LABORATORY 1

INTRODUCTION TO MICROSCOPY
LEARNING OBJECTIVES
When you have completed this lab, you will be expected to:
1. List the major branches of microscopy and briefly describe their advantages and
disadvantages.
2. Identify the various components of a compound light microscope, describe what
they do mechanically, and describe their function in microscopy.
3. Prepare a slide of a specimen.
4. Use a compound light microscope to magnify a specimen and bring the image into
fine focus using all objective lenses.
5. Define field of view, magnification, resolution, working distance, field diameter,
and depth of field.
6. Describe the relationship between magnification and working distance,
magnification and field diameter, and magnification and depth of field.
7. Estimate the size of objects viewed through the microscope.

INTRODUCTION
The microscope is a fundamental tool in biological research. It is used to view a
magnified image of a specimen. All microscopes rely on one or more lenses to magnify
images. Three important considerations in microscopy are 1. the degree of
magnification, 2. The degree of resolution, and 3. whether the microscope can
produce a 3-dimentional image or simply a 2-dimentional image of the specimen.

Magnification: Magnification is the ratio of an object’s image to its real size. Expressed
a factor such as 40 times (40X).

Resolution: Resolution is the ability to see fine details of structure. Limit of resolution
refers to how far apart adjacent objects must be to be distinguished as separate entities.
Expressed as a distance such as 500 nm.

There are various types of microscopy. Two main branches of microscopy are light
microscopy, which uses visible light to produce a magnified image, and electron
microscopy, which uses a beam of electrons. One major advantage of light microscopy
is that it can be used to view live specimens. In the case of electron microscopy, the live
specimen is killed during the procedure. However, electron microscopes have much
higher magnification and resolution powers. The most powerful electron microscopes so
far have magnification powers of 100,000X and resolution powers of 0.1 nm whereas the
most powerful light microscopes have magnification powers of 1,000X and resolution
powers of 200 nm. Within each of these branches of microscopy there are several types
of microscopes and techniques, each with certain advantages and disadvantages
leading to specific applications.

Light Microscopy
General Biology I Laboratory Exercises Page 1
Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

In Part A of these lab exercises, we will be using the compound light microscope. In
Part B we will be using a stereoscopic (dissecting) light microscope in addition to the
compound light microscope. Our compound light microscope offers higher maximum
levels of magnification than our stereoscopic microscope (400X compared to 40X).
However, the stereoscopic microscope provides a 3-dimensional view of a specimen that
the compound light microscope does not. The compound light microscope is the
instrument of choice when a high magnification is required (e.g., viewing a cell) whereas
the stereoscopic microscope would be a useful tool when low magnification and a 3-D
image are required (e.g., dissecting an insect).

The Compound Light Microscope

The compound light microscope (various views illustrated in Fig. 2) is designed to pass a
beam of condensed visible light through a thin specimen placed firmly on the sample
stage, through a series of lenses that magnify the image before it enters the eyes. Each
component carries out a different mechanical function and overall function in producing a
clear, sharp magnified image of your specimen.

When viewing a specimen such a cell under a compound light microscope, it is common
to use stains. A stain is a colored chemical that will bind to certain parts of the specimen
and allow you to highlight structures compared to the background, thus enhancing
contrast. In Part B, we will be using iodine to highlight the cell wall and nucleus of onion
cells. We will also be using methylene blue to highlight the cytoplasm and nucleus of our
own epithelial cells (see Fig. 1). Most stains are semi-specific for certain structures or
chemicals. Fluorescent-based labels can also be used with the appropriate light
microscope. These fluorescent stains can be designed to bind to specific structures and
molecules in specimens. They are important tools and widely used in biology.

Figure 1. Epithelial cells stained with

methylene blue.

General Biology I Laboratory Exercises Page 2

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

Figure 2. The Nikon YS100 compound light microscope.

Table 1 lists the major components of a compound light microscope and their functions
in microscopy.

General Biology I Laboratory Exercises Page 3

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

TABLE 1. COMPONENTS OF A COMPOUND LIGHT MICROSCOPE AND THEIR

FUNCTIONS IN MICROSCOPY

Microscope Component Function

Label in Fig. 2
Arm Supports the body tube and lenses. Use the arm to
Not labeled carry your microscope.

Base Supports the entire microscope. Broad and heavy,

Not labeled the base gives the instrument stability.

Light Source, Light Intensity Provide illumination of the specimen. The intensity of
Knob, and On/Off switch the light can be adjusted using the light intensity
H; D; C knob near the light source on/off switch.

