Appendix X - Light Microscopy
A. Compound Light Microscope
Compound light microscopes (Figure 1) are used to view minute details of a variety of biological specimens at a
relatively high magnification, usually up to 1,000x total magnification. Compound microscopes have two sets
magnifying lenses, the ocular and objective lenses. The ocular lenses, the ones which you will look though (hence
their name), magnify objects to 10x their size. The objective lenses are those closest to the specimen and have a range
of magnification from 4x to 100x (printed on the side of each lens). These lenses are located on the revolving
nosepiece which allows the user to easily change the magnification as needed. The total magnification at which any
specimen is being viewed is the product of the ocular lens magnification and the magnification of the objective lens in
use. Detailed information, including magnification power, is printed on the side of every ocular and objective lens.
Ocular Lenses
Interpupillar Distance Scale
Observation Tube
Diopter Ring
Microscope Body
Revolving Nosepiece
Objective Lens
Slide Holder
Light Switch
Aperture Iris
Rheostat
Stage
Condenser
Fine Focus
Light Source
Course Focus
Field Iris
X-axis Knob
Y-Axis Knob
Figure 1. Diagram of a typical compound light microscope with major components identified (adapted from
Olympus, 2005).
As you switch from one objective lens to another, you will notice that the working distance, the clearance between
the lens and the specimen, decreases with increasing power (see Figure 2). When using the higher power objective
lenses (40x or 100x), only the fine focus adjustment knob is used to minimize the risk of contact between the lens and
the specimen which can damage both the specimen and the delicate glass of the objective lens.
Also shown in Figure 2 if the diminishing field of view, the illuminated area and contents that are seen when looking
through the ocular lenses, as the objective power increases; this is due to a smaller and smaller aperture at the bottom
of the lens through which light enters. This means that things are harder to find on a slide when you are using high
power since only a small fraction of the slide can be seen, and less light enters your eye making everything appears
darker. As a consequence, you will learn to switch back to a lower power objective lens when you want to 'scan'
Objective
scanning
low power
high power
Magnification
4x
10x
40x
Working Distance
25 mm
8.3 mm
0.5 mm
Slide
Field of View
(diameter)
4.6 mm
1.8 mm
0.46 mm
around the slide, and manipulate the amount of light coming into the lens to see the objects more clearly.
Figure 2. The working distance (above) and the field of view (below) change
with magnification of the objective lens.
A light source is used to provide proper illumination of the sample which is crucial for maintaining contrast and
resolution at higher magnifications. Light is collected and focused by the condenser located underneath the stage, to
be transmitted through the specimen. As magnification is increased adjustments to lighting will be required which can
be accomplished by use of the rheostat (controls light intensity), field iris (controls contrast), and aperture iris
(control of resolution and contrast).
Basic Use
Uncover and plug in your microscope. The cord should have been wound carefully around the lower portion of the
body and the light source casing; unwind it taking care that you do not damage the condenser and aperture iris
controls beneath the stage. Note which objective lens is in position and how close it is to the stage. The lens that
should be in position is the smallest one: the 4x scanning lens. Make sure you have this lens in place when you finish
using the microscope.
Turn on the microscope and adjust the interpupillary distance, distance between your pupils, of the ocular lenses so
that you see a single illuminated field, even if you move your head very slightly from side to side. If side-to-side
movements of your head causes one field to black out, the interpupillary distance is set incorrectly. Use the diopter
ring to compensate for vision differences between your own eyes by first bringing a specimen into focus using only
your right eye and then look at the specimen with only your left eye and turn the diopter ring to focus as needed
(assuming the microscope is equipped with a diopter ring on the left ocular eyepiece only). You should be able to
clearly see the specimen with both eyes in a comfortable manner; if you find yourself squinting or using only one eye
then it is likely the interpupillary distance or diopter ring has not been set correctly, readjust the settings as needed.
