Page 170 - Fundamentals of Light Microscopy and Electronic Imaging

CHAPTER
10

DIFFERENTIAL INTERFERENCE

CONTRAST (DIC) MICROSCOPY

AND MODULATION CONTRAST

MICROSCOPY

OVERVIEW

In Chapter 7 we examined phase contrast microscopy, an optical system that converts
optical path differences in a specimen to contrast differences in the object image. In this
chapter we examine two optical systems for viewing gradients in optical path lengths:
differential interference contrast (DIC) microscopy and modulation contrast microscopy
(MCM). The images produced by these systems have a distinctive, relieflike, shadow-
cast appearance—a property that makes the image appear deceptively three-dimensional
and real (Fig. 10-1). The techniques were introduced within the past few decades and
have gained in popularity to the point that they are now widely used for applications
demanding high resolution and contrast and for routine inspections of cultured cells.
Although the ability to detect minute details is similar to that of a phase contrast image,
there are no phase halos and there is the added benefit of being able to perform optical
sectioning. However, there is no similarity in how the images are made. The DIC micro-
scope uses dual-beam interference optics based on polarized light and two crystalline
beam-splitting devices called Wollaston prisms. The generation of contrast in modulation
contrast microscopy is based on oblique illumination and the placement of light-obscuring
stops partway across the aperture planes.

THE DIC OPTICAL SYSTEM

The DIC microscope employs a mode of dual-beam interference optics that transforms
local gradients in optical path length in an object into regions of contrast in the object
image. As will be recalled from Chapter 5 on diffraction, optical path length is the prod-
uct of the refractive index n and thickness t between two points on an optical path, and
is directly related to the transit time and the number of cycles of vibration exhibited by
a photon traveling between the two points.
In DIC microscopy the specimen is sampled by pairs of closely spaced rays (coher-
ent wave bundles) that are generated by a beam splitter. If the members of a ray pair tra-
verse a phase object in a region where there is a gradient in the refractive index or
thickness, or both, there will be an optical path difference between the two rays upon 153