Oculars (Eyepieces) Magnifies the image (10X) and puts it in a form and
A position suitable for viewing. Can be adjusted to fit
the user’s interpupillary distance and correct for
vision differences (except astigmatism) in the user’s
eyes.

Revolving Nose Piece Allows the user to change objective lenses without
M damaging them. The objectives should not be
pushed or pulled to change lenses.

Objective Lenses Supplies the initial magnified image of the specimen.

L The magnification of each objective is indicated on
its side.

Mechanical Stage (with slide Clips a slide into place and allows the user to move
holder) the slide in the x-plane (left to right) using the
B appropriate stage control knob. A Vernier scale
tracks movement of the specimen in the x-plane.

Sample Stage Provides a platform for the specimen and allows the
K user to move the specimen in the y-plane (front to
back) using the appropriate stage control knob. A
Vernier scale tracks movement of the specimen in
the y-plane. The sample stage can also be moved
up and down to focus (see coarse and fine focusing
knobs).

Coarse and Fine Focus Adjusts the height of the sample stage relative to the
Adjustment Knobs objective to focus the specimen. The Coarse Focus
O; G Knob is very sensitive (in terms of the change in
height as the knob is rotated) and allows for the
initial focusing (only used with the 4X objective.) The
Fine Focus Knob is much less sensitive and allows
for fine focusing when using any objective.

General Biology I Laboratory Exercises Page 4

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

Stage Control Knobs Move the mechanical stage in the x-plane and the
E; F sample stage in the y-plane.

Condenser Collects and concentrates light onto the specimen

J and projects the image. Can be controlled to
optimize resolution and contrast of the image viewed
and reduce glare. It’s controlled using the Condenser
Adjustment Lever and the Iris Diaphragm Lever.

Condenser Adjustment Lever Controls the distance between the condenser to the
N sample stage.
Lever down®Condenser up;
Lever up®Condenser down.
As a rule of thumb, the lower the magnification, the
higher the condenser should be positioned in the
optical path.

Iris Diaphragm Lever Controls the amount of light entering and leaving the
I condenser. This control should NOT be used to
adjust the light intensity. The light intensity knob
(near the light source on/off switch) can be used to
adjust the intensity at the light source.

Lab Exercise 1: Identification of Compound Light Microscope

Components
1. Your instructor will ask you to get a microscope from the storage cabinets. When
called upon, use both hands (one holding the base and the other holding the
arm) to carry the microscope to your bench. Take a moment to observe how the
microscope was put away and note the number of your microscope. Do not remove
the plastic tie used to wind-up the excess power cord.

Record the microscope number observed on the label on the back of the
microscope. My microscope number is: ______.

You will be asked to complete a sign-out sheet. Clearly write your name next to the
microscope number. This microscope will be the one you will use in all subsequent
labs that require a microscope.

2. Once you have your compound light microscope in front of you, try to identify the
various parts listed in Table 1. For each part, determine A. What they are doing
physically B. what role this plays in seeing a sharp, magnified image of a specimen.

3. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

General Biology I Laboratory Exercises Page 5

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

Lab Exercise 2: Using your Compound Light Microscope to View

a Specimen
In this exercise, you will learn how to correctly set-up your microscope, prepare a
specimen on a slide, and produce a sharp magnified image of your specimen. This
exercise will also allow you to observe the way in which the lenses of the compound light
microscope operate.

Exercise 2-1
Setting-up your microscope and finding your field of view

4. Plug in your microscope.

5. Put the 4X objective in place. Rotate the 4X objective using the revolving
nosepiece, ensuring that the objective clicks into place. DO NOT ROTATE
OBJECTIVES BY GRABBING ONTO THE OBJECTIVES THEMSELVES AS THIS
WILL BEND AND DAMAGE THEM.

6. Turn on the light source. Raise the condenser by gently pulling down on the
condenser control level until it’s in the down position.

7. Look through the oculars and adjust the light intensity to a comfortable level.

8. Adjust the distance between the oculars to accommodate your interpupillary

distance (distance between the two eyes). Adjust the distance by using the
baseplate of the oculars (as illustrated in Fig. 2) until you see only one circle of
light when using both eyes. This circle of light is your field of view. While you are
adjusting the microscope, try varying how close your eyes are to the ocular lenses.

Figure 2. Adjusting the microscope for interpupillary distance.

General Biology I Laboratory Exercises Page 6

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

Exercise 2-2
Preparing a specimen on a slide

1. Cut out a square containing the letter “e” out of the paper provided.

2. Using forceps, place it right side up on a clean microscope slide.

3. Place a drop of water near the letter on the slide.

4. Lower a coverslip carefully so that one end touches the slide near the letter at
roughly a 45-degree angle. Drop the other end of the coverslip onto the specimen
using your finger or a toothpick (illustrated in Fig. 3). Using a toothpick will avoid
adding oil to the coverslip or contaminating your specimen. The water should spread
evenly over the paper specimen. This procedure minimizes the number of air
bubbles caught under the coverslip. If any bubbles obscure your view of the letter,
gently tap the coverslip with the forceps to force them out.

