Page 190 - Fundamentals of Light Microscopy and Electronic Imaging

MODULATION CONTRAST MICROSCOPY 173

Exercise: DIC Microscopy

• Review of procedures for aligning optical components for DIC microscopy.
In a sketch, indicate the proper sequence and orientation of the polarizer and
analyzer, as well as the condenser and objective DIC prisms. Indicate the pat-
tern and orientation of interference fringes seen with a Bertrand lens when
the microscope is set at extinction and when the condenser or objective DIC
prism has been removed.
• Examination of diatom shells by DIC microscopy. Examine the diatom test
plate at 100 with DIC optics and determine if the striae and pores in Frus-
tulia and Amphipleura can be resolved. Alternatively, examine the myofibrils
of striated muscle in the thorax flight muscles of a fruit fly, Drosophila.
Anesthetize one or two flies, remove the head and abdomen, and place the
thorax in a 30–50 L drop of water between a microscope slide and cover-
slip, and squash the thorax to disperse the muscle. Blot off excess water, dry,
and seal the edges of the coverslip with nail polish to prevent evaporation.
Allow to dry thoroughly (!), and clean the glass surfaces before examination.
To obtain the highest resolution and contrast for these demanding specimens,
oil both the objective and the condenser. Adjust the microscope for Koehler
illumination and use the Bertrand lens to check that the condenser diaphragm
masks some of the peripheral light, but does not significantly block the aper-
ture. Does the spacing you observe approach the theoretical Abbe limit for
resolution in the light microscope?
Since DIC microscopy uses polarized light, it can be used in combination
with a compensator such as a full-wave plate to examine interference colors
in phase objects. The high-NA optics available for DIC mean that the speci-
men can be examined at very high resolution and with optical sectioning.
Insert a red-I plate somewhere in the light path between the two crossed
polars to observe the effect of optical staining. Be sure you have the correct
orientation (axes of the refractive index ellipsoid of the plate at 45° to the
axes of the polarizers). Note the colors of the background and of the edge of
the shell and uniform portions of the shell itself using a 40 objective.
Notice that each edge of the specimen is composed of a double band of inter-
ference colors. On the opposite side of the diatom, the same double-color
band is observed, only in reversed order. The exact color observed depends
on the optical path difference intrinsic to the specimen and the bias retarda-
tion introduced by the DIC prism. Although the interference colors indicate
optical path differences in the specimen, the DIC microscope is primarily a
qualitative instrument.
• Examination of buccal epithelial cells. Buccal epithelial cells are excellent
specimens for practicing optical contrasting with the DIC prism and for
examining the effect of the condenser diaphragm on image contrast and opti-
cal sectioning. The cells contain a centrally placed nucleus and have a mod-
ified plasma membrane that preserves the angular edges of their original
polygonal shape in the epithelium, as well as patterns of multiple parallel
ridges and grooves that exhibit an interridge spacing of 0.4 m. Gently