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:
- 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.
- 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.
- 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
- 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
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".
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.
use of the
and oil immersion
lenses and STOP.
Place a small
oil on the
the oil droplet,
the lens "picks
up" the drop
of oil, and
lens on the
its tip is
in oil. Be
oil by mistake
on any of
and do not
of the lenses
- 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.
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.