Page 114 - Fundamentals of Light Microscopy and Electronic Imaging

CHAPTER
7

PHASE CONTRAST MICROSCOPY
AND DARK-FIELD MICROSCOPY

OVERVIEW

Unstained objects such as cells present a unique problem for the light microscopist
because their images generate very little contrast and are essentially invisible in ordi-
nary bright-field microscopy. As we have seen, this is even true for transparent periodic
specimens such as diffraction gratings and diatoms. Although transparent objects induce
phase shifts to interacting beams of light due to scattering and diffraction, they remain
nearly invisible, because the eye cannot detect differences in phase. In this chapter we
examine two optical methods for viewing such objects: phase contrast microscopy,
which transforms differences in the relative phase of object waves to amplitude differ-
ences in the image; and dark-field microscopy, where image formation is based solely
on diffracted wave components. Phase contrast microscopy produces high-contrast
images of transparent specimens such as cells and micro-organisms, tissue slices, litho-
graphic patterns, and particles such as organelles. Living cells in tissue culture can also
be examined directly, without fixation and staining (Fig. 7-1).

PHASE CONTRAST MICROSCOPY

In the case of stained, histological preparations or specimens with naturally occurring
pigments, specific wavelengths are absorbed by dyes or pigments, allowing objects to
appear in color when illuminated with white light. With monochromatic illumination
using a color filter complementary to the color of the specimen—for example, a blue
object examined through a yellow filter—object rays are significantly reduced in ampli-
tude, resulting in a high-contrast image. Such objects are called amplitude objects
because they directly produce amplitude differences in the image that are detected by the
eye as differences in the intensity (Fig. 7-2). Although most transparent biological spec-
imens do not absorb light, they do diffract light and cause a phase shift in the rays of light
passing through them; thus, they are called phase objects (Fig. 7-2). The retardation
imparted to a plane-wave front is shown in Figure 7-3. Phase contrast microscopes fea-
ture an optical design that transforms differences in the phase of object-diffracted waves
to differences in the image, making objects appear as if they had been optically stained. 97