Optical Microscopy

binocular microscopes with image-erecting prisms, and the first stereomicroscope (14). Early in the twentieth century, microscope manufacturers began parfocalizing objectives, allowing the image to remain in focus when the microscopist exchanged objectives on the rotating nosepiece. In 1824, Zeiss introduced a LeChatelier-style metallograph with infinitycorrected optics, but this method of correction would not see widespread application for another 60 years. Shortly before World War II, Zeiss created several prototype phase contrast microscopes based on optical principles advanced by Frits Zernike. Several years later the same microscopes were modified to produce the first time-lapse cinematography of cell division photographed with phase contrast optics (14). This contrast-enhancing technique did not become universally recognized until the 1950s and is still a method of choice for many cell biologists today. Physicist Georges Nomarski introduced improvements in Wollaston prism design for another powerful contrast-generating microscopy theory in 1955 (15). This technique is commonly referred to as Nomarski interference or differential interference contrast (DIC) microscopy and, along with phase contrast, has allowed scientists to explore many new arenas in biology using living cells or unstained tissues. Robert Hoffman (16) introduced another method of increasing contrast in living material by taking advantage of phase gradients near cell membranes. This technique is now termed Hoffman Modulation Contrast, and is available as optional equipment on most modern microscopes. The majority of microscopes manufactured around the world had fixed mechanical tube lengths (ranging from 160 to 210 millimeters) until the late 1980s, when manufacturers largely migrated to infinity-corrected optics. Ray paths through both finite tube length and infinity-corrected microscopes are illustrated in Figure 1. The upper portion of the figure contains the essential optical elements and ray traces defining the optical train Introduction