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CTS Home > Laboratory Exercises > Microscope Use

Use of the Light Microscope

It is essential that you familiarize yourself with the operation of the light microscope provided to you in order to maximize your success in the laboratory. Please systematically work your way through the material presented here. Additional information on the light microscope is provided in the course textbook [Gardner and Hiatt, p. 3]. If you have any questions about your understanding of this material or the use of any of the components of your microscope, please seek assistance from one of the lab instructors. Your understanding and proper use of the light microscope is critical to success in the laboratory component of this course.

Locate and handle the various parts of your microscope and be sure that you know their functions. In particular, make sure that you can locate and use the field diaphragm, the condenser diaphragm, the condenser centering knobs, stage controls, and the condenser focusing knob.

Each time you use the microscope, you should set it up for proper ("Koehler") illumination. You will also need to adjust the eyepieces for comfortable viewing with both eyes.

Use the following steps to adjust the eyepieces, establish Koehler illumination, and acquaint yourself with use of the several objectives:

  1. Always move the microscope with two hands; you will have to tip the microscope to remove it from the cabinet. You may have to attach the black power cord (found behind the scope in the cabinet). Set the microscope on the counter top with its single foot and the binocular viewing body towards you. Turn on the lamp with the amber switch in the right rear foot and adjust its intensity with the black circular knob in the base (on the left). Place any slide from your set on the microscope stage. Swing the 4x (scanning) objective into the optical axis, focus the image of the slide using the focus knobs (labeled "1" in diagram), and then switch to the 10x objective and refocus.

  2. Match the eyepieces of the microscope to the distance between your eyes by rotating the two parts of the binocular body inward or outward. Compensate for any difference in visual acuity between your two eyes by first blocking vision through the eyepiece with the knurled ring and focusing the microscope objective through the other eyepiece. Then, without changing the focus of the microscope objective, look at the slide through the knurl-ringed eyepiece and obtain the sharpest image by rotating the ring on that eyepiece. You may need to repeat these adjustments each time you use the microscope.

  3. Now, close the FIELD diaphragm of the microscope (labeled as #4 in the diagram, large knurled ring around the light exit window in the microscope base) and focus the condenser by turning the black knurled knob on the left side of the microscope until the smallest circle of light is observed and the edge of the field diaphragm is in sharpest focus. This will also produce the least colorful range of the fringes around the diaphragm image.

  4. Open your field diaphragm until it approaches the edge of the visual field and center it by using the two small knurled screws on the front sides of the condenser (# 6 in the diagram). Then open the field diaphragm until the entire field of view is evenly illuminated.

  5. Next, fully open the APERTURE diaphragm on the condenser (large knurled ring in plane of item 7 in the diagram) so that the maximum amount of light comes through.  Now close this diaphragm slowly until you can just detect an effect upon the visual field. This should provide the proper aperture adjustment for this lens. Note that you should NOT attempt to control the brightness of the illumination with the condenser aperture diaphragm; you may use it, however, to change contrast in the image. Closing the condenser aperture diaphragm will eliminate stray light and increase the contrast in the image. Overall brightness should be controlled by adjusting the light rheostat, using the black circular knob within the left side of the base.

    These several steps to focus the condenser and adjust the field and aperture diaphragms will produce light best adjusted for proper microscope use, identified as "Koehler illumination".

  6. Change to your high-dry (40x) lens and readjust both field and aperture diaphragms as necessary to reproduce Koehler illumination with this objective. As you will observe, when the objective is changed, reestablishment of proper illumination requires slight changes in field and aperture diaphragms as well.

  7. Now practice use of the oil immersion (100x) lens. Turn the objective nosepiece half-way between the high-dry and oil immersion lenses and STOP. Place a small drop of immersion oil on the slide over the spot of light from the condenser. Swing the oil immersion lens into the oil droplet, note that the lens "picks up" the drop of oil, and focus the lens on the slide. This lens cannot be focused sharply unless its tip is immersed in oil. Be careful to avoid getting oil by mistake on any of the other objectives, especially the high-dry (40x) lens, and do not clean any of the lenses with the solvent in the brown glass bottles.

  8. When you have finished viewing with the 100x lens, lower the stage to break the connection with the oil droplet and gently but thoroughly wipe all oil from the lens and the stage using lens paper. The same cleaning procedure can be used if you accidentally get immersion oil on any other objective lens. Lenses can be irreparably damaged if significant amounts of oil are inadvertently left on them after use. Clean the slide with lens paper and the solvent provided in the brown glass bottles. Use the 100X oil immersion lens sparingly.

Summary: Whenever you first use the microscope or when you change objectives, do a realignment of the microscope: 1) focus on the specimen, 2) close the field diaphragm, 3) adjust position of condenser lens to obtain a sharp image of the field diaphragm, 4) open the field diaphragm, 5) adjust contrast using the condenser aperture diaphragm.