5. If the slide only has a portion of the specimen submerged in water, you can add
more water by placing a drop of water on the edge of the coverslip near the region
without water. The water will be sucked under the coverslip by capillary action. This
procedure can introduce air bubbles however. Ask your instructor for assistance to
avoid introducing air bubbles.

Figure 3. Preparing a specimen on a slide.

Exercise 2-3
Viewing your specimen at scanning magnification

Scanning magnification is the lowest magnification. It allows a user to view the specimen
and identify an area of interest to further magnify. Note that whenever you want to
magnify a specimen, you should start by creating a sharp image at the scanning
magnification. Even if you intend to magnify at the highest level, you should always start
with the scanning magnification first. In addition, if you are viewing an image at the
highest magnification level and you lose your image for whatever reason, you should
always return to the scanning magnification and start again.

General Biology I Laboratory Exercises Page 7

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

1. Check that your microscope has the 4X objective in place and that the condenser is
up close to the stage (lever down). Bring the sample stage down to its lowest
position.

2. Place your slide on the sample stage and use the mechanical stage to clip in into
place. Use the stage control knobs to move the specimen around in the x/y-plane.
Place the specimen in the path of light coming from the condenser.

3. While looking through the oculars, raise the specimen using the coarse focus knob
until you get a rough outline of the “e” shape. Use the fine focus knob to sharpen
the image.

4. Use the stage control knobs to center the image of the “e” in the field of view.

5. Use the iris diaphragm lever to adjust contrast.

As a result of the multiple (compound) lens system inside a compound microscope, the
orientation of the image in the field of view (as seen through the oculars) appears
differently than the actual orientation of the specimen.

6. Move the specimen in the x/y-plane and determine how the movement of the image
compares to the movement of the specimen.

7. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

Exercise 2-4
Further magnifying your specimen

1. Put the 10X objective into place using the revolving nosepiece and bring the
condenser down (lever up).

2. If you have not touched the focusing knobs since having a sharp image in In-Lab
Exercise 2-3, the image should be coarsely focused. The microscope objectives are
designed to have different lengths that will roughly be the appropriate distance
between the specimen and lens for the specimen to be in focus. Do not use the
coarse focus knob at this point! The distance between the specimen and the
objective is too close and you may hit the objective lens against the specimen.
Sharpen the image using the fine focus knob. Only use the coarse focus knob
when the scanning (4X) objective is in place. If you are having trouble remembering
which is the coarse focusing and which is the fine focusing control knob, the right
hand side only has a fine focus control knob.

3. Use the iris diaphragm lever to adjust contrast.

4. Repeat the process for the 40X objective.

General Biology I Laboratory Exercises Page 8

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

Note that as you magnify your specimen, you are zooming into a smaller and
smaller section of your specimen. If you are having trouble seeing black ink in this
exercise as you magnify your specimen, you may have to move your specimen in the
x/y-plane using the stage control knobs to make sure that the ink is in your field of view.

5. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

Exercise 2-5
Calculating total magnification

1. Calculate the total magnification when each of the objectives is used.

2. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

Lab Exercise 3: Relationship Between Magnification and Other

Variables
In this exercise, you will explore the relationship between magnification and other
variables in microscopy.

Exercise 3-1
Relationship between magnification and working distance

The working distance is the distance between the top of the specimen and the lens of
the objective when an image of the specimen is in focus.

1. With the ruler provided, estimate the working distance for each objective when
your specimen is in place.

2. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

Exercise 3-2
Relationship between magnification and field diameter

The field diameter is the diameter of the area viewed on the slide (field of view).
Although the diameter of your field of view does not change, the field diameter does as
you increase the magnification. A quick and easy way to measure the field diameter is to
simply use a ruler, which can be viewed when the working distance is large enough to
accommodate the width of the ruler. This can only be done using the 4X objective. You
can then use a formula to calculate the field diameter when using the other objectives.

1. Bring the sample stage down, remove your slide from the microscope. Do not
dispose of your specimen, you will be using it again. Put it in a safe place. Put the
4X objective in place. Place the ruler (cut to the dimensions of a slide) on the
microscope and clip it into the mechanical stage.

