Lab 2: Analysis of Cells by Microscopy
Introduction
"Micro" refers to tiny, "scope" refers to view or look at. Microscopes are tools used to enlarge
images of small objects so as they can be studied. Microscopes range from a simple magnifying
glass to the expensive electron microscope. The compound light microscope is the most common
instrument used in education today. It is an instrument containing two lenses, which magnifies,
and a variety of knobs to resolve (focus) the picture. It is a simple piece of equipment to
understand and use. In this lab, we are going to learn the proper use and handling of the
microscope.
Objectives
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Demonstrate the proper procedures used in correctly using the compound light
microscope.
Prepare and use a wet mount.
Determine the total magnification of the microscope.
Develop a checklist to insure the proper handling of the microscope.
Materials
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Compound microscope
Glass slides
Cover slips
Eye dropper
Beaker of water
The letter "e" slide
Colored thread slide
Toothpick
Methyl blue
Various living samples: Elodea, yeast, red blood cells, cheek cells
Proper Handling of the Microscope
1. Carry the microscope with both hands --- one on the arm and the other under the base of
the microscope.
2. One person from each group will now go over to the microscope storage area and
properly transport one microscope to your working area.
3. Remove the dust cover and store it properly. Plug in the scope. Do not turn it on until told
to do so.
4. Examine the microscope and study the function of each of the parts found below.
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Eyepiece Lens: the lens at the top that you look through. They are usually 10X or 15X power.
Tube: Connects the eyepiece to the objective lenses
Arm: Supports the tube and connects it to the base
Base: The bottom of the microscope, used for support
Illuminator: A steady light source (110 volts) used in place of a mirror. If your microscope has
a mirror, it is used to reflect light from an external light source up through the bottom of the
stage.
Stage: The flat platform where you place your slides. Stage clips hold the slides in place. If
your microscope has a mechanical stage, you will be able to move the slide around by turning
two knobs. One moves it left and right, the other moves it up and down.
Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can
be rotated to easily change power.
Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost
always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common)
eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. The
shortest lens is the lowest power, the longest one is the lens with the greatest power. All quality
microscopes have parfocal lenses.
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Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen.
Condenser lenses are most useful at the highest powers (400X and above). Microscopes with in
stage condenser lenses render a sharper image than those with no lens (at 400X).
Condensor Diaphragm: Many microscopes have a rotating disk under the stage. This diaphragm
has different sized holes and is used to vary the intensity and size of the cone of light that is
projected upward into the slide. There is no set rule regarding which setting to use for a
particular power. Rather, the setting is a function of the transparency of the specimen, the
degree of contrast you desire and the particular objective lens in use.
Field Diaphragm: At the light source, a diaphragm controls the diameter of the light that is
allowed to emit. With each objective, the field diaphragm should be controlled to permit the light
to circumscribe the field of vision.
Köhler Illumination: Principles of Light Microscopy and Factors Related to Resolution
The light microscope is a critical tool in studies ranging from subcellular structure and function
to pathology, embryology, gene expression, and gene mapping. For many of these purposes, the
limits of resolution of the light microscope must be exploited to the fullest potential. For optimal
results in a given application, the microscope should be equipped with high-quality optics
(objectives, eyepieces, and condensers), be precisely aligned, and make use of the appropriate
light sources, filters, and contrast enhancement devices (e.g., phase contrast).
The first and most critical step in setting up a microscope for optimal resolution involves the
mechanics of Köhler illumination. Köhler illumination was first described in 1893 by August
Köhler, a young zoologist in Giessen, Germany, who later joined Carl Zeiss. It provides
efficient, bright, and even illumination in the specimen field, minimizes internal stray light, and
allows for control of contrast and depth.
The advantages of Köhler illumination are listed below.
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Only the specimen area viewed by a given objective/eyepiece combination is illuminated;
no stray light or "noise" is generated inside the microscope.
