe free areas. Sometimes it is
helpful to add a tiny drop of oil or other nontoxic, immiscible liquid to the preparation.
This clear drop can provide an area for calibrating the instrument and taking
background images in a preparation that is otherwise dense with birefringent structures.

15.4.3 Combining Polarized Light with DIC and Fluorescence Imaging

Polarization and DIC microscopy both rely on the use of polarized light for creating contrast
in images of phase objects. While polarized light imaging highlights changes of refractive