Department of Molecular Biology and Genetics / Fundamentals of Biology Laboratory Course

Duration: 2 hr

LAB 4 - Use of the Microscope

Introduction
In this laboratory you will be learning how to use one of the most important tools in biology - the compound
light microscope - to view a variety of specimens. You will also use a slightly different type of light
microscope called a stereoscopic dissecting microscope.

The first lens used to magnify things was developed in the first century A.D. These were pieces of glass
shaped in a convex form - thicker in the middle and tapering off to the sides - and were the first magnifying
glasses that could increase the image of an object about 10 - 20 X. The creation of glass lenses improved
dramatically at the end of the 16th century, vastly improving the magnifying power. By 1609, Galileo Galilei
refined the methods of lens making in an effort to view objects in the sky.

About half a century later, the Dutchman Anton van Leeuwenhoek further improved the art of lens making,
allowing him to view objects in pond water that had never been viewed by humans - microorganisms - life
at a tiny level. At the same time, an English physicist named Robert Hooke improved the technology of van
Leeuwenhoek and confirmed the existence of tiny organisms in pond water. He also famously examined a
piece of cork and observed tiny boxes arranged in such a way that they looked like the "cells" (rooms) in a
monastery if you removed the roof and looked in from above.

Today the best compound light microscopes are able to magnify objects up to 2,500X without losing their
resolution - the sharpness of the image itself.

Part 1: THE COMPOUND LIGHT MICROSCOPE

The Parts of the Compound Light Microscope

Exercise 1A - Getting familiar with the microscope

You will first get acquainted with the major parts of the compound light microscope before learning the
proper way to use it. Get a microscope from the cabinet below your lab bench, being sure to handle it by
the arm and base (refer to image on page 2), and place it on the bench in front of you. Remove the cover
and place it below, out of the way, and then plug in the microscope. The ocular lens (eyepiece) and stage
should be facing you. Read the description of each part of your microscope on the next two pages being
sure to follow all instructions, and then complete the matching exercise on your worksheet.
 Objective •

Power switch

Light intensity
regulator

Coarse focus

S 'Fine focus Mechanical

stage knobs

OCULAR LENS (eyepiece) - Your microscope will have either one (monocular) or two (binocular) ocular
lenses. These are the lenses you will look through when examining a specimen with the microscope. Take a
look at the side of your ocular lens and you will notice a label of "10X". This indicates that each ocular lens
magnifies the image by a factor of 10 or 10X.

OBJECTIVE LENSES - Notice the set of objective lenses on the revolving nosepiece. These lenses allow you to
change the degree of magnification. Some of our microscopes have four objective lenses while others have
only three. The degree of magnification for each objective lens is indicated on its side. Let's take a look at
each progressing from the shortest to longest objective lenses, being sure to rotate the revolving nosepiece
to click each objective lens into position above the stage before examining it:

4X - This objective magnifies the image by a factor of 4. It is referred to as the "scanning

objective" since it is used to scan the slide to locate the specimen before viewing it at
higher magnification. Your microscope may not have this objective lens, in which case
you can begin with the 10X objective.
10X - This objective magnifies the image by a factor of 10 and is referred to as the "low power"
objective.
40X - This objective magnifies the image by a factor of 40 and is referred to as the "high power"
objective.
100X - This objective magnifies the image by a factor of 100. It is referred to as the "oil
immersion objective" since it requires a drop of immersion oil on the slide to provide
good resolution. You will not be using this objective lens.

For now, make sure that the low power objective is clicked into position above the stage, and keep in mind
that you will only be using the low power and high power objectives. Also keep in mind that the total
magnification of any image you see through the ocular lens is the product of the objective and ocular lens
magnifications (for example, when using the lower power lens the total magnification is: 10X ocular x 10X
low power objective = 100X).
STAGE and STAGE CLIP - The stage is the flat surface upon which you will place each slide you will examine.
Notice that there is a moveable stage clip that can be used to secure the slide on the stage. Open and close
the stage clip to see how it will snugly hold your slide in position.

MECHANICAL STAGE KNOBS - To move the slide on the stage when it is secured in the stage clip, you will use
the mechanical stage knobs on the underside of the stage to move the slide backward/forward and right/left.
Adjust each knob to see how one knob controls backward/forward movement and the other knob controls
right/left movement.

COARSE FOCUS and FINE FOCUS KNOBS - In order for a specimen on a slide to be in focus, the distance
between the specimen and the objective lens must be just right. The coarse focus knob, the larger of the
two, will move the stage or objective lens (depending on the microscope) up and down quickly and quite
visibly, altering the distance between them. It is very important that the coarse focus knob is only used with
the low power or scanning objective lenses, otherwise the microscope or objective lenses could be damaged.
Adjust the coarse focus knob to observe how quickly the focal distance changes. In contrast, the fine focus
knob will move the stage or objective lens such a small amount that it is hardly noticeable to the naked eye.
This is the knob you will use to get the perfect focal distance so the image will be crystal clear.

