Page 32 - Fundamentals of Light Microscopy and Electronic Imaging

CHAPTER
2

LIGHT AND COLOR

OVERVIEW

In this chapter we review the nature and action of light as a probe to examine objects in
the light microscope. Knowledge of the wave nature of light is essential for understand-
ing the physical basis of color, polarization, diffraction, image formation, and many
other topics covered in this book. The eye-brain visual system is responsible for the
detection of light including the perception of color and differences in light intensity that
we recognize as contrast. The eye is also a remarkably designed detector in an optical
sense—the spacing of photoreceptor cells in the retina perfectly matches the require-
ment for resolving the finest image details formed by its lens (Fig. 2-1). Knowledge of
the properties of light is important in selecting filters and objectives, interpreting colors,
performing low-light imaging, and many other tasks.

LIGHT AS A PROBE OF MATTER

It is useful to think of light as a probe that can be used to determine the structure of
objects viewed under a microscope. Generally, probes must have size dimensions that
are similar to or smaller than the structures being examined. Fingers are excellent
probes for determining the size and shape of keys on a computer keyboard, but fail in
resolving wiring patterns on a computer’s integrated circuit chip. Similarly, waves of
light are effective in resolving object details with dimensions similar to the wavelength
of light, but generally do not do well in resolving molecular and atomic structures that
are much smaller than the wavelength. For example, details as small as 0.2 m can be
resolved visually in a microscope with an oil immersion objective. As an approximation,
the resolution limit of the light microscope with an oil immersion objective is about one-
half of the wavelength of the light employed.
Visible light, the agent used as the analytic probe in light microscopy, is a form of
energy called electromagnetic radiation. This energy is contained in discrete units or
quanta called photons that have the properties of both particles and waves. Photons as
electromagnetic waves exhibit oscillating electric and magnetic fields, designated E and
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