With the lowest power object lens in position, place a slide on the stage and center the portion you wish to view over
the condenser. Using the coarse focus adjustment knob, raise the stage to its highest position while watching from the
side to ensure there is no contact between the slide and the objective lens. Most compound microscopes have a
mechanical stop to prevent such contact for the lower power objective lenses but extra care goes a long to prevent
damage to the microscope. Bring the specimen into focus by using the coarse focus to slowly lower the stage while
looking through the ocular lenses and finish the focusing with the fine focus adjustment knob. Magnification may
then be increased by turning the nosepiece to bring the next highest power objective lens into place. The microscope
is parfocal, meaning that no adjustments to the focus should be required as magnification is increased once the
specimen has been brought into focus with the low power objective lens. However, slight adjustments, using only the
fine focus, are usually needed when switching between the various objective lenses.
The light intensity will need to be increased as magnification is increased to compensate for the reduced amount of
light entering the higher power objective lenses. Each objective lens has a specified numerical aperture (NA) value
which is printed on the side of the lens, the aperture iris is set to 70 - 80% of the NA value for the three lower power
objective lenses and set to 0.9 when using the 100x objective lens (the aperture iris scale is partially hidden under the
stage). Properly setting the aperture iris is critical for good resolution and contrast. Contrast can also be further
enhanced by opening the field iris until its image is just slightly larger than the field of view which reduces the
amount of stray light entering the objective lens.
If the image is muddy, lacks contrast after such adjustment, or is overlaid by blotches that do no move when the slide
is moved back and forth with the stage, then some cleaning is needed. Cleaning of any of the lenses or other glass
components should only be carried out with proper lens paper moistened with lens cleaner (60% ethanol, or greater).
If the field is blotchy, turn each ocular lens to see if the blotches are on the oculars. Next, examine the the field of
view through each of the object lenses, noting whether the field is clear or blotchy and finally by moving on to the
condenser and glass over the light source. If you are unable to obtain a clear, evenly illuminated field of view, notify
your instructor for further assistance.
When you are finished with your microscope;
 ensure the light is switched off and the rheostat is at the lowest setting
 the 4x objective is in place,
 the stage is lowered,
 the cord is wrapped neatly around the base,
 the dust cover is replaced, and
 immersion oil, if used, has been properly cleaned off the 100x objective lens.
Oil Immersion
As light passes from one medium to another with a differing refractive indexes, it bends and light of different
wavelengths bends differently (e.g. a prism effect). As compound light microscopes use white light, which is
compromised of multiple wavelengths, distortion results at magnifications over 400x as the light passes from the glass
slide to the air and then to the glass objective lens. This problem can be eliminated by filling the air gap with
immersion oil, formulated to have a refractive index similar to glass. Use of immersion oil is restricted to objective
lenses that have been specifically designed for this purpose, generally only the 100x lens (again, this information is
printed on the side of the lens). Using immersion oil with an incorrect lens will, at the very least, require extensive
cleaning of the lens (as the oil can get inside the lens housing itself) and in some cases damage the lens.
Oil immersion microscopy is relatively simple, first focus on your sample as you normally would with the 40x
objective lens (or highest power, non-oil, objective lens on your microscope). Turn the revolving nosepiece so that
your specimen lies half way between the 40x and 100x objective, add a single drop of immersion oil to your
specimen, and move the 100x objective lens in place. Be extremely careful when adjusting the fine focusing as the
working distance is very small. Remember, you can not go back to 40x objective lens if you lose sight of your
specimen and searching for a specific point on the slide using the 100x objective lens can be extremely tedious. When
you are finished clean the immersion oil from the lens and slide using lens paper and lens cleaner.
B. Dissecting Microscope
Dissecting microscopes (Figure 3), also known as stereoscopic microscopes, provide stereo images of larger fields of
view but at a lower magnification as compared to compound microscopes. These microscopes use one objective lens
for each eye to provide depth perception of the specimen being examined. The main use for such microscopes if for
viewing larger surface areas or even whole organisms while providing the user with ability to precisely manipulate the
specimen during examination.
Zoom Adjustment
Eyepiece
Focus Adjustment
Clamping Screw
Microscope Body
Stage
Stage Plate
Figure 3. Diagram of a typical dissecting (stereoscopic) microscope with major components
identified (adapted from Olympus, 2010).
