Page 116 - Fundamentals of Light Microscopy and Electronic Imaging

PHASE CONTRAST MICROSCOPY 99

Reference
a)
wave

Amplitude Reduced
b)
object amplitude
Phase Retarded
c)
object phase

Figure 7-2
Effects of amplitude and phase objects on the waveform of light. (a) Reference ray with
characteristic amplitude, wavelength, and phase. (b) A pure amplitude object absorbs energy
and reduces the amplitude, but does not alter the phase, of an emergent ray. (c) A pure
phase object alters velocity and shifts the phase, but not the amplitude, of an emergent ray.

In the 1930s, Frits Zernike, a Dutch physicist at the University of Groningen, created
an optical design that could transform differences in phase to differences in amplitude.
The development of phase contrast optics is a brilliant example of how basic research in
theoretical optics led to a practical solution for viewing unstained transparent objects in
the light microscope. The Zeiss optical works in Jena introduced phase contrast objec-
tives and accessories in 1942, which transformed research in biology and medicine. For
his invention and theory of image formation, Zernike won the Nobel prize in physics in
1953 (Fig. 7-4). Excellent descriptions of the technique are found in Bennett et al. (1951),
Françon (1961), Slayter (1976), Slayter and Slayter (1992), and Pluta (1989).
In this chapter we first examine the process of image formation using the terminol-
ogy and diagrams that are commonly employed to explain phase contrast optics. Then
we examine the relative contributions of diffraction and differences in optical path
length in generating the phase contrast image.

The Behavior of Waves from Phase Objects
in Bright-Field Microscopy

WAVE TERMINOLOGY AND THE IMPORTANCE OF COHERENCE
Upon transit through a phase object, an incident wavefront of an illuminating beam
becomes divided into two components: (1) an undeviated (0th-order) wave or surround
wave (S wave) that passes through the specimen, but does not interact with it, and (2) a

Wavefront of
Object
transmitted light

Figure 7-3
Disturbance by a phase object to an incident planar wavefront.