Chapter 1: Fundamentals of Light Microscopy

Abstract

In this chapter, we examine the optical design of the light microscope and review pro- cedures for adjusting the microscope and its illumination to obtain the best optical performance. 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 fi xed, but vary in their location depending on the focus position of the objective and condenser lenses. Therefore, an important fi rst step is to adjust the microscope and its illuminator for Koehler illumination, a method of illumination 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 fi lters, prisms, and diaphragms are located at precise physical loca- tions 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 procedure for determining magnifi cation, and measure the diameters of cells and nuclei in a tissue sample.