Use and Care of Light Microscopes
Compound and dissecting light microscopes provide magnification ranges from the macroscopic to the limits
afforded by light—from a few times actual size to 1000×. These microscopes must be treated with the care
and respect that such precision instruments require. To get the most out of the microscopes, follow these
precautions and instructions for use.
Precautions
Avoid getting fluids on the microscopes. Wipe any dishes and slides that have specimens in fluid
with a towel before placing them on the microscopes. Never mount a slide on the compound
microscope unless its specimen is covered by a coverslip. Sea water is especially corrosive,
but other physiological fluids contain salts that can corrode microscope parts as well, and
organic solvents can erode cements that hold the lenses in place; some can corrode lenses or
their optical coatings. If spills do reach a microscope, wipe it dry as soon as possible; use only
lens tissue or cotton-tipped applicators on the lenses themselves. Before putting a microscope
away, blot up any water on it and then wipe it down with a second towel dampened with
fresh water and dry it.
Don’t touch the lenses. Occasional cleaning to remove oil or grease may be necessary; this should
be done only with special lens tissue or cotton-tipped applicators, in some cases dampened
with solvents. Because different microscopes have different cements holding their lenses, each
may need to be treated differently. Please consult the instructor before attempting cleaning.
Please do not loosen any set screws, such as the screw holding the head of the microscope in its
mounting ring. (Please do not turn the head to show lab partners views under the scope;
keep the microscope fully stationary and make them move to look in it.)
Compound microscope
Turning the illumination on and off should be done gradually by using the rheostat appropriately.
Before turning on the rocker switch, make sure the rheostat is turned all the way down to 0;
then turn on the rocker switch and dial the rheostat slowly up until some illumination can
be seen. Similarly, in turning the illuminator off, turn the rheostat down to 0 before turning
off the rocker switch. This gradual on-and-off is important for long life of the bulb. Do
not run the illuminator at full throttle (6) except briefly when making observations at high
magnification; this setting is higher than the voltage rating of the bulbs.
Bright-field Köhler illumination. Optimum resolution is obtained only with appropriate setting
of the lenses and apertures—specifically a condition called Köhler illumination in which the
conjugate field planes and conjugate aperture plans are properly aligned. This illumination
minimizes stray light that would degrade the image and balances resolution against contrast
to afford the sharpest possible image, especially for photography. Köhler illumination can be
set this way:
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1. Focus roughly on a specimen (starting with a low-magnification objective—i.e., the
4×) and then changing to at least the 10× objective. If the two eyepieces are not set
so that both of your eyes are focused on the specimen, adjust them by turning the
knurled knobs on them. (Most microscopes will have just one independently focusable
eyepiece; focus first on a small, crisp detail in the specimen with just the one eye
looking through the non-focusable eyepiece [e.g., tilt your head so that only your right
eye sees the specimen through the right eyepiece]; then look through just the other
eyepiece [e.g., tilt your head so that only your left eye sees the specimen through the
left eyepiece] and adjust the knob on it until the same detail can be seen clearly with
that eye. Make sure both eyes see the same field of view completely once the eyepieces
are adjusted; adjust the interocular distance as necessary to accomplish this.
Use of light microscopes
2. Check that the lamp filament is focused on the front aperture of the condenser. Close
that aperture fully and see that the image of the filament is projected onto it. If a
focusing knob is provided for the lamp, adjust to sharpen the image and center it on
the aperture.
3. Pick the lens (10× – 100×) that you will be using and focus on the specimen with
it. (Köhler illumination needs to be set independently for each objective lens, so you
will need to run through the following steps for whichever lens you use for critical
observation or photography before you start with that lens.)
4. Close down the field-stop diaphragm until you can see its edge as you look through
the eyepieces. Center the image of the edge (the diaphragm aperture) in the field of
view using the alignment screws on the condenser lens.
5. Focus the image of the edge of the field-stop diaphragm using the knob on the condenser lens and recenter it if necessary.
6. Expand the field-stop diaphragm until its aperture goes just beyond the edge of the
field of view, no further.