2. Bring the ruler markings into fine focus.

General Biology I Laboratory Exercises Page 9

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

3. Move the ruler in the x-plane so that one of the graduation lines is at the edge of
your field of view. Count the number of millimeters you can see. Estimate the
fraction of the last millimeter. Total: _________ mm. This is the field diameter in mm
using the 4X objective.

4. Use the following formula to calculate the field diameters at the higher
magnifications.

Formula:
Total mag.1 x Field diameter1 = Total mag.2 x Field diameter2

5. Put your specimen back onto the microscope and bring it into fine focus using the
4X objective. Use the field diameter measured to estimate the diameter of the “e”
(Hint: ask yourself how many times the letter “e” fits into the field diameter).

6. Remove the slide and dispose of it in the designated disposal bin.

7. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

Exercise 3-3
Relationship between magnification and depth of field

Depth of field refers to the thickness of the plane of focus, a two-dimensional (2-D)
plane at which an image appears to be in focus. For example, when you are looking at a
picture of a person in a movie scene, there is a certain area in front of and behind the
person that will appear in focus; that area is the depth of field. One of the challenges in
using the compound light microscope is to extract a three-dimensional (3-D) shape from
the 2-D images generated by this microscope. Veteran compound light microscope
users constantly move the sample stage up and down using the fine focus adjustment
knob when observing a specimen. By doing this, they can look at multiple slices of the
specimen and build a mental 3-D image of the specimen.

A prepared slide containing three silk threads of different colors will be used in the
following exercise to highlight the relationship between magnification and depth of
field and building a metal 3-D image of a specimen.

It is up to you to determine which thread is on the top, middle, and bottom. Using the
highest magnification to zoom into the points where the threads intersect, you will slowly
raise the sample stage from a position where the whole sample is out of focus and
determine which thread is on top (the first one that comes into sharp focus), which
thread is in the middle (the second thread to come into sharp focus), and which thread is
on the bottom (the third thread to come into sharp focus).

1. Bring the stage all the way DOWN, put the 4X objective in place, and put a
prepared slide containing the threads in the microscope. Note that these slides are
already prepared so you do not need to add a cover slip.

2. Bring the region where all three threads intersect into fine focus. Put the 10X
objective in place and bring the image into fine focus.

General Biology I Laboratory Exercises Page 10

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

3. Put the 40X objective in place and bring the image into fine focus. Use the FINE
focus knob to slowly bring the specimen DOWN until all threads are out of fine
focus.

4. While observing through the oculars, slowly turn the fine focus knob in the opposite
direction (bring the specimen up) and note which threads come into fine focus first,
second, and third.

5. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

N.B. Do not throw out these prepared slides! Remove the prepared slide and put
it back into its container.

Lab Exercise 4
Putting Away your Microscope and Cleaning your Bench Area

Since many people will be using these microscopes, it is good lab etiquette to put a
microscope (or any common equipment) back clean and in a correct manner. In addition,
these instruments contain many fragile components, so putting a microscope back
properly will avoid damage.

1. Turn the light intensity knob to its lowest position and turn off the light source.
Unplug the microscope.

2. Bring the sample stage all of the way down and remove your slide.

3. If your microscope has a slot in the revolving nosepiece where no objective is

installed, put this blank slot into place. Otherwise, put the shortest (scanning)
objective into place.

4. Move the oculars close together and center the mechanical stage.

5. Look for any obvious areas to clean up (e.g., specimen spill on the sample stage)
and use water and a tissue to clean up any messes. Use the lens cleaner and lens
tissues provided to gently clean the objective and ocular lenses.

6. Put the wound-up region of the power cord around ONE of the oculars (positioned
close together to hold it is place) and replace the plastic bag over the microscope.

7. COMPLETE THE CHECKLIST IN THE LAB REPORT SECTION

8. With TWO hands, bring the microscope to the storage cabinets and put it back
objectives and oculars facing the BACK of the cabinet. Make sure to put the
microscope back in the space dedicated for your specific microscope (match the
numbers on the microscope and storage area in the cabinet).

9. Make sure you clean up your bench area and station. Put all materials back to
where you got them from and clean your lab bench with water from the sinks at the
side benches and paper towels.

General Biology I Laboratory Exercises Page 11

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

General Biology I Laboratory Exercises Page 12

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

NAME: ______________________________ LAB SECTION: ____

LABORATORY I
INTRODUCTION TO MICROSCOPY
LAB REPORT

INTRODUCTION

1. Microscopes allow users to see specimens in the micrometerm) and

nanometer (nm) size range. Complete the table by carrying out the size
conversions.

millimeters (mm) micrometersm) nanometers(nm)

0.4
5.2
0.003

2. Complete the table to summarize the two main branches of microscopy.

Branch Main Advantage Main Disadvantage

3. Which specific type of microscope will we be using during this lab period?

_____________ _____________ microscope

4. What is the major advantage of this type of microscope (listed in question 3)

when compared to a stereoscopic light microscope?

