LABORATORY EXERCISE 1

Care and Use of the Compound Light Microscope

1. Learning Outcomes
When you complete the activities, you should be able to:
1. Identify the parts of a compound light microscope and explain their functions.
2. Demonstrate the proper method for viewing a specimen with the compound microscope.
3. Describe the principle of inversion of image.
4. Measure the diameter of the field of view and estimate the size of structures in a tissue
section.

2. Introduction
The microscope allows us to view tissue and cellular structures that are too small for
examination with the naked eye. Clinically, microscopes have become essential diagnostic tools
and are used regularly during surgical procedures.
Light microscopes use optical lenses to bend or refract the light waves that pass through an
observed object (e.g., a section of tissue). As a result, the image of the object is magnified,
typically 40 to 1000 times. The two basic types of light microscopes are the simple microscope,
which has a magnifying system that utilizes only one lens, and the compound microscope,
which uses a series of lenses in combination.
The microscopes you will be using in the laboratory are monocular or binocular compound
microscopes. A monocular microscope has only one ocular (eyepiece) lens; thus, only one eye
can be used for viewing. A binocular microscope has two ocular lenses, one for each eye. The
light microscope is one of the most important tools you will use to study medical biology.

3. Learning the Parts of a Light Microscope

The microscopes that you will be using are expensive precision instruments. They can be
damaged if they are not handled with prudence and care. When carrying a microscope, hold it
firmly in front of you, with one hand on the arm and the other hand supporting it under the base.
Always set it down gently, without sliding it, onto the table.
Before you use the light microscope for the first time, identify the locations and study the
functions of its parts.
1. Plug in the electric cord and switch on the substage light source built into the base. There
may be a rolling on/off light switch that will allow a range of brightness. If so, always start
with a high light intensity and adjust the brightness with the iris diaphragm. The lever of the
iris diaphragm can be seen under the microscope stage. Alternatively, some microscopes have
an iris diaphragm composed of a wheel that can be rotated between different-sized openings.
Question 1. Look through the ocular (eyepiece) lens and move the lever to open and close
the diaphragm. Describe what you observe.
2. The microscope stage is the platform on which a microscope slide is placed. The stage has a
central opening or aperture through which light passes to reach the specimen. Typically, the
stage is equipped with a clamping device, called a mechanical stage, that holds the slide
securely and is used to position the object to be viewed directly over the stage aperture.
Below the stage, on the side of the microscope, identify the two mechanical stage control
knobs.
Question 2. Describe how the mechanical stage moves as you turn each knob.
3. Locate the condenser lens, between the stage and the iris diaphragm. After light passes
through the aperture (opening) of the iris diaphragm, it travels through the condenser lens.
The condenser lens will concentrate the light before it passes through the tissue specimen.
4. Identify the nosepiece, located at the base of the head. The nosepiece is a revolving structure
that holds two to four objective lenses. After traveling through the condenser lens and the
tissue specimen, light passes through an objective lens and magnifies the image that you see.
The magnification of each objective lens, often called the “power,” is stamped on its rim.
The typical laboratory microscope will be equipped with at least three objective lenses: a
low-power (10×) lens, a high-power (40× to 45×) lens, and an oil immersion (100×) lens (×
stands for “times”). Some microscopes may also have a scanning lens with a magnification
of 4×.
5. Identify the ocular or eyepiece lenses. As light passes through these lenses, the image is
magnified again before it reaches your eyes. For most laboratory microscopes, the ocular
lenses are 5×, 10×, or 15×. For the microscopes that you will be using, the magnification is
probably 10×. Similar to the objective lenses, you should find the magnification stamped on
the rims of the lenses.
6. On each side of the base of the microscope are two knobs, the larger coarse adjustment knob
and the smaller fine adjustment knob. You use these knobs to bring the specimen into focus.
Question 3. With the lowest-power objective lens in place, describe what you observe when
you turn the coarse adjustment knob in a clockwise direction and then in a counterclockwise
direction.