7. Adjust the condenser diaphragm: while looking at a specimen, open the diaphragm
fully, then close it down slowly until the brightness of the image just starts to dim. (If
an eyepiece telescope or Bertrand lens is available, view the condenser aperture with
it, and adjust the aperture so that it blocks a quarter of the width of the full width of
the aperture of the lens [i.e., when about 3 /4 of the maximum diameter of the aperture
remains illuminated]. In the absence of such visual aids, closing the condenser just
to that point at which the image first begins to dim accomplishes that quarter-width
closure.) With specimens of high contrast, the aperture can be opened further to
achieve better resolution; closing it any further, however, reduces resolution.
8. Adjust illumination: adjust brightness with the rheostat on the illuminator as you
view specimens, not with the condenser aperture. (It is also possible, in the absence
of lamp-brightness control, to use neutral-density filters to reach desired brightness.)
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This is Köhler illumination. It is the optimum for observing detail as you study a specimen
and the optimum, especially, for photomicrography. As you change objective lenses, you
will need to adjust the apertures and condenser lens for each, starting with step 4 above, to
achieve Köhler illumination with it.
Contrast can potentially be enhanced on transparent specimens by closing down the condenser
aperture, but realize that by doing so you sacrifice resolution—you are leaving Köhler illumination. Contrast can also be enhanced (again sacrificing resolution, but not as severely)
by making the illumination oblique—e.g., placing a narrow strip of paper across the substage
illuminator. (It is best to use phase contrast or differential interference contrast to view
transparent specimens—i.e., special illumination and lenses.)
Use the dissecting microscope to find and mount specimens on slides for the compound microscope.
Specimens on slides must be covered with a coverslip (never use the microscope to examine a
specimen without a cover slip.) Live specimens also usually need to be compressed with the
cover slip but not squashed by it. To achieve the correct compression, place small wax or
clay feet on the corners of the coverslip by picking each corner into a piece of wax or clay and
getting just a very small fleck on one side at the corners. Then lower the coverslip onto the
specimen by resting first one edge on the slide and then lowering the other gradually with a
dissecting needle, thus minimizing trapping of air bubbles on the specimen. Compression can
be adjusted by pressing gently on the wax/clay feet of the coverslip and by wicking out fluid
from under the coverslip with bibulous paper or filter paper (or other absorbent paper).
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Use of light microscopes
Substage controls on the Olympus microscope:
2: Focusing knob for objective lens
3: Field-stop aperture
3a: Condenser aperture
4: Focusing knob for condenser lens
5: Centering knobs for condenser lens
Setting the condenser aperture:
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Views of field-stop aperture during setup:
3: out of focus and uncentered
4: in focus, still uncentered
5: in focus and nearly centered
6: expanded but not quite centered
7: centered and expanded just beyond field of view
Use of light microscopes
Dissecting microscope
The eyepieces (oculars) adjust in focus independently and should be adjusted when you first start
work on the microscope. The spacing between the eyepieces (interpupilary spacing) is also
adjustable and should be set so that both eyes see the specimen simultaneously. It is best to
focus on a specimen with the large focus knob of the microscope while looking first through
the right eyepiece (only) with your right eye; then look through the left eyepiece (only) with
your left eye and turn the knurled ring on it until the same object you saw with your right
eye in the other eyepiece is in focus.
Magnification is changed by rotating the knurled knob on the right side of the head. To obtain
parfocal zoom in magnification (i.e., so that the image stays in focus at all magnifications),
first focus on a specimen at high magnification, then zoom to low magnification and adjust
focus with the eyepieces only.
Illumination. For reflected illumination, place the fiber-optic heads relatively high above the
specimen and facing downward. Their position should not be so close to the specimen that
they heat it up unnecessarily, and their angle should be adjusted to avoid producing glare on
your eye. Adjust the substage mirror to give a high-contrast background (dark or light). For
transmitted illumination, aim one of the fiber-optic heads horizontally through the rear hole
on the stage and adjust the mirror to give a fully illuminated field of view.
The binocular head should be firmly clamped in its support ring. (Please do not loosen the set
screw on this ring and turn the head to show lab partners views under the scope.)
Putting microscopes away
Remove all specimens from the microscope.
Return the magnification to the lowest lens/setting available.
Ensure that no sea water or other fluids are on the microscope. Wipe off sea water with a towel
dampened in fresh water and then dry the spot.
Turn off the illumination.
Replace the plastic dust cover on the microscope.
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