Microscope Introduction
Introduction
A laboratory tool that has become almost synonymous with biology is the microscope. As
an extension of your eyes, the microscope is one of the most important biological tools
that you will use. Get to know it, how it works, and what its limitations are.
A light microscope is really only a sophisticated arrangement of magnifying lenses,
constructed for convenient observation of small objects and using light as a source of
illumination. There are several common types of microscopes, differing primarily in their
magnification ranges, types of light used, physical structures, and applicability to the
materials under study. Whatever type of microscope you will be using, proper care and
maintenance will enable you to see the objects of study more clearly. All the lenses of
your microscope should be cleaned before every use. Use only lens paper specifically
manufactured for this purpose, to avoid scratching the surface of the lens. The eyepiece
may often become smeared with mascara, dust, oil, or dirt. The objective lenses should be
cleaned to remove any residue that may have coated them during previous use. Should a
lens accidentally contact the object you are observing, immediately dry and clean the lens
with lens paper. If seawater has contaminated a lens or the stage of the microscope, clean
and dry it using distilled water and lens paper.
The Compound Microscope
A compound microscope is a delicate and expensive instrument and must be treated
gently.
Familiarize yourself with the parts and operation of your instrument, referring when
necessary to figure B.1.
Remove your assigned compound microscope from its cabinet by the arm. Holding it
upright and supporting the base with your free hand, take the instrument to your desk and
put it down gently. (A microscope should always be carried in this fashion with both
hands.)
The arm supports the body of the microscope, which in turn supports the magnifying
elements. At the top of the body tube is the ocular, or eyepiece, one of the magnifying
elements. It is usually removable. The other magnifying elements are screwed into a
revolving at the bottom of the body tube. These magnifying elements are called
objectives. Together, the ocular and the objectives constitute the magnifying system of
your microscope.
Below the body tube and nosepiece is a flat plate, the stage, where the objects to be
examined are placed. Directly beneath the stage opening you will notice a system of
lenses, the condenser lens, that serves to concentrate light from the mirror or light source
below. On most instruments, one side of the mirror is concave and the other is flat. When
the light source is your desk illuminator, always use the concave side of the mirror.
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Light plays an extremely important role in the operation of a compound microscope.
Light is directed up through the stage opening, passes through the specimen, and then
into the body tube, ultimately forming an image on the retina, the light-sensitive portion
of the eye. The critical importance of light necessitates careful adjustment, and several
controls are available for this purpose. The iris diaphragm may be found attached below
the condenser. Look up through the stage opening, find the iris diaphragm lever, a small
handle at one side of the diaphragm. Push it back and forth, noting how the size of the iris
opening changes to regulate the amount of light passing through it. This control is one of
the most important on your instrument. Open the diaphragm fully and reposition your
microscope; replace the ocular.
Just as with a magnifying glass, viewing an object with the microscope requires that the
lens be a certain specified distance from the object. That distance is a property of the lens
system and is constant for each objective; it is called the working distance. At the
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working distance from an object, an objective is in focus. Changes and adjustments in the
focus, which are necessary when an object is first placed on the microscope, are essential
in order to obtain a precise image. Focusing is accomplished by means of coarse and fine
adjustment knobs, usually located on the arm. Try turning each of these knobs, noting
carefully how each affects the position of the objectives. The coarse adjustment is used to
obtain an approximate focus and the fine adjustment to obtain an exact and clear focus.
Magnification
The magnification of most objectives and oculars is engraved on them. On the ocular, the
marking can be found on the smooth cylinder that fits inside the body tube. On the
objectives, the magnification is engraved on the side of the cylinder. The marking means
that the lens yields an image 10 times larger in each dimension than the object being
viewed.
Remember that with a microscope of this kind you are using two sets of magnifiers. On
low power (10X), for example, the objective forms an image (inside the body tube) that is
10 times as large as the viewed object; the 10X ocular then magnifies this primary image
another 10 times. The image that finally reaches your eye has been enlarged 100X the
size of the object. The total magnification for any combination of objective and ocular
can be computed simply by multiplying the magnification of each lens.
When using the microscope we will need to measure the size of certain objects.
Remember that 1mm. = 1,000µm. Since our scopes do not have ocular micrometers we
will need to measure our field of view for each objective and then estimate the size of a
given specimen.
The common unit of length used in microscopy is the micrometer. One micrometer
equals 0.001 millimeter (mm), or 1/25,400 inch. The calibrations on the fine adjustment
knob are in micrometer units. By using this scale, you can determine the thickness of a
microscopic object.
Oil Immersion
Modern microscopes commonly have three or four objective lenses. Most microscopic
observations use objective lens magnifications up to 45x power. However, when
observing bacteria, you must use objective lenses up to 100x power. Owing to the small
lens diameter of this power, special means must be employed to ensure that light coming
up through the stage is not scattered and lost. Directing light into the objective lens is
accomplished by the technique of adding light-transmitting oil to the slide and lowering
the objective lens until it comes in contact with the oil. This keeps light from scattering
out of the pathway of the objective lens. As stronger objective lenses are used, the
focusing distance between the lenses and the slide gets smaller and smaller (fig. B.2).
We will not be using the oil immersion lens in this class.
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The Dissecting Microscope
The dissecting microscope is useful for observing organisms larger than those for which
the compound microscope is used. The magnification is not as great, but there is much
more distance between the objective lens and the stage, allowing for manipulation and
dissection of specimens. Most dissecting microscopes are binocular, providing a
stereoscopic view, and are equipped with either a zoom lens or multiple objective lenses
for variable magnification. Total magnification is computed in the same manner as for the
compound microscope. There is no fine adjustment knob on these instruments.
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Magnifications
Ocular
_____________X
Red Objective (Low Power)
_____________X
Yellow Objective (Medium Power)
_____________X
Blue Objective (High Power)
_____________X
Total Magnification of ocular and objective
Red
____________X
Yellow
____________X
Blue
____________X
1. Measure the field of view with a ruler using the Red objective (Round to the nearest mm.).
____________mm
___________µm
2. Measure the field of view with a ruler using the Yellow objective (Round to the nearest mm.).
____________mm
___________µm
3. Calculate Blue field of view by using the ratio of yellow/blue magnification to yellow/blue
diameter.
____________mm
___________µm
Example:
Low magnification
Med. Magnification
Organism
Mag
Diameter
(µm)
=
X
Low diameter in microns
Times
Across
Size (µm)
Foraminifera
Diatom
Radiolaria
Sponge Spicule
Dinoflagellate
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Compound Microscope Quick Reference
Initial Set-up
1.
2.
3.
4.
5.
Unwrap scope and clean lenses using lens paper only.
Turn light source on to intermediate brightness
Open iris diaphragm
Move condenser to the base of the stage, then make a ¾ turn down
Set interpupillary distance
Focus
1.
2.
3.
4.
5.
Place specimen properly on the stage and secure using stage clip
Use both of your eyes!
USE THE LOW POWER OBJECTIVE to center the image with the stage controls
Obtain focus using the coarse and then the fine focus adjustment
Switch to the next power, center the image and focus using FINE FOCUS
ADJUSTMENT ONLY
6. Do not use the oil immersion lens (100X)
Rules in using the microscope
1.
2.
3.
4.
Always carry with both hands
Clean lenses before and after each use
Never leave any water on the stage or scope
Always remove slides before storing the scope
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