Marine Invertebrate Zoology
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.
Remove the 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.
Familiarize yourself with the parts and operation of your instrument, referring when necessary to the
microscope schematic on the following page.
Microscope Introduction
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Microscope Introduction
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Parts of the Microscope
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 (see pictures 10 & 11 below). It is
usually removable. The other magnifying elements are screwed into a revolving nosepiece at the bottom of
the body tube (see picture 8 below). 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 (see
picture 5 below). Directly beneath the stage opening you will notice a system of lenses, the condenser lens
that serves to concentrate light from the light source below. 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 (see picture 9 below). 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.
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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 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 (see picture 12 below). 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. Objects can be moved while on
the stage using the x and y adjustments. Remember, that a mirror is used to get the image from the objectives
to the ocular, which means that the specimen will appear inverted. Additionally, an adjustment on the x plane
to the ‘north’ will result in the apparent movement of the specimen to the ‘south’.
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 larger in each dimension than the
object being viewed.
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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 (see calculations on last page).
When using the microscope we will need to measure the size of certain objects.
Remember that 1mm = 1,000µm (micron), or 1/25,400 inch. 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
(see calculations on last page).
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.
\
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. Refer to Figure B3 on the following page
for Dissecting Microscope parts.
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Scanning Electron (SEM) and Transmitting Electron Microscopes (TEM)
Electron microscopes are used to overcome many of the limitations found when using standard light
microscopy. Electron microscopes fire a finely concentrated beam of electrons at a specially prepared
specimen greatly increasing the magnification and resolution of the resulting image (Magnification can
reach over 500,000X).
The use of these instruments requires specially prepared rooms (no overhead electrical wiring, concrete
room foundations that are deeper and poured separate from the attached building) and expertise much
greater than standard microscopy.
For more information on electron microscopy, UNCW’s microscope laboratory has both and SEM and
TEM in use. Visit: http://uncw.edu/bio/faculty_dillaman.htm for more information.
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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.
6.
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
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
Other Adjustments Necessary for Optimum Resolution and Contrast.
Adopted from Richard Fox Lander University Copyright 2001
To Customize the Diopter Setting
1.
2.
3.
4.
5.
6.
7.
The focal lengths of the two eyepieces must be adjusted to conform to differences, if any,
between your two eyes so that the images in both eyepieces are in focus simultaneously. Each
eyepiece is equipped with a diopter adjustment ring by which it can be focused independently
of the other eyepiece.
Adjust the interpupillary distance until it is correct for you.
With a test slide in position on the stage, rotate the 10X ocular lens into position.
Rotate the dipopter adjustment ring (=tube length adjustment ring) of the left eyepiece until its
numerical value is the same as your interpupillary distance.
Close your right eye and bring an object into clear sharp focus using the coarse and fine
adjustment, as needed.
Close your left eye and bring the same object into focus using the diopter ring of the right
eyepiece.
Record the two diopter numerical values so you do not have to repeat this process every time
you use the microscope:
Left = _________________
Microscope Introduction
Right = _________________
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To Adjust the Height of the Condenser
The condenser height should be set correctly to optimize light quality and resolution. The position of
the condenser is often accidentally changed and you need to know how to return it to the proper
position. Height is adjusted using the condenser adjustment knob (=substage focusing control). These
are small black plastic knobs located on the both sides of the condenser mount, below the stage. Turn
either knob to move the condenser up or down.
1 Place a slide on the stage of the microscope and position it for viewing. There must be a slide on
the stage!
2. Turn on the substage lamp and increase the voltage until its indicator lamp glows green.
3. Rotate the turret so that the 10X lens clicks into position.
4. Focus on the specimen using the coarse and fine adjustment as needed.
5. Open the condenser iris diaphragm fully.
6. While looking through the eyepieces, rotate the field iris adjustment knob until you can see the
outline of the field diaphragm in the eyepiece. It will be a 10-sided polygon.
7. Use the condenser control knob to raise or lower the condenser until the field diaphragm is in
sharp focus.
8. This is the proper height for the condenser and it should not be moved from this setting. If it is
inadvertently moved, go through the adjustment procedure again to restore its proper position.
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Name ________________________________________
Magnifications & Field of View Calculations
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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