Page 78 - Fundamentals of Light Microscopy and Electronic Imaging

CHAPTER
5

DIFFRACTION AND INTERFERENCE
IN IMAGE FORMATION

OVERVIEW

This chapter deals with diffraction and interference in the light microscope—the key
principles that determine how a microscope forms an image. Having just concluded a
section on geometrical optics where image locations and foci are treated as points, lines,
and planes, it is surprising to learn that in the microscope the image of a point produced
by a lens is actually an extended spot surrounded by a series of rings and that a focal
plane is contained in a three-dimensional slab of finite thickness. These properties are
due to the diffraction of light (see Fig. 5-1). In the microscope, light from the illumina-
tor is diffracted (literally broken up in the sense of being scattered or spread) by the
specimen, collected by the objective lens, and focused in the image plane, where waves
constructively and destructively interfere to form a contrast image. The scattering of
light (diffraction) and its recombination (interference) are phenomena of physical optics
or wave optics. We study these processes, because they demonstrate how light, carrying
information from an object, is able to create an image in the focal plane of a lens. With
a working knowledge of diffraction, we understand why adjusting the condenser aper-
ture and using oil immersion techniques affect spatial resolution. Diffraction theory also
teaches us that there is an upper limit beyond which a lens cannot resolve fine spatial
features in an object. In studying diffraction, we see that complex optical phenomena
can be understood in mathematically precise and simple terms, and we come to appre-
ciate the microscope as a sophisticated optical instrument. Readers interested in the
physical optics of diffraction and interference of light can refer to the excellent texts by
Hecht (1998) and Pluta (1988).

DEFINING DIFFRACTION AND INTERFERENCE

Diffraction is the spreading of light that occurs when a beam of light interacts with an
object. Depending on the circumstances and type of specimen, diffracted light is per-
ceived in different ways. For example, when a beam of light is directed at the edge of an
object, light appears to bend around the object into its geometric shadow, a region not
directly illuminated by the beam (Fig. 5-2a). The situation reminds us of the behavior of 61