Page 18 - Fundamentals of Light Microscopy and Electronic Imaging

CHAPTER
1

FUNDAMENTALS
OF LIGHT MICROSCOPY

OVERVIEW

In this chapter we examine the optical design of the light microscope and review proce-
dures for adjusting the microscope and its illumination to obtain the best optical per-
formance. The light microscope contains two distinct sets of interlaced focal
planes—eight planes in all—between the illuminator and the eye. All of these planes
play an important role in image formation. As we will see, some planes are not fixed, but
vary in their location depending on the focus position of the objective and condenser
lenses. Therefore, an important first step is to adjust the microscope and its illuminator
for Koehler illumination, a method introduced by August Koehler in 1893 that gives
bright, uniform illumination of the specimen and simultaneously positions the sets of
image and diffraction planes at their proper locations. We will refer to these locations
frequently throughout the book. Indeed, microscope manufacturers build microscopes
so that filters, prisms, and diaphragms are located at precise physical locations in the
microscope body, assuming that certain focal planes will be precisely located after the
user has adjusted the microscope for Koehler illumination. Finally, we will practice
adjusting the microscope for examining a stained histological specimen, review the pro-
cedure for determining magnification, and measure the diameters of cells and nuclei in
a tissue sample.

OPTICAL COMPONENTS OF THE LIGHT MICROSCOPE

A compound light microscope is an optical instrument that uses visible light to produce
a magnified image of an object (or specimen) that is projected onto the retina of the eye
or onto an imaging device. The word compound refers to the fact that two lenses, the
objective lens and the eyepiece (or ocular), work together to produce the final magnifi-
cation M of the image such that

M M M .
final obj oc
1