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 type used in this course is a bright-field microscope, where the specimen
appears darker against a bright background

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 16 th
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.
Resolving Power- ability of a lens system to separate fine detail
Resolution- smallest resolvable distance between two objects
 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.
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.

Iris 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)
 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.

Working distance

The distance between the objective lens and specimen when the specimen is in focus. The
objective lenses vary in lengths, the working distance will change as you switch from one
objective lens to the other.
The magnification increase, the W.D will decrease.

Field of View

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:
 Magnification field of view

4X
10X
40X
100X

A good analogy is to imagine yourself viewing the Earth from space as you gradually move
closer and closer to Mission College, 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 ( DFV) at a particular magnification, since you can use this information
to estimate the size of the specimen you are viewing. The at diameter of the field of view
( DFV ) OF low power for your microscope (100X) is ~1.8 mm. Using this FOV diameter, you
can calculate the FOV diameter at other magnifications. You can use this relation:-

M1 X DFV1 = M2 X DFV2

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

1.8 mm x 100X = 500X x DFV2 = 0.36 mm = 360 µm

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 µm. 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 µm = 90 µm WIDTH = 1/10 x 360 µm = 36 µm

~90µ m
360

~36 m

500X 500X 500X

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 thick and the specimen you a 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.

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.
 Part 4: THE STEREOSCOPIC DISSECTING MICROSCOPE

Up until now you have been exclusively using a compound light microscope. While it
is ideal for viewing tiny microbes that can be mounted on a slide, there are biological
specimens that are too large and/or thick to be mounted on a slide and viewed with the
compound microscope (yet too small for the naked eye). In this case you will want to use
the stereoscopic dissecting microscope or “dissecting microscope” for short. Two
advantages of this microscope are 1) you can manipulate your specimen (turn, flip,
dissect) using your hands or tools while viewing it under magnification (hence term
“dissecting”), and 2) by looking through both oculars you can see the image in three
dimensions (“stereoscopic”).
The dissecting microscope has objective lenses that allow you to view your specimen from
0.7 x to 4.5 x . While the total magnifications possible on this microscope are low, they
provide the advantages of a very large field of view and a very thick depth of focus. This
will allow you to see most, if not all, of your specimen clearly and in three dimensions.
Your dissecting microscope contains a single focus knob and two different light sources controlled
by knobs on either side of the arm of your microscope. Turn them on and you will notice that one
light source is below the stage and the other is above the stage. The light below the stage produces
light that will pass through a transparent specimen, what we call transmitted light. The light above
the stage produces light that will bounce or reflect off the specimen. We refer to this as reflected
light, which is used to illuminate a non-transparent specimen from above.

You will first examine some 3-dimensional biological objects to get used to using the dissecting
microscope, and then you will examine some samples of the fruit fly Drosophila. Drosophila has
been an incredibly valuable organism for over a century in the study of genetic inheritance and
embryological development.
Exercise 4 – Using the stereoscopic dissecting microscope
Examine the samples indicated below, and as you do so, adjust the lighting to give you the best image, and
answer the corresponding questions on your worksheet.

1) Examine the letter “e” slide at 30X, noting its orientation viewed with the microscope relative to
your naked eye.

2) To become more familiar with viewing specimens under the dissecting microscope, examine the
objects provided on your bench at both 15X and 30X with transmitted and/or reflected light.

3) Using the tweezers provided, carefully and very gently place one Drosophila fruit fly from
4) each container onto the stage, and note the eye color of each fly