LAB 1:
LIGHT MICROSCOPY IN CELL BIOLOGY
I. Pre-lab preparation.
In addition to reading this lab and preparing your lab notebook, be sure to read Ch. 6, p. 94-97 of
your textbook. Also, note that you should bring the hand-out distributed last time: “Care and Use of
the Olympus CH30 Compound Microscope”.
II. Objectives
In this lab you will reinforce your understanding of the use of compound microscopes as tools in
studying cell and molecular biology and gain experience in the care and use of the Olympus compound
scopes we will use in Bio 2. In addition, you will be introduced to the use of differential interference
microscopy (DIC) and fluorescence microscopy and their applications in biology. By the end of this
lab, you should:

Be able to identify the parts of the Olympus CH30 microscope and describe the
function of each

Understand and be able to describe the basic types of compound microscopes and
their uses

Be able to prepare and view wet mounts and prepared slides

Be able to utilize a stage micrometer to calibrate ocular micrometers and then use
the ocular micrometer to measure organisms you are viewing

Understand and be able to explain the basic principles of DIC microscopy and
fluorescence microscopy

Gain exposure to the techniques used to culture animal cells and

Be able to recognize animal cells growing in culture using an inverted compound
microscope
I. Safety Considerations
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
In this lab, there are no toxic chemicals used, biohazards to be handled, or other specific
hazards.

