Microscopy
Many organisms and structures in biology are too small to be seen with the naked eye and require
the use of a microscope to be visible. In this course, you will be using two types of microscopes: the
compound light microscope and the dissecting microscope (also called stereomicroscope).
The compound microscope is used to magnify objects found on prepared microscope slides or
temporary wet mount slides. This microscope can magnify objects from 40x to 1000x their original
size. The dissecting microscope has a smaller magnification range and is useful for viewing details
on larger specimens; the dissecting microscopes in our lab magnify objects 7x to 45x their original
size. Specimens do not need to be mounted on a slide when using the dissecting microscope; three
dimensional objects can be placed directly on its stage.
Learning Objectives:
1. Properly use a compound and dissecting microscope, and determine the appropriate
microscope to use when viewing a specimen.
2. Identify the parts of compound and dissecting microscopes and describe the function of
each part.
3. Describe properties of microscopy, such as total magnification, inversion, parfocal, field of
view, and depth of field.
4. Properly make a wet mount slide to view living cells.
I. Parts of the Compound Light Microscope
Compound microscopes are named for having two sets of lenses: ocular lenses and objective lenses.
The ocular lens is located at the top of the microscope in the eyepiece and contains a magnification
of 10x. Compound microscopes may have one ocular lens (monocular) or two ocular lenses
(binocular). The second lens system functions to further magnify an object and includes four
different objective lenses: scanning (4x), low power (10x), high power (40x), and oil immersion
(100x). These objective lenses are located on the revolving nosepiece, which allows a user to
switch from one objective to another. The lenses and attached parts are supported by the arm on
the side and the base at the bottom of the microscope.
When viewing an object, the slide is placed on the stage, the flat platform below the objective
lenses. Stage clips located on the stage hold the slide securely in place. The stage can be raised or
lowered by using the two adjustment knobs on the arm. The larger knob is called the coarse
adjustment knob and makes large adjustments to the stage to bring the slide into focus. The
smaller fine adjustment knob makes small adjustments to the stage to bring the slide into sharper
focus. The stage can also be moved to the left or right by using the X-Y stage control knobs.
These knobs are located under the stage and function to center a slide under the objective lens.
The light source of the compound microscope comes from the illuminator, which is located on the
base. The amount of light released from the illuminator can be controlled by adjusting the rheostat,
which is usually located on the arm or base. The light is focused onto the slide by the condenser,
which is directly under the stage. On the condenser is the iris diaphragm, the lever that adjusts the
amount of light that hits the object.
Procedure
1. Label the following structures on the microscope below: arm, base, ocular lens, objective
lens, revolving nosepiece, stage, stage clips, coarse adjustment knob, fine adjustment knob,
X-Y stage control knobs, illuminator, rheostat, condenser, and iris diaphragm.
© Kara Rosch
II. Using the Compound Light Microscope
Because the compound light microscope contains two sets of lenses, it is necessary to multiple the
magnification of each lens (ocular x objective) to calculate the total magnification when viewing a
specimen. Since the ocular lens has a magnification of 10x, the total magnification can be
determined by multiplying the objective magnification by a factor of ten.
When viewing a slide through the compound microscope, the image will appear flipped both
horizontally and vertically. This property of the compound microscope is referred to as inversion.
When centering your specimen under the microscope, you will need to move the slide to the left to
move the image to the right and vice versa.
Another property of the compound microscope is that the objective lenses are parfocal, which
means that the image remains in focus when switching objective lenses. When viewing a specimen,
always start on the lowest magnification, the scanning objective lens. Once the image is in focus,
you can move to a higher magnification. It is only necessary to use the fine adjustment knob to
bring the image into sharp focus.
The entire circular area that is visible when looking through a microscope is referred to as the field
of view. As you increase magnification, the field of view decreases. By determining the diameter of
the field of view, you can estimate the size of a cell that you are observing under the microscope.
The vertical distance that remains in focus on the slide is known as the depth of field. The depth
of field also decreases as magnification increases.
To properly use the compound light microscope, follow the steps below.