General Biology I Laboratory Exercises Page 13

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

5. Use the letter labels in the

figure to the right to match a
microscope component to the
following list of functions.

a) Lens that collects light from the

light source and concentrates it onto
the specimen. Can be controlled to
optimize resolution and contrast of
the image viewed and reduce glare.
_____

b) Supplies the initial magnified

image of the specimen.
Magnification level can be varied by
choosing which of these to use.
_____

c) Clips a slide into place. Can be controlled to move the slide. _____

d) Magnifies the image at a fixed level of 10X and puts it in a form and position suitable
for viewing. _____

e) Adjusts the height of the sample stage relative to the objective to focus the specimen
with precision. Used to bring the image into fine focus. _____

General Biology I Laboratory Exercises Page 14

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

LAB EXERCISES

Exercise 1
Identification of Compound Light Microscope Components

1. Complete the table.

Microscope
Physical Action Role in Microscopy
Component
Moves the sample stage and Can be used to view different
Sample stage
the specimen on it in the y- areas in the specimen along
control knob
plane. the y-plane.

Mechanical stage
control knob

Coarse focus knob

Fine Focus knob

[Describe the INDIVIDUAL [Describe the role of the

action of each component (1-3)] system as a WHOLE]
Condenser System:

1. Condenser
Lenses
2. Condenser
Control Lever
3. Iris Diaphragm
Lever

General Biology I Laboratory Exercises Page 15

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

Exercise 2-3
Viewing your specimen at scanning (low) magnification

1a. Draw the orientation of the actual specimen and the magnified image you see below.

Actual Image

1b. How does the orientation of the image compare to the actual orientation of the
specimen on the sample stage? Be specific.

2. Fill in the blank. When the slide is moved to the right, the image in the field of view
moves to the ______________.

3. Fill in the blank. When the slide is moved away from you, the image in the field of view
moves ________________________.

Exercise 2-4
Further magnifying your specimen

Provide ONE (1) main reason you should NOT use the coarse focusing knob when using
the 10X or 40X objective.

Exercise 2-5
Calculating total magnification

1. Complete the table.

Total magnification when using the various objectives of our microscope.

Objective Magnification Ocular Magnification Total Magnification

4X

10X

40X

General Biology I Laboratory Exercises Page 16

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

Exercise 3-1
Relationship between magnification and working distance

1. Indicate your estimation (using the ruler provided) of the working distance for each
objective when your specimen was in place.

4X _________ mm 10X _________ mm 40X _________ mm

2. Describe the relationship between magnification and working distance by filling in

the following:

As magnification increases, the working distance _________________. Therefore,

magnification and working distance are [directly / inversely] _________________
proportional.

Exercise 3-2
Relationship between magnification and field diameter

1. Describe the relationship between magnification and field diameter by filling in the
following:

As magnification increases, the field diameter _________________. Therefore,

magnification and field diameter are [directly / inversely] _________________
proportional.

2. Complete the following table by listing the field diameters in both millimeters (mm) and
micrometers (m).

Exercise 3-2. Field diameter when using the various objectives of our microscope.

Objective Field Diameter (mm) Field Diameter (m)

4X

10X

40X

3. Estimate the diameter of the “e” specimen in micrometers.

________ m

SHOW how you calculated this diameter.

General Biology I Laboratory Exercises Page 17

Dawson College
Fall 2023
 Lab 1-Microscopy (Part A)

Exercise 3-3
Relationship between magnification and depth of field

1. Describe the relationship between magnification and depth of field by filling in the
following:

As magnification increases, depth of field _________________. Therefore, magnification

and depth of field are [directly / inversely] _________________ proportional.

2. Prepared Slide (Letter): ________

Color of thread on: Top ________Middle ________ Bottom ________

3. Why is this exercise (building a 3D-image of this specimen) best done at the highest
magnification?

Exercise 4: Putting away your microscope

Task Done (ü)

Light intensity knob turned all the way down and light switched off
Rotate the nosepiece to the 4X objective lens
Sample stage brought down to lowest position (use coarse adjustment
knob)
Slide removed and placed in appropriate disposal bin
Sample and mechanical stages centered
Microscope observed for spilled liquid or other mess and cleaned if
necessary
Wound-up region of the power cord around one of the oculars, oculars
together to hold the power cord in place
Plastic bag replaced

General Biology I Laboratory Exercises Page 18

Dawson College
Fall 2023