While there are no specific hazards to highlight, as always, be aware of all general lab safety
considerations—handle glass carefully, do not allow microscope cords to hang loose to trip
others, etc.
II.
Introduction
Microscopy has been, and continues to be, one of the most important techniques used to understand
cell biology. The two major types of microscopes, light microscopes and electron microscopes, allow
us to view cells and cellular structures that we cannot see with the naked eye. Light microscopes are
able to magnify up to about 1000x so that we can see objects that are 0.1 micrometers (µm), or 100
nanometers (nm) in diameter and transmission electron microscopes allow us to view objects that are
0.5 nm in diameter or 1/200,000 th the size of objects that we can see with the naked eye.
Our ability to see objects with a microscope does not depend simply upon the magnification we can
achieve but depends upon our ability to resolve what we are seeing. Resolution or resolving power
(RP) is defined as the smallest distance between two points that allows the two points to be
distinguished as being separate. In its simplest form, RP can be expressed as:
RP= /2 NA
where  = the wavelength of light and NA = the numerical aperture of the lens.
This formula helps us understand that large difference in resolution of a light microscope and an
electron microscope.
Using a filter that allows just blue light ( = 400 nm) to illuminate our
specimen, and assuming an NA of 1, RP = 400 nm/2 = 200 nm. Therefore two mitochondria that are
200nm apart could be resolved as separate objects. Using an electron microscope ( = 0.005 nm), RP
= 0.005 nm/2 = 0.0025 nm! How does this compare to the thickness of the plasma membrane?
The NA of each lens is indicated on it. NA is essentially the light gathering ability of the lens. A high
NA lens collects light across a greater angle than does a lower NA lens. Lenses with increasing NA’s
increase in cost.
One limitation in the ability of lenses to capture light across a broad angle results
from the refraction of light as it passes from the sample into air. Oil immersion lenses increase the NA
by replacing the air between the sample with oil which decreases the amount of diffraction since oil
and glass transitions cause less refraction than occurs at glass/air transitions.
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III. TYPES OF MICROSCOPY
Much of what we are able to accomplish in cell and molecular biology using microscopes depends upon
combining the correct type of scope with some additional method to enhance the sample that we are
viewing to bring out features that are of interest to us. In some instances, no enhancement is
necessary. As most, if not all, of you have done, we can simply prepare a wet mount and observe our
sample without staining. Doing this with pond water, for example, allows us to see the wide array of
microscopic organisms that are present. Very often, though we choose to stain cells or label particular
cells or structures and then use the appropriate type of scope and illumination to be able to view our
target well. Chapter 6, p. 94-97 provides a nice overview of this.
Let’s start working with our scopes to illustrate some of the key differences between scopes and their
basic uses.
IV. Things To Do
A. LIGHT MICROSCOPES
1. Stereo (dissecting) microscopes. Several of these are set up on the side bench.
What are three key differences between this type of compound scope and compound
scopes such as the Olympus CH30 that you will begin using in the next section?
i. Hints: How is a stereo image generated? How is the lighting of the object
being observed accomplished?
ii. Record your answers in your lab notebook.
2. Compound scopes: Olympus CH30
i. This is the scope that we will use routinely. Retrieve a scope (one for each
student) from the shelves on the side of the room. Handle with both hands.
ii.
Use the separate hand-out “Care and Use of the Olympus CH30 Compound
Microscope” and complete exercises #1 and #2.
NOTE on eyeglasses and the use of microscopes: If you are nearsighted or farsighted, it
is not necessary to wear your glasses as the scope can be focused to correct for this. The
scope cannot correct for astigmatism, though.
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3. Retrieve a slide of the letter “e” from the front bench. Hold the slide so that the e is in
the correct orientation for reading. Now place the slide on the stage and observe it
through your microscope.
i. What orientation is the letter when viewed through the scope?
ii. How would it appear in a dissecting scope?
iii. Looking from the side, move the stage to the right.
iv. Now, repeat this looking through the oculars. What direction does the stage
appear to move?
4. Next, grab a slide with colored threads and observe these at each magnification
beginning with the lowest power objective progressing to the highest power objective.
How does the brightness of the field of view change?
i. What is a reasonable explanation for this?
5. Return to the lowest power objective and focus on the point at which the threads
cross. Repeat the transition from low to high power. At each magnification, note the
depth through which the sample is in focus.
i. How does depth of focus change with increasing magnification?
ii. By assembling successive planes that are in focus it is possible to assemble a
three-dimensional view of the specimen. Confocal microscopes are coupled to
a computer to collect and assemble successive “optical sections” to produce an
image that is in focus across a wide depth.
6. Measuring cells and structures viewed through compound microscopes.
i. Retrieve a stage micrometer from the front bench--these are protected in
individual cases and look like microscope slides. Each has a microscopic ruler
etched on it. Hold this up to the light and you’ll be able to see it.
ii. Also retrieve an ocular micrometer.
This looks like the ocular of your scope.
Replace the non-focusing ocular of your microscope with this ocular.
iii. Place stage micrometer on the stage and bring it into focus at the lowest
power.
Rotate the ocular micrometer and adjust the stage so that the
divisions of the ocular micrometer line up with those of the stage micrometer.
Once you have completed this, calculate the size of the divisions of the ocular
micrometer by determining the length of 10 divisions of the ocular micrometer
using the stage micrometer and then divide this length by 10.
1. Record the calculations and length of one division of the ocular
micrometer in your notebook
2. Repeat this process at each magnification
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iv. Prepare a wet mount of several of your hairs as follows:
1. Place one or several hairs on a microscope slide
2. Pipet a small volume of water onto the hair (s)
3. Drop a cover slip over the sample capturing as few air bubbles as
possible.
To do this, place edge of the cover slip on the slide and
gently lower it over the sample. This should force most bubbles out
