Reflected light microscopy

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.

Open

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.

Open

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.

Darkfield reflected light microscopy:

Darkfield reflected light illumination is especially useful for revealing fissures, pores, and grain boundaries in semi-opaque specimens. In darkfield illumination, only the wavefronts arising to stray reflection by structural elements in the specimen are able to enter the objective. Those wavefronts reflected by surface elements positioned perpendicular to the microscope optical axis do not reach the objective and these areas remain dark. The instrument configuration features an opaque occluding disk that is placed into the light path in the vertical illuminator so that only the peripheral wavefronts reach the deflecting mirror above the objective. Waves reflected by the mirror pass through a hollow collar surrounding the objective and serve to illuminate the specimen at highly oblique angles.

Polarized reflected light microscopy:

Polarized reflected light microscopy is a technique that is suitable for examining surfaces containing structures that alter the state of polarization during the reflection process. For example, structural grains in ore samples and a number of metallic alloys and thin films can be readily examined using this method. The linearly polarized light waves are focused onto the specimen surface and reflected back into the objective. After leaving the objective aperture as a parallel bundle of wavefronts, the light is then projected onto a second polarizer (the analyzer) oriented at 90 degrees with respect to the polarizer. Only depolarized or shifted wavefronts are able to pass through the analyzer to reach the tube lens. An auxiliary lambda plate can also be inserted just prior to the analyzer in the optical train to determine the sign of birefringence or to add color (changing gray to color contrast). In cases where objectives of very low magnification are used in reflected polarized light, a rotatable optical plate (termed an Antiflex cap) consisting of a one-quarter wavelength lambda plate is placed on the objective front lens element to block reflections from the objective itself.

DIfferential Interference Contrast (DIC) reflected light microscopy:

In the optical configuration, a birefringent prism (also known as a Wollaston or Nomarski prism, depending upon design) is placed in the infinity space just above the objective and a polarizer is installed in the vertical illuminator (similar to polarized light). The prism splits the polarized light wavefronts into two orthogonal polarized beams, oriented at 45 degrees in reference to the polarizer, on their way to the specimen. These perpendicular light beams impact the specimen to create a lateral displacement in regions where surface relief exists. If the surface is completely flat, no features are observed. However, if there is, for example, a small step between the two wavefronts, one of the beams must travel a path that is longer and is assigned this path difference. Once the parallel beams have returned to the microscope after passing back through the objective and prism, they pass through a second polarizer (the analyzer) where interference produces an intermediate image where path differences are translated into gray values that can be seen by the eye. Similar to polarized light microscopy, a lambda plate can be positioned beneath the analyzer to shift gray values into colored hues.

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