Reflected light microscopy is primarily used to examine opaque specimens that are inaccessible to conventional transmitted light techniques. A material is considered opaque if a thin (polished or not) section about 25 micrometers in thickness is non-transparent in the visible light spectrum range between 450 and 650 nanometers.
Figure 1: An unidentified native metal occurs within galena which has complex intergrowths with chalcopyrite and sphalerite. A single crystal of tetrahedrite (green-blue, centre top left) is present. Polished thin section, plane polarized light, x 160, oil.
A typical microscope configured for both types of illumination is illustrated in Figure 2 (the transmitted light source and optical pathway is not shown in this illustration). The optical pathway for reflected light begins with illuminating rays originating in the lamp housing for reflected light (the upper housing in Figure 2). This light next passes through the collector lens and into the vertical illuminator where it is controlled by the aperture and field diaphragms. After passing through the vertical illuminator, the light is then reflected by a beamsplitter (a half mirror or elliptically shaped first-surface mirror) through the objective to illuminate the specimen. Light reflected from the surface of the specimen re-enters the objective and passes into the binocular head where it is directed either to the eyepieces or to a port for photomicrography.
Figure 2: Reflected light pathway.
In reflected light microscopy, absorption and diffraction of the incident light rays by the specimen often lead to readily discernible variations in the image, from black through various shades of gray, or color if the specimen is colored. Such specimens are known as amplitude specimens and may not require special contrast methods or treatment to make their details visible. Other specimens show so little difference in intensity and/or color that their feature details are extremely difficult to discern and distinguish in brightfield reflected light microscopy. The latter specimens behave much like the phase specimens so familiar in transmitted light work, and are suited for darkfield and reflected light differential interference contrast applications.
Reflected light objectives:
Objectives for reflected light can be recognized by the Epi or similar inscription on the decorative outer barrel (see Figure 3). They differ from objectives for transmitted light in two ways. Reflected light objectives feature lens surfaces that are particularly well coated with anti-reflection layers to prevent the illuminator light from being reflected towards the eyepiece. Such reflections would be superimposed on the image and have a disturbing effect. The second difference is that these objectives are designed and optically corrected for specimens lacking a coverslip. The vast majority of samples in the materials sciences (where reflected light microscopes are most heavily used) are usually viewed without a cover slip. Therefore, higher numerical aperture objectives require a different optical computation than do transmitted light objectives.
Figure 3: Objectives for reflected light.
References:
http://www.atlas-of-ore-minerals.com/plate27e.htm
http://zeiss-campus.magnet.fsu.edu/articles/basics/reflected.html
http://zeiss-campus.magnet.fsu.edu/articles/basics/reflectedcontrast.html