1. If the scanning objective is not in place, move to this objective by turning the revolving
nosepiece. You must always start with the scanning objective when first viewing a
slide.
2. Lower the stage by using the coarse adjustment knob.
3. Place your slide on the stage and secure it with the stage clips. Make sure that the stage clips
are resting against the slide and NOT on top of the slide.
4. Turn on the microscope by using the on/off switch, which is usually located in the back. If
the light does not turn on, you may need to adjust the rheostat.
5. Center the slide under the objective lens by using the X-Y stage control knobs.
6. While looking through the ocular lens, slowly raise the stage by using the coarse adjustment
knob until you see the object. Most of the time, you are looking for a specimen that is
stained, such as pink or blue.
7. If your microscope contains two ocular lenses, you should have both eyes open when using
the microscope and see a single circle in your field of view. If you do not see a single circle,
adjust the distance between the ocular lenses at the base of the eyepieces.
8. When you see your object in the field of view, center it using the X-Y stage control knobs.
Use the fine adjustment knob to bring the object into sharp focus. Adjust the light by using
the rheostat or iris diaphragm, if necessary.
If you want to increase magnification, continue with the following steps.
9. The cell or structure that you want to focus in on must be centered in your field of view.
Move to the low power objective.
10. Use the fine adjustment knob to bring your object into focus. From now on, you will only
be using the fine adjustment knob; the coarse adjustment knob should only be used
under the scanning objective.
11. If you want to increase magnification further, center the object again and move to the high
power objective. Bring your object into sharp focus using the fine adjustment knob.
12. You may need to adjust the amount of light as you increase magnification.
13. Your microscope may be equipped with an oil immersion lens; it is named because
immersion oil is required when using this lens. We will not be using the oil immersion lens
in this class. Do not view your slide under this lens as you can damage the microscope or
slide.
When you are finished using the microscope, follow the steps below.
1.
2.
3.
4.
Remove the slide from the stage.
Return to the scanning objective lens.
Turn off the power and wrap the cord around the arm.
Return the microscope to its correctly numbered location.
Procedure
1. Fill in the table below to determine the total magnification when viewing a specimen under
each objective.
Name of Objective
Lens
Scanning
Low Power
High Power
Oil Immersion
Magnification of
Objective Lens
Magnification of
Ocular Lens
Total
Magnification
2. Obtain a letter “e” slide, focus it under the compound microscope so the entire “e” is visible
in the field of view, and draw what you see below.
Total
Magnification _____
3. What property of the compound microscope is apparent when viewing the letter “e”?
4. Obtain a 1 millimeter (mm) grid slide and focus it under scanning power. Measure the
diameter of the field of view by counting the number of squares visible. Each square equals
1 mm; measure to the nearest tenth of a mm.
Diameter of field of view under scanning objective = __________
5. Once you know the diameter under scanning power, you can calculate the diameter of the
other objectives by using the following formulas:
Low Power Diameter = Scanning Diameter x
Scanning Magnification
Low Power Magnification
High Power Diameter = Scanning Diameter x
Scanning Magnification
High Power Magnification
Diameter of field of view under low power = __________
Diameter of field of view under high power = __________
6. What happens to the field of view as you increase magnification?
7. Obtain a cross threads slide. Focus on the overlapping region under scanning, low power,
and then high power. Notice how the depth of field decreases as magnification increases.
Under high power, determine the order of the colored threads. You can do this by slowly
raising the stage until the top color comes into focus; continue to raise the stage until the
middle thread and then the bottom thread come into focus.
Top color: __________ Middle color: __________
Bottom color: __________
III. Prepared Mounts and Wet Mounts
Prepared mounts contain specimens that have been fixed and mounted to result in a permanent
slide. The specimens on these slides have usually been stained to increase the contrast when
viewing, and thus, they may not represent the true coloration as when the cells were living. Before
viewing a prepared slide under the microscope, it is necessary to obtain a perspective of the
specimen that is mounted. Specimens that have not been sectioned (sliced) prior to mounting are
mounted as a whole mount (abbreviated w.m. on the slide label). Whole mounts are generally used
when viewing single cells or very small organisms. When larger organisms or structures are
mounted, they must be thinly sectioned to fit on a microscope slide. These specimens are usually
mounted as a cross-section (c.s.) or longitudinal section (l.s.). In a cross-section, the specimen is
cut at a right angle to its main axis. Cross-section mounts often appear circular, such as when
viewing an earthworm or plant stem cross-section. In a longitudinal section, the specimen is cut
parallel to its main axis, and the resulting mount is often long and rectangular.
