Microscope Activities, 16: Darkfield Illumination

In the past, Hooke College of Applied Sciences offered a microscopy workshop for middle school and high school science teachers. We thought that these basic microscope techniques would be of interest not only for science teachers, but also for homeschoolers and amateur microscopists. The activities were originally designed for a Boreal/Motic monocular microscope, but the Discussion and Task sections are transferable to most microscopes. You may complete these 36 activities in consecutive order as presented in the original classroom workshop, or skip around to those you find interesting or helpful. We hope you will find these online microscope activities valuable.

EXPERIMENT 16: Darkfield Illumination

Goal

To make darkfield stops for the brightfield substage condenser, thereby converting the microscope for making observations by the oldest contrast method.

Level

Basic

Materials Needed

8½” x 11” acetate sheet protector; paper punch (~ ¼”)

Procedure

Using your 10X objective, focus on any specimen; say, a preparation of Radiolaria. Cut out a corner of an acetate sheet protector, so as to produce a piece approximately 1½” x 3”, which hinges on the 1½” slide. Remove the black paper insert, and punch out a ¼” diameter black circle. Place the ¼” black disc in between the two acetate layers, about ½” from the folded end, as illustrated in Figure (16-1).

Slip this “black dot sandwich” between the two ground glass discs at the lower end of the substage condenser, in such a way as to center the black disc in the light path. This is illustrated in Figure 16-2, but the lower ground glass has been temporarily swung out of the light path for clarity in showing the centralized location of the black disc, which is now properly referred to as a “darkfield stop.”

Next, it is vital that you OPEN THE SUBSTAGE APERTURE DIAPHRAGM FULLY!

Now, look at your specimen of Radiolaria again, and adjust the height of the substage condenser, while observing the specimen, until a good darkfield image is obtained.

Discussion

Darkfield—or, darkground, as it is sometime called—is the oldest contrast method in microscopy, going back about 350 years. It is called so because the diameter of the “stop” is selected so as to just block out all central, axial light going to the objective (thus darkening the background field of view), but allowing a cone of light from the periphery of the condenser to impinge on, and cross through, the plane of the specimen, without entering the objective. The sample is illuminated by highly oblique rays, and is seen lighted, but against a black background.

Darkfield was the most important technique for observing and studying bacteria at the end of the nineteenth century, because their small size and transparent, colorless nature made them impossible to see otherwise in the live state. There seems to be today an inexplicable neglect of this useful technique.

Dedicated darkfield condensers may be purchased from microscope manufacturers, but, with the exception of very high numerical aperture condensers, simple homemade stops, such as those made in this experiment, yield results that are at least equivalent to commercial units.

One has to realize, however, that a different diameter stop is required for each different numerical aperture. As higher numerical apertures involve higher angles of illumination, it stands to reason that the diameter of the darkfield stop must be increased with increased numerical aperture, in order to block out the central, axial rays. Strictly speaking, you should make three different diameter stops for your three objectives.

How do you know what diameter to make the stops? You could find a working diameter by trial and error. If the diameter is too small for a given numerical aperture, the field of view will not be entirely dark; there will be a ring of light at the periphery; if the stop is too large, it will give a darkfield effect, but light is lost that could have been used to illuminate the specimen. The ideal way to determine the diameter of the darkfield stop is to place a transparent metric rule in the plane where your stop will be placed, and then read the number of millimeters in your objective back focal plane. This elegant method does not work with your Boreal/Motic microscope because of the fixed ground glass above the plane of the stop.

Task

Make a darkfield stop for your 10X objective as described in the Procedure section, and view your slide preparation of Radiolaria. Try another stop, but his time cut out of black paper using scissors, and eyeballing the diameter. Does it work?

1. Close the aperture diaphragm completely while observing the sample, and explain the results.
2. Try using your other objective magnifications with the same stop.

Note that you could mount the darkfield stop on a glass or acetate disc, place it on the lower ground glass in the center, and then just swing it in and out, as needed.

A quick-and-dirty darkfield and/or oblique illumination can be achieved by opening the aperture diaphragm fully, and then deliberately sticking your little finger in the light path; the little finger in the light path can often be manipulated to get a darkfield or oblique illumination effect that will help elucidate a particular structure.

See Experiment 17, Rheinberg Illumination, for a color modification and extension of this contrast method.