7. The total magnification of the microscope with a particular objective lens in place can be
calculated as follows:
Total Magnification = (Ocular Lens Magnification) × (Objective Lens Magnification)
Question 4. Suppose you are viewing a section of lung with a 10× ocular lens and a 40×
objective lens. Calculate the total magnification of the section that you are viewing.
Question 5. Do people who normally wear eyeglasses need to wear them while using a
microscope?
4. Viewing a Specimen with a Compound Microscope
Objects that are magnified by a light microscope will have more clarity of detail only if
resolution is also increased. Resolution, also referred to as resolving power, is the ability to
distinguish close objects as separate and distinct. The unaided human eye has a resolving power
of about 0.1 millimeter (mm). This means that a person can distinguish two objects that are 0.1
mm apart as distinct entities. If they are less than 0.1 mm apart, they are perceived as being a
single object. A good compound microscope can increase resolution to 0.001 mm, so the
resolving power is 100 times greater than that of the unaided eye.
Before you view a slide, the microscope and its objective lens must be in the correct position.
1. Place the microscope on your lab bench so that the ocular lenses are pointing toward you.
Switch on the light source and immediately check the brightness. If the light appears too
bright, make an adjustment with the on/off switch or the iris diaphragm.
2. Rotate the lowest-power objective lens into position by manually moving the nosepiece until
the lens snaps into place. Using the coarse adjustment knob, move the nosepiece up (or move
the stage down) to make room for placing a slide in position. The distance between the
 objective lens and the microscope stage is called the working distance. Before you insert or
remove a slide, always make sure the working distance is at its maximum.
3. Clean microscope lenses regularly so dirt or blurred images are not confused with cell
structures. Always use lens paper for cleaning microscope lenses. Other types of tissues, such
as paper towels, can scratch the lenses. To remove an oily smudge, moisten the lens paper
with lens cleaner before cleaning. Avoid touching the lenses with your fingers because oils
from your skin can damage the lenses. If you accidentally touch a lens, clean it immediately
with lens paper moistened with lens cleaner.

Obtain a prepared microscope slide of any type of sectioned tissue and place it on the
microscope stage.
1. Make sure the slide is secured in position by the mechanical stage. Adjust the position of the
slide with the two mechanical stage control knobs so that the tissue you are about to view is
centered over the stage aperture.

2. Turn the coarse adjustment knob to bring the objective lens and stage as close as possible.
Notice that the coarse adjustment knob will stop turning before the objective lens and slide
come into contact. This will only occur when a scanning (4×) or low-power (10×) objective
lens is in position. Therefore, to guard against damaging a lens or breaking a slide, always
begin with the lowest power objective lens that is on your microscope.
With a scanning or low-power objective lens and the stage in their correct positions, you can
now begin to focus the tissue section.
1. Look into the microscope through the ocular lenses. If you are using a binocular microscope,
which has two separate eyepieces, you may have to adjust the ocular lenses by moving them
closer together or farther apart. You can do this by turning the adjustment knob, located just
below or between the ocular lenses, or by manually moving the lenses closer or farther apart.
The adjustment is correct when the images from both eyes fuse into one circular image.
2. The illuminated area that you are viewing is called the field of view. Adjust the iris
diaphragm so that enough light passes through the tissue section.
3. While looking into the ocular lenses, focus the image by slowly turning the coarse
adjustment knob. When the image is approximately in focus, use the fine adjustment knob
to bring it to exact focus.
 Select an area on the tissue section that you would like to view at a higher magnification.
1. Move the slide with the mechanical stage control knobs, so that the selected area is in the
center of the field of view.
2. Rotate the nosepiece so that a higher-magnification (40× to 45×) objective lens is in position.
3. Make sure that the lens will not hit the slide before moving it into position. (The 100× oil
immersion lens will not be used regularly for the required microscopic work in this course.
If it is used, your laboratory instructor will teach you the correct method for viewing
structures with this lens.)
4. Look into the ocular lenses to see if any focusing adjustments must be made. If your
microscope is parfocal, then once the initial focus is made with a low-power objective lens,
the image will remain in focus when switching between objective lenses. If your microscope
is not parfocal, make any focusing adjustments with the fine adjustment knob. Do not
attempt to focus with the coarse adjustment knob under high power
Question 6. When you move to a higher magnification, what change occurs in the overall size
of the field of view?

Question 7. As you move to a higher magnification, you may have to adjust the iris diaphragm
to allow more light to reach the specimen. Explain why.

