Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers. Much like the fluorescence microscope, in reflected brightfield microscopy the sample is illuminated from above through the objective. The Köhler illumination principle applies in cases where the objective with its pupil plane is also utilized as the front lens of the condenser. This interactive tutorial explores the optical pathways in a typical reflected light microscope.
The tutorial initializes with a labeled cut-away diagram of a typical reflected light microscope appearing in the window. In order to operate the tutorial, use the Field Diaphragm and Aperture Diaphragm sliders to open and close these iris apertures and observe the effects on the light beam traversing the optical train. The amount of light produced by the lamphouse can be adjusted using the Illumination Intensity slider.
Today, many microscope manufacturers offer advanced models that permit the user to alternate or simultaneously conduct investigations using both vertical and transmitted illumination. The optical pathway for reflected light begins with illuminating rays originating in the lamp housing for reflected light. 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, passes through the beamsplitter again and enters the binocular head where it is directed either to the eyepieces or to a port for photomicrography. Reflected light microscopy is frequently the domain of industrial applications, especially in the rapidly growing semiconductor arena, and thus represents a most important segment of microscopical studies.
A typical upright compound reflected light microscope has two eyepiece viewing tubes and often a trinocular tube head for mounting a digital or video camera system. Standard equipment eyepieces are usually of 10x magnification, and most microscopes are equipped with a nosepiece capable of holding four to six objectives. The stage is mechanically controlled with a specimen holder that can be translated in the x- and y- directions and the entire stage unit is capable of precise up and down movement with a coarse and fine focusing mechanism. Built-in light sources range from 20 and 100 watt tungsten-halogen bulbs to higher energy mercury vapor or xenon lamps that are used in fluorescence microscopy. Light passes from the lamphouse through a vertical illuminator, which is either integrated into the stand or interposed above the nosepiece but below the underside of the viewing tube head. The specimen's top surface is upright (usually without a coverslip) on the stage facing the objective, which has been rotated into the microscope's optical axis. The vertical illuminator is horizontally oriented at a 90-degree angle to the optical axis of the microscope and parallel to the table top, with the lamp housing attached to the back of the illuminator. The coarse and fine adjustment knobs raise or lower the stage in large or small increments to bring the specimen into sharp focus.
Inverted reflected light microscope stands incorporate the vertical illuminator within the body of the microscope. Many types of objectives can be used with inverted reflected light microscopes, and all modes of reflected light illumination may be possible: brightfield, darkfield, polarized light, differential interference contrast, and fluorescence. Some of the instruments include a magnification changer for zooming in on the image, contrast filters, and a variety of reticules. Because an inverted microscope is a favorite instrument for metallographers, it is often referred to as a metallograph. Manufacturers are largely migrating to using infinity-corrected optics in reflected light microscopes, but there are still thousands of fixed tube length microscopes in use with objectives corrected for a tube length between 160 and 210 millimeters.
On the inverted stand (similar in basic construction to the inverted tissue culture style microscope frames commonly employed in biology), the specimen is placed on the stage with its surface of interest facing downward. The primary advantage of this design is that samples can be easily examined when they are far too large to fit into the confines of an upright microscope (such as large rock samples and industrial materials). Also, only the side of the specimen facing the objectives need be perfectly flat. The objectives are mounted on a nosepiece under the stage with their front lenses facing upward towards the specimen and focusing is accomplished either by moving the nosepiece or the entire stage up and down.
In the vertical illuminator, light travels from the light source, usually a 12 volt 50 or 100 watt tungsten-halogen lamp, passes through collector lenses, through the variable aperture iris diaphragm opening and through the opening of a variable and centerable pre-focused field iris diaphragm. The light then strikes a partially silvered plane glass reflector, or strikes a fully silvered periphery of a mirror with elliptical opening for darkfield illumination. The plane glass reflector is partially silvered on the glass side facing the light source and anti-reflection coated on the glass side facing the observation tube in brightfield reflected illumination. Light is thus deflected downward into the objective. The mirror is tilted at an angle of 45 degrees to the path of the light travelling along the vertical illuminator.
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.
Contributing Authors
Rudi Rottenfusser - Zeiss Microscopy Consultant, 46 Landfall, Falmouth, Massachusetts, 02540.
Sunita Martini and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.