Page 176 - Fundamentals of Light Microscopy and Electronic Imaging

THE DIC OPTICAL SYSTEM 159

Formation of the DIC Image

Both ray tracing and wave optics are useful for explaining image formation in the DIC
microscope. By tracing the trajectories of rays from polarizer to image plane, we
observe the actions of optical components and understand the DIC microscope as a
double-beam interference device. By examining the form and behavior of wavefronts,
we also come to appreciate that optimal image definition and contrast are affected by the
amount of phase displacement (bias retardation) between the two wavefronts introduced
by the operator. (Note, however, that properly adjusted DIC equipment can only ensure
good visibility of details that are within the resolution limit of the optics of the bright-
field microscope.) Although complex, these details will help you to understand where
essential actions occur along the microscope’s optical path, allow you to align and trou-
bleshoot the optics, and help you to use the microscope effectively. Before proceeding,
keep in mind that the potential spatial resolution is limited, but not guaranteed, by the
NA of the objective and condenser lenses.
Ray tracing of the optical pathway shows that an incident ray of linearly polarized
light is split by the condenser prism into a pair of O and E rays that are separated by a
small distance (Figs. 10-3, 10-4, and 10-5). The E vectors of the two rays vibrate in
mutually perpendicular planes. Between the condenser and objective lenses, the trajec-
tories of the ray pair remain parallel to one another and are separated by 0.2–2 m—the
shear distance—which is as small or smaller than the spatial resolution of the micro-
scope objective being employed. In fact, as the shear distance is reduced, resolution
improves, although at some expense to contrast, until the shear distance is about one-
half the objective’s maximum resolution. Thus, every point in the specimen is sampled
by pairs of beams that provide dual-beam interference in the image plane. Notice that
there is no universal reference wave generated by an annulus and manipulated by a
phase plate as in phase microscopy, where the distance separating the object and back-
ground rays can be on the order of millimeters in the objective back aperture.
In the absence of a specimen, the coherent O and E waves of each ray pair subtend
the same optical path length between the object and the image; the objective prism
recombines the two waves, generating waves of linearly polarized light whose electric
field vectors vibrate in the same plane as the transmission axis of the polarizer; the
resultant rays are therefore blocked by the analyzer and the image background looks
black, a condition called extinction (Fig. 10-5a, b). Thus, the beam-splitting activity of
the condenser prism is exactly matched and undone by the beam-recombing action
of the objective prism. Note that the axes of beam splitting and beam recombination of
both DIC prisms are parallel to each other and fixed at a 45° angle with respect to the
transmission axes of the crossed polarizer and analyzer. This axis is called the shear axis
because it defines the axis of lateral displacement of the O and E wavefronts at the spec-
imen and at all locations between the specimen and the image.
If, however, the O- and E-ray pair encounters a phase gradient in an object, the two
beams will have different optical paths and become differentially shifted in phase. We
treat the situation the same as we do in standard light microscopy: Waves emanating
from the same object particle in the specimen meet at their conjugate location in the
image plane, with the difference that the waves must first pass through the objective DIC
prism and analyzer. These waves emerge from the prism as elliptically polarized light
(Fig. 10-5c). The E vector of the resultant ray is not planar, but sweeps out an elliptical
pathway in three-dimensional space. These rays partially pass through the analyzer,
resulting in a linearly polarized component with a finite amplitude. This information