5. Inversion of Image: Viewing the Letter ‘e’

When viewing a tissue section with a microscope, the image that you observe is both inverted
(turned upside down) and reversed (turned from side to side). The following activity
demonstrates this principle.
1. With the coarse adjustment knob, maximize the working distance on your microscope. Place
the low-power (10×) objective lens in position.
2. Position the condenser lens as far up as it will go. Adjust the iris diaphragm to produce a
brightly illuminated field of view. Focus the letter ‘e’ with the coarse and fine adjustment
knobs. Place a prepared microscope slide with the letter e on the stage in the right-side-
uposition and centered over the aperture. Do not view the slide through the ocular lens yet.
Question 8. In the space below, draw the letter e as viewed with the unaided eye.
3. Position the condenser lens as far up as it will go. Adjust the iris diaphragm to produce a
brightly illuminated field of view. Focus the letter e with the coarse and fine adjustment
knobs. Place a prepared microscope slide with the letter e on the stage in the right-side-up
position and centered over the aperture. Do not view the slide through the ocular lens yet. In
the space below, draw the letter e as viewed with the unaided eye. . Observe the letter e in
the field of view.
Question 9. Draw the e as it actually appears under low power. Describe the changes in the
orientation of the letter e when viewed with a microscope.
 4. While looking through a microscope, use the mechanical stage control knob to move the
slide to the right.
Question 10. In which direction does the e move in the field of view? Now use the
mechanical stage control knob to move the slide away from you. In which direction does the
e move in the field of view?

6. Determining the Diameter of the Field of View

When you switch to a higher-power objective lens to increase the total magnification, the
diameter of the field of view (field diameter) will decrease proportionately. We can express the
relationship between total magnification and field diameter with the following equation:
M1D1 = M2D2
where M1 is the initial total magnification, D1 is the field diameter at M1, M2 is the second total
magnification when the objective lens is changed, and D2 is the field diameter at M2.
This relationship is useful for predicting the field diameter when the total magnification is
increased (or decreased). For example, suppose that at a total magnification of 100×, the
diameter of the field of view is 4.0 mm. If you increase the total magnification to 200×, you can
estimate the field diameter at the greater magnification as follows:
M1 = 100×; D1 = 4.0 mm; M2 = 200×; D2 is unknown
(100×)(4.0 mm) = (200×)(D2)
D2 = (100×)(4.0 mm)/200× = 2.0 mm
If the diameter of the field of view is known, you can estimate the size of structures in tissue
sections. For example, consider a structure (e.g., a sweat gland in skin) that extends across one-
tenth of the diameter of the field of view. If the field diameter is known to be 2.0 mm, then the
diameter of the object will be one-tenth of 2.0 mm, or 0.2 mm.

Estimating the Diameter of the Field of View

1. Place a clear millimeter ruler across the aperture on the microscope stage. With the scanning
(4×) objective lens in position, focus the ruler lines and make sure that they are crossing the
widest portion of the field of view.
2. Align the beginning of a millimeter interval with the left edge of the field of view as shown
in the figure on the left.
3. Estimate the diameter in millimeters (mm).
4. Record the magnification and field diameter in Table 1.
5. Repeat steps 1–4 with the low-power (10×) and then the high-power (40× to 45×) objective
lenses in position.
 Table 1. Measuring the Diameter of the Field of View
Diameter of the
Type of Magnification of Magnification of Total
Field of View
Objective Lens Objective Lens Ocular Lens Magnification
(mm)
Scanning (4x)
Low Power
(10x)
High Power
(40x)

Question 11. How does the diameter change as you increase the magnification of the objective
lens?
Estimating the Size of a Structure in the Field of View
1. Place a prepared slide with any type of tissue on the microscope stage and view it with the
scanning (4×) objective lens. When you use this objective lens, the total magnification will
be 40×.
2. Select a specific structure in the tissue that you are viewing. Estimate the proportion of the
field diameter across which the structure extends.
Question 12. Use your earlier measurements of the field diameter (Table 1) to estimate the
structure’s size (i.e., diameter of the structure). Size of structure (mm):

3. Move the slide so that the structure is centered.

4. Switch to the low power (10x) objective lens and view the structure at a higher magnification,
The total magnification is now 100x.
5. Once again, estimate the proportion of the field diameter across which the structure extends.
 Question 13. Predict the size of the structure by using the data recorded in Table 1. How
does this calculation compare with your earlier estimate of structure size at the lower
magnification?
6. View the structure with the highpower (40× or 45×) objective lens so that the total
magnification is 400x or 450× and repeat the previous steps to predict the size of the structure.
Question 14. How does this calculation compare with your first two estimates of structure
size with the scanning and low-power objective lenses?