CONDENSER LENS - Just underneath the stage is the condenser lens. This lens serves to capture and focus
light from the lamp below onto the slide mounted on the stage. On many microscopes the condenser lens
can be adjusted up or down with a knob beneath the stage. Examine the condenser on your microscope to
see if it is adjustable. If so, be sure to adjust it as high (close to the stage) as possible since, for our purposes,
this is where it should be set.

DIAPHRAGM - The diaphragm is located within the condenser and is one of the most important pieces of the
microscope, though it is often neglected by many students. The diaphragm allows you to adjust the amount
of light passing through the slide by adjusting the diaphragm lever. Most of the time the diaphragm will be
all the way open to allow the maximum passage of light. However it is important to adjust the diaphragm at
times to reduce the amount of light passing through your specimen should the image be too bright or dim,
and also to increase the contrast to allow you to see the specimen more easily against the background. For
now, open the diaphragm all the way, and when using the microscope, do not forget to use the diaphragm.

LAMP - The lamp emits light to illuminate the specimen so that you can actually see something.

BASE and ARM - The base is the bottom of the microscope that sits on the table, and the arm is the vertical
framework ascending from the base along the back of the microscope. When handling the microscope always
hold the arm while supporting the base with your other hand.

Proper Use of the Compound Light Microscope

Exercise 1B - Steps to follow when using the microscope
If you really want to be able to see a specimen on a slide, you must follow the steps on the next page every
time you look at a new slide. The microscope will be your friend if you always use the following steps in their
proper order. Before you begin, be sure your microscope is plugged in and the power is "on". Before you
start, clean all of the lenses with special lens paper which is soft enough to not scratch
the lens. Do not use anything else for this purpose (paper towel, shirt, backpack .... ) or you will scratch the
lenses.
Step 1. Get a slide of the letter "e" from the tray on the side counter. This an example of a prepared
slide, a slide that is already made for you and meant to be reused.
(i.e., don't dispose of it, please return it to the tray when you are finished!)
Step 2. Use a piece of lens paper to clean any smudges (fingerprints, grease, etc.) off the slide.
Place the slide on a white piece of paper find the specimen (the letter "e") on the
slide with your naked eye, noticing its location and orientation.

Step 3. Lock the low power objective lens into place (it should "snap" into place) if you have not already
done so. You will always (always, always, always .......) start with either the low power or
scanning objective when you want to view a slide.

Step 4. Use the coarse focus knob to move the stage (or objective lens) so that they are as far apart from
each other as possible. Open the stage clip and place the slide snugly in the corner of the stage
clip (make sure the slide is completely flat) before releasing the clip to hold the slide firmly in
place. Then use the mechanical stage knobs to position the slide so that the specimen (i.e., letter
"e") is centered over the condenser and the light that passes through it.

Step 5. Next, using the coarse focus knob once again, move the slide and objective lens as close
together as the knob will allow.

(NOTE: To this point, you have not yet looked into the oculars. This may be surprising,
but this is the proper way to use a microscope so that you will actually see
something!)

Step 6. Now, look into the ocular lens(es). Using the coarse focus knob, SLOWLY increase the distance
between the slide and objective until the specimen is in focus.

If the light is too intense, adjust the diaphragm lever (or dial near the lamp if present) until the light
level is comfortable before trying to locate the specimen.

If you have difficulty locating and focusing on your specimen (the letter "e"), make sure that it
is properly centered and you may need to adjust the course focus more slowly. If you still can't
locate it, ask your instructor for assistance.

Step 7. Adjust the diaphragm lever so there is sufficient contrast between the specimen and the
background, closing it no more than is necessary. This step is especially important for live
specimens since you may not be able to see them otherwise.

Step 8. Now use the fine focus knob to get the specimen in proper focus. You should now be able to see
the object clearly. Before going to the next step (increasing the magnification), be sure to center
your specimen in the field of view as best you can.

Step 9. Now that you have centered and focused the object as best you can at low power, rotate the high
power objective into place over the slide being sure it "clicks" into position. Use the fine focus
knob (NOT the coarse focus) to bring the object into perfect focus.

(NOTE AGAIN: You should only use the coarse adjustment knob with the low power objective)

FOLLOW THESE STEPS EVERY TIME YOU WANT TO VIEW A NEW SLIDE AND YOU WILL BECOME A GOOD
MICROSCOPIST!
 Part 2: PROPERTIES OF LIGHT MICROSCOPY
In this section we will focus on some of the key properties relating to light microscopy. To help you
understand each property you will first read an explanation and then do an exercise to illustrate that
particular property. Let us begin with the property of magnification...