To make a temporary mount of cells or tissue, a wet mount can be made. Because specimens under
a wet mount are not fixed, they can be used to view living cells. In a wet mount, cells are placed in a
drop of water between a microscope slide and a cover slip. If cells are already located in an aqueous
environment, such as in pond water, there is no need to add an additional drop of water to the slide.
When making a wet mount, it is important to prevent air bubbles by slowly lowering the cover slip
at a 45 ͦ angle when placing it on top of your liquid.
Procedure
1. Obtain a Euglena preserved slide and draw it below under high power.
Total
Magnification _____
2. What kind of prepared mount is this Euglena slide (whole mount, cross-section, or
longitudinal section)?
3. What is the estimated length of each Euglena? You will need to refer to the diameter of the
field of view you calculated earlier.
4. Obtain a corn (Zea mays) stem prepared slide. This slide contains both a cross-section and
longitudinal section. Focus on each section under scanning and draw them in the
appropriate circle below. If the entire section does not fit completely within the field of view,
just draw the portion that is visible.
Total
Magnification _____
Zea mays cross-section
Total
Magnification _____
Zea mays longitudinal section
5. Make a wet mount of a drop of pond water. Obtain a clean microscope slide, place a drop
of pond water on the slide, and then slowly lower a cover slip on top of the pond water.
Wipe off any excess water from the slide before placing the slide on the stage. Find living
organisms on the slide and draw them below. Refer to the pond water posters to identify
your organisms.
Total
Magnification _____
IV. Parts of the Dissecting Microscope
The dissecting microscope also has two sets of lenses: two ocular lenses are located in the
eyepieces and two objective lenses are internal. The magnification can be controlled by adjusting the
magnification control knob on the head of the microscope. On the arm of the microscope is the
focus adjustment knob that brings the object into focus.
Unlike the compound microscope, the dissecting microscope contains two different light sources. A
reflective light source is located on the arm of the microscope. This light source hits an object
from above and is reflected off of the object, illuminating thick or large specimens. If a transparent
object is being viewed, the transmitted light source can be used to transmit light through the
specimen from below. The transmitted light source is located under the glass stage plate on the
base of the microscope. The lights can be turned on and off by adjusting the lighting control
knob on the base of the microscope.
Procedure
1. On the microscope below, label the following parts: ocular lens, head, arm, base,
magnification control knob, focus adjustment knob, reflective light source, transmitted light
source, stage plate, and lighting control knob.
© Kara Rosch
V. Using the Dissecting Microscope
The dissecting microscope is used to magnify larger, three-dimensional specimens. It is also useful
to magnify specimens on microscope slides that are too large or too thick for the compound light
microscope.
To properly use the dissecting microscope, follow the steps below.
1. Place your specimen on the stage.
2. Turn on the appropriate light source. The reflective light source is used most often.
However, the transmitted light source may be used if viewing a transparent object, such as a
microscope slide.
3. Adjust the magnification using the magnification control knob. The total magnification can
be determined by multiplying the number found on this knob times the ocular magnification
(10x).
4. Bring the object into focus by using the focus adjustment knob.
Procedure
1. There are various specimens on display for practice using the dissecting microscope, such as
insects, lichens, and plastomounts. Obtain a specimen, focus it under the dissecting
microscope, and draw it below.
Total
Magnification _____
2. For each of the following specimens, determine what light source (reflective or transmitted)
would be appropriate to use.
a. Crab exoskeleton –
b. Liver fluke prepared slide –
c. Jellyfish plastomount –
d. Live starfish –