Page 182 - Fundamentals of Light Microscopy and Electronic Imaging

THE DIC OPTICAL SYSTEM 165

well defined or is misoriented, the condenser prism may need to be rotated to
optimize the alignment. Usually this adjustment is made by the manufacturer
and remains fixed (or can be locked down with a set screw), so it does not need
to be altered. If it is not properly displayed, contact the manufacturer and get
instructions on how to reset the alignment. The image field as seen through the
eyepieces again looks bright and featureless.
4. Mount and focus a specimen (such as buccal epithelial cells) on a slide and set
Koehler illumination with both DIC prisms and polarizers in position with the
optics set at extinction. The image field looks very dark gray at extinction, while
a sharply defined interference fringe or band is seen running in a northeast-
southwest direction across the diameter of each refractile object particle (Fig.
10-6, top). The orientation of the interference fringe is shifted by 90° compared
with the fringe orientation seen in aperture views of the individual condenser
and objective DIC prisms already described, and is the correct orientation for the
fringe in the image at extinction. While viewing the image, note that advancing
the objective DIC prism (or rotating the analyzer a few degrees to either side of
the extinction position) moves the interference fringe bisecting particles or
organelles along an axis oriented in a northwest to southeast direction (the shear
axis), causing one side of the organelle to look dark and the opposite side to look
bright. For a given microscope, only one of these elements is adjusted (prism or
polarizer or analyzer) to introduce bias retardation. Adjusting the bias retarda-
tion brightens the background, improves image appearance and contrast, and is
an essential final step in the adjustment of DIC optics. In addition to the presence
of discrete light and dark intensities at opposite edges of each organelle along
the shear axis, a broad and indistinct field fringe is sometimes observed, a gra-
dient of light across the entire field of view. With well-designed optics, the field
fringe is so broad that the entire image background appears a uniform medium
gray. More commonly, some evidence of the fringe remains, so after introducing
bias retardation (by adjusting the DIC prism or rotating the analyzer), the field
exhibits a shallow gradient of light intensity from one edge to the other.
5. When the back aperture of the objective is examined with an eyepiece telescope
with the DIC prism set at extinction, the central region should look dark gray and
uniform, but possibly with some brightening at four quadrants at the periphery,
giving the appearance of a very broad extinction cross similar to the one observed
in the back aperture of a polarizing microscope. The brightening represents an
artifact due to partial depolarization of light at lens elements of the condenser and
objective. Image contrast can be greatly improved if these regions are masked out
by partly closing down the condenser aperture, leaving 75% of the aperture diam-
eter clear. If the optics are in perfect adjustment at extinction, the cross stands
upright and is seen to be composed of two broad interference fringes, each bent
in the shape of a right angle and meeting in the center of the aperture. On most
microscopes the fringe pattern can be adjusted to make the central region of the
aperture darker and more uniform. This is done by loosening and rotating the con-
denser DIC prism a small amount, or by slightly rotating the polarizer or analyzer.
Now secure the components, leaving only one for adjusting bias retardation. As a
final adjustment and check, move the condenser focus very slightly out of the
Koehler position to determine if extinction at the back aperture can be improved
still further. However, too great a movement will bring the conjugate interference
planes of the prisms too far apart and degrade optical performance.