the side.
a.
Before looking at the slide in the scope, predict how thick you
predict a hair will be: _____________ (fill in your prediction
here or in your lab notebook).
4. Using your ocular micrometer, measure the thickness of a hair and
record that in your notebook. _____________ (fill in your prediction
here or in your lab notebook).
5. Compare your measurement with those of three others in the lab and
record those measurements here ____________ or in your notebook.
6. Record any other interesting observations about the structure of a hair
in your notebook.
7. Are hairs made of cells? Can you answer this by examining them
microscopically?
v. Examining Elodea
1. Retrieve a single Elodea leaf from the sprigs that are provided.
They’re submerged in water since this is an aquatic plant. These were
collected here on the American River.
2. Prepare a wet mount of this leaf as you did in viewing hair:
a.
Place the leaf on a microscope slide
b.
Pipet a small volume of water onto the leaf
c.
Drop a cover slip over the sample capturing as few air bubbles
as possible.
To do this, place edge of the cover slip on the
slide and gently lower it over the sample. This should force
most bubbles out the side.
d.
Before looking at the slide in the scope, predict how big you
expect an Elodea cell will be: _____________ (fill in your
prediction here).
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e.
How big do you predict a chloroplast will be: ___________?
f.
Examine your wet mount at several magnifications.
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g.
Identify the chloroplasts and note their distribution. Sketch
their distribution in several cells.
h.
Are the chloroplasts moving?
i.
Using your ocular micrometer, measure the dimensions of four
or more cells. What are the average dimensions of the cells?
How does this compare with your prediction?
j.
Now, using the ocular micrometer again, measure the sizes of
several chloroplasts. How does this compare with your
prediction?
k.
Sketch their position again. Have their positions changed
significantly?
l.
If they have changed position, why do you think this occurred?
m. How could you test this experimentally?
n.
How do you think cells are able to move chloroplasts around
inside the cell?
What cellular components do you think are
necessary for this?
7. Differential Interference Microscopy (DIC) and Phase Microscopy--Demo
i. We have set up a scope that is capable of DIC here in the lab. In both DIC
and phase microscopy compound scopes are used as they are in brightfield
microscopy that you have just been doing. In order to carry out these types
of microscopy several components are added to the scope that enhance the
image that we see. Essentially, the key in both of these types of microscopy is
to illuminate the sample with polarized light. The interference pattern
generated as this polarized light hits the sample exaggerates some features
which enhances the image.
ii. We will use an Elodea wet mount as an example of the enhancement of the
image compared to your brightfield image. As the scope becomes available
take a turn looking through this scope to observe the image. A camera
mounted on the scope will also allows us to project images to the class for all
to see.
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8. Inverted Compound Microscopy--Demo
i. In the microscopy that we have done so far, we have placed our samples on
microscope slides and covered them with cover slips. In order to observe
them, it was necessary to lower the objective into close proximity to the cover
slip. In many instances, we would like to view samples that don’t fit
conveniently onto a slide.
Often, these are samples of living organisms
growing under sterile conditions in containers that won’t fit on an upright
scope.
ii. One example of living cells used extensively in cell and molecular biology are
animal cells growing in culture. A wide variety of these are grown, including
stem cells. We will discuss what we have learned from cultured cells and how
they are used in biology in a number of instances during the semester. This is
a chance to look at them first hand and see how they are grown.
iii. A variety of containers are used to grow cells. The flasks that we will look at
today are one common type of container used. As you examine the flask
you’ll see liquid in the flask. This is growth medium that contains the
components necessary for the cells to grow. You’ll also notice that the flasks
can be sealed. This allows the flasks to be prepared or treated under sterile
conditions in a laminar flow hood and then closed before being removed from
the sterile conditions of the laminar flow hood.
iv. Cultures are started with small numbers of young cells that then grow across
the bottom surface of the flask.
v.
To observe the cells we can use an inverted scope. This type of scope allows
the objective to approach the sample from below. Since cells growing in
culture are on the lower surface of the flask they are readily observed with an
inverted scope.
vi. We’ll step across the hall and observe flasks of growing cells.
vii. Based on the measurements you made earlier at each magnification, about
how big do you think these cells are?
9. Fluorescence Microscopy
i. Of course, in many instances we’ll enhance our samples by using a variety of
staining or labeling techniques that will help us identify specific cell types and
subcellular structures and measure important biological activities such as
enzyme activity and gene expression.
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ii. Many of the most important techniques in cell and molecular biology use stains
or tags that are fluorescent. Using these techniques we are able to label cells
or structures with molecules that are fluorescent and then use microscopy to
observe the results. For fluorescence of the tag to be observed, the sample is
illuminated with light of the correct wavelength to be absorbed by the
fluorophore (molecule capable of fluorescing). Upon absorption of light of the
correct wavelength electrons in the fluorophore abaorb enough energy to
move to a higher orbital. As the electron drops back to its original orbital light
of a longer wavelength emitted as fluorescence.
iii. Fluorescent scopes are able to supply light of the correct wavelengths to excite
fluorophores and then use filters to collect the fluorescence while excluding the
exciting wavelengths.
iv. We’ll use one of our fluorescence scopes across the hall from our lab to
observe a fluorescently labeled sample.
v.
Observe the sample. Record your observations in your lab notebook. Can you
determine what types of cells these are and what structures are labeled?
V. Lab Clean-Up
Before leaving lab today:

Rinse off used microscope slides and set them out to dry on the trays provided.

Return microscopes to their cabinets

Tidy up everything at your lab bench for the next lab section.

Wash your hands.
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