Most living specimens of suitable size will be viewed by reflected rather than transmitted light, with the illumination
coming from the side or above the object being viewed. The stage plate in the stage is removable, and can be
positioned so that a black or a white surface faces upwards. If the disk cannot be easily removed, check with your
instructor. Develop the habit of changing the disk back and forth to determine which background gives you better
contrast for a particular observation. Adjusting both the intensity and the angle of the light source can also enhance
contrast.
Sometime thick or tall specimens require the microscope body to be raised. This can be done by loosening the
clamping screw, but the microscope body must be carefully suspended to keep it from dropping unexpectedly when
the clamping screw is loosened. On some microscopes there is a failsafe ring, which can be tightened just below the
position of the head on its vertical shaft. Also, you must take care that the objective lenses are in proper position, or
fit exactly in their click stops. If you see a double image when the magnification setting is in its proper position, the
microscope is out of alignment. Call your instructor for help.
To view a specimen using a dissecting microscope, first place on the stage plate, and adjust the light source so that the
beam of light falls directly upon the specimen. Starting with the lowest magnification, focus on the specimen using
the focus adjustment knob. Then increase the magnification as necessary and make your observations. As with the
compound light microscope, please that you store the dissecting microscope properly. The body of the microscope
should be raised well above the stage, the cord must be wrapped tightly around the microscope, and the dust cover
replaced. If the light source the specific microscope has a separate control unit, its cord should be wrapped up neatly
and place with the control unit on the stage of the microscopic.
C. Wet Mounts
Preparation of wet mount slide allows you to view living microscopic organisms. To prepare a wet mount slide you
simply place your specimen with a drop or two of water on the center of the slide. You then place a cover slip on the
slide, touching the edge of the cover slip to the slide first away from the water drop then dragging it up to the drop at a
45-degree angle. When the cover slip is touching the water, begin to carefully lower it into place over the specimen.
Placing the cover slip this way reduces the chance of air bubbles being trapped which can obscure the specimen.
While some specimens are large enough to be manipulated with your fingers or forceps, unicellular organisms need to
be retrieved from a culture and transferred to your slide using a Pasteur pipette, or similar device. If you hold your
rubber bulb-tipped pipette so that the sides of your thumb and index finger press through the sides of the rubber bulb
against the upper end of the glass tube of the pipette, you can control the pressure on the bulb very precisely by means
of slightly rolling your index finger and thumb over the top of the pipette tube. The narrow end of the pipette can be
held steady by allowing the barrel to pass between your little finger and ring finger. With practice, this will allow you
to pick up and deliver single unicellular organisms to a slide. Remember to use the iris diaphragm to adjust for the
optimal contrast when looking at specimens under the compound microscope.
Vaseline mounting is a preparation method that allows you to work with a slide for a longer period of time (up to a
week) than with traditional wet mounts. To prepare a Vaseline mount, make an open spiral of Vaseline about 1 cm in
diameter on a slide using a “Vaseline gun” (a syringe loaded with Vaseline). This requires a fair bit of pressure and a
steady hand. The spiral should be in contact with the slide and not looped up into the air in places. If your first
attempt is unsuccessful, practice one or two additional spirals on different spots of the same slide. When you have
achieved a neat open spiral, place a small drop of culture liquid inside the spiral away from the open end. Do no fill
the whole spiral with liquid, as you want the culture to fill and just flow into the narrow channel between the open
arms of the spiral when you lower the cover slip into place. Before you add the cover slip you can use a dissecting
microscope to check that you do have some organisms on your slide. Then hold the cover slip to the slide at the
closed edge of the spiral and from a 45-degree angle lower it slowly and gently into place. Do not press the cover slip
down into the Vaseline. Diagrams on display in the lab will help you prepare your slide appropriately.
References
Olympus (2005). Olympus CX31 Biological Microscope manual. Olympus, Center Valley, PA, U.S.A.
Olympus (2010). Olympus SZ61 Zoom Stereo Microscope manual. Olympus, Center Valley, PA, U.S.A.