Even, uniform illumination of the specimen area is achieved by distributing the energy of
each source point over the full field.
Full control of the illumination aperture provides for best resolution, best contrast, and
optimal depth of field.
Adjusting the microscope for Köhler illumination will be demonstrated by your instructor.
Specific details are provided below.
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Focus on any specimen feature.
Close down the field diaphragm.
Focus the condenser until the image of the field diaphragm is sharp.
Center and focus the light source.
Fill the circular area with the image of the light source.
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6. Slowly close condenser aperture diaphragm for best contrast and resolution. The optimal
setting for this condenser diaphragm is largely dependent on contrast features inherent in
the specimen.
Examination of the letter "e”.
1. Turn on the microscope and place the slide on the stage; making sure the "e" is facing the
normal reading position. Using the course focus and low power, move the body tube down until
the "e" can be seen clearly. Draw what you see in the space below.
2. Describe the relationship between what you see through the eyepiece and what you see on the
stage.
3. Offer an explanation of why this happened.
4. Looking through the eyepiece, move the slide to the upper right area of the stage. What
direction does the image move?
5. Now, move it to the lower left side of the stage. What direction does the image move?
6. Re-center the slide and change the scope to high power. You will notice the "e" is out of focus.
Do Not touch the coarse focus knob, instead use the fine focus to resolve the picture.
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Determining Total Magnification:
1. Locate the numbers inscribed on the eyepiece and the low power objective and fill in the
blanks below.
Eyepiece magnification
______________
(X)
Objective magnification
=
______________
Total Magnification
_____________X
2. Do the same for the high power objective.
Eyepiece magnification
______________
3.
(X)
Objective magnification
=
______________
Total Magnification
_____________X
Write out the rule for determining total magnification of a compound microscope.
Depth of Field
The depth of field is the distance through which you can move the specimen and still have it
remain in focus.
1. Obtain a prepared slide of three crossed colored threads. Once you have the threads in focus,
use the fine focus know to focus with the4x, 10x and 40x objective.
How many threads are in focus?
At which magnification is it the most difficult to focus the threads?
2. Using the 40x objective, determine the order of the three threads mounted on the slide. Record
the results.
3. Remove and put away the slide.
Examining Cells: preparing a wet mount.
1. Using a Pasteur pipette, place one drop of yeast culture on a microscope slide.
2. Place one end of a glass coverslip to the right of left of the specimen so that the rest of the
slips held at a 45o angle over the specimen.
3. Slowly lover the coverslip to avoid trapping air bubbles.
4. Observe the wet mount, first at low magnification and then at higher power. Draw what you
see. Clean the slide.
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Yeast:
5. Repeat the process to visualize Elodea, red blood cells, various Paramecium (add Protoslo- a
methyl cellulose solution that slows down swimming microorganisms) and cheek cells with and
without methyl blue.
Elodea
red blood cells
Ptotozoa 1
Protozoa 2
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Cheek cells without methyl blue
Cheek cells with methyl blue
Remove the slides and clean it. Turn off the microscope. Place the low power objective in place
and lower the body tube. Cover the scope with the dust cover. Place the scope back in its original
space on the storage cart.
A variety of microscopic techniques exploit light properties to enhance contrast
Contrast mode
Mechanism
Comments
Bright field
contrast depends on light
absorption
usually used in conjunction with
histological stains to boost contrast
Phase contrast
converts optical path
differences to intensity
differences
contrast proportional to local "phase
dense" objects including mitochondria,
lysosomes, chromosomes, nucleoli, and
stress fibers
Differential
interference
contrast (DIC)
converts rate of change of
optical path across specimen
cell and organelle edges where optical
path abruptly changes stand out in relief
Dark field
scattered light observed
produces images of cell and organelle
edges
Polarization
detects birefringence caused
by supramolecular
organization below optical
resolution
used to study oriented arrays such as
cytoskeletal structures (e.g., microtubules
in the mitotic apparatus and stress fibers);