Page 184 - Fundamentals of Light Microscopy and Electronic Imaging

THE DIC OPTICAL SYSTEM 167

Finally, note that the intensity of shadows and highlights is greatest along the direc-
tion of the shear axis. If we examine the contrast at the edges of a spherical particle
along diameters taken at different azimuths, we observe that the contrast between the
particle and the background gradually decreases and reaches zero at 90° along a line
defining the axis of the interference fringe. At this position, the irregularities in profiles
of the O and E wavefronts are exactly aligned, so subtraction of the two wavefronts can-
cels out any retardations and gives a positive value that exactly matches that of the back-
ground.
The best amount of bias retardation is that giving optimum contrast to the object
image and is unique for each object. Since the field of view usually includes many phase
objects of different size and refractive index, the best overall bias setting is a compro-
mise. The following guidelines are useful in performing this adjustment:

• The amount of bias retardation required to maximally darken one slope or edge of
an object also gives the maximum possible contrast between the object and the
background. Thus, for any given object, there is an optimal amount of bias retarda-
tion that requires a particular prism setting.
• If a bias retardation is chosen that is greater than the minimum amount required, the
contrast will be reduced.
• Thick light-scattering objects may require a higher bias compensation setting in
order to obtain extinction of one edge (gradient slope) of the object.
• When the condenser aperture exceeds about 75% of the objective aperture, light scat-
tering in the optical system increases significantly and contrast becomes reduced.

The Use of Compensators in DIC Microscopy

Although the DIC microscope is largely a qualitative instrument, a compensator can be
used to manipulate the amount of bias retardation between O- and E-wave pairs more
precisely and give more control to adjusting the contrast of specimen details in the
image. The action of compensators as contrasting and measuring devices is described in
Chapter 9. The compensator is placed in a specially designated slot between the crossed
polars and introduces a known amount of retardation.
A full-wave plate or plate, such as the red-I plate with a retardation of 551 nm,
can be used to color the image by introducing a spectrum of interference colors. The col-
ors at the edges of objects and their immediate background can be compared using a
Michel Lèvy color chart to estimate the magnitude of the optical path difference.
The Sénarmont compensator contains a fixed /4 wave plate and a rotating ana-
lyzer, and is frequently used with DIC optics to introduce a known amount of bias retar-
dation to a specimen. This might be needed in certain semiquantitative applications or
simply as a monitor of DIC optical alignment. Allen (1985) and Inoué (1989) used the
technique to introduce a precise amount of retardation to optimize the contrast of micro-
tubules imaged by video-enhanced DIC microscopy. When using this technique, the /4
wave plate and analyzer (adjusted for extinction) are inserted into the optical path and
the objective DIC prism is adjusted to give extinction. The analyzer is then rotated to
give the desired amount of bias retardation and background intensity (for details on Sén-
armont compensation, refer to Chapter 9). The degrees of rotation can be noted for
future reference.