Total Magnification
The total magnification of an image is quite simple - it is the product of the ocular lens magnification times
the magnification of the objective lens you are using:

magnification of ocular x magnification of objective = total magnification

For example, if the ocular lens magnifies the image by a factor of 10 (10X), and the objective lens magnifies
the image by a factor of 50 (50X), the total magnification of the image is 500X:

10X x 50X = 500X

Many students make the mistake of adding the two magnifications, so remember that total magnification is
the product (multiplication) of the ocular and objective lens magnifications.

Exercise 2A - Determining total magnification

On your worksheet, calculate the total magnifications for the examples given, then calculate the total
magnification when using each of the objective lenses on your own microscope.

Field of View (optional)

The field of view (FOV) is the actual "circle" you see when looking in the microscope. Although this circular
field of view appears to be the same no matter which objective lens you are using, this is not the case. The
circular area you are actually viewing will decrease as you increase the magnification:
field of view
total magnification
40X
100X
450X
1000X
A good analogy is to imagine yourself viewing the Earth from space as you gradually move closer and closer
to Mission College. Initially your field of view is the entire western hemisphere, but as you approach the
Earth's surface your field of view will progressively shrink to encompass the western United States, Southern
California, the San Fernando Valley, Sylmar, etc. Although your field of view is shrinking, the image in your
field of view is becoming increasingly magnified. This is really no different than looking into your microscope
at increasing levels of magnification.

It is also useful to know the diameter of the field of view (FOV diameter) at a particular magnification, since
you can use this information to estimate the size of the specimen you are viewing. The FOV diameter at low
power for your microscope (100X) is ~1.8 mm. Using this FOV diameter, you can calculate the FOV diameter
at other magnifications. This is done by multiplying by the ratio of the magnifications:

known FOV diameter x total mag. (known FOV) = unknown FOV diameter total mag. (unknown FOV)

If you want to know the FOV diameter at 500X, you could calculate it as follows:

1.8 mm x 100X/500X = 1.8 mm x 1/5 = 0.36 mm = 360 um

Once you know the FOV diameter, you can estimate the dimensions of your specimen. For example, assume
you are viewing the specimen below at 500X total magnification and, based on your calculation above, you
know FOV diameter to be 360 um. It appears that ~4 of your specimens would fit across the FOV end to end
(i.e., length = 1/4 of FOV), and ~10 side to side (i.e., width = 1/10 of FOV). Thus you would estimate the
dimensions of your specimen to be:

LENGTH = 1/4 x 360 um = 90 urn WIDTH = 1/10 x 360 um = 36 um

Exercise 2B - Field of view and estimating size (optional)
Before you can estimate the size of a microscopic specimen, you must first determine the diameter of the
field of view at the magnification you are using. Once you have that information you are prepared to estimate
the size of any specimen you observe at that magnification:

1) Calculate the FOV diameter for each possible total magnification on your microscope given
the FOV diameter at low power (100X) is 1.8 mm.
2) Examine a prepared slide of Paramecium at low power and estimate the length and width of
a single Paramecium.
3) Examine a prepared slide of Euglena at high power and estimate the length of a single
Euglena.

Depth of Focus (optional)

Once you have a specimen in focus under the microscope, if you adjust the fine focus knob up and down the
specimen will come in and out of focus. Thus, there is a range in the vertical dimension in which the specimen
on your slide will appear in focus. The "thickness" of the vertical range in which the specimen remains in
focus is referred to as the depth of focus. As it turns out, the depth of focus decreases as the magnification
increases as illustrated below:

total magnification depth of focus

40X 100X 450X 1000X

To make sure this concept is clear, imagine the range in which you can adjust the distance between the
objective lens and the slide (via the focus knobs) to be a loaf of bread standing on end. The image produced
in your microscope will only be in focus if the objective lens is positioned within a particular slice of that loaf
of bread. This slice of bread is the depth of focus, and it will get thinner as you increase the magnification.

This property of microscopy becomes very noticeable if the specimen you are examining is actually thicker
than the depth of focus at the magnification you are currently using. For example, if the depth of focus is
only 1 0m thick and the specimen you are observing is 2 Elm thick, there will always be a portion of the
specimen outside the depth of focus. This portion will thus be out of focus and cause the image to appear
blurry no matter how carefully you adjust the fine focus knob.

observed specimen magnification depth of focus image

high power
blurry

low power focused

In this example, the image will look blurry when viewed at high power magnification no matter what you
do. To get a focused image in this case you will have to increase the depth of focus and thus lower the
magnification.

To help you understand and appreciate the concept of depth of focus, complete the exercises that follow:

Exercise 2C - Depth of focus in the vertical dimension (optional)

Obtain prepared slides of Paramecium and "colored threads" and observe them as follows:

1) Observe a single Paramecium at low power (100X) and then at high power (400X),
and answer the corresponding questions on your worksheet.

2) Examine the colored thread slide at low power (100X), and determine the vertical order (top
to bottom) of the three colored threads as you slowly adjust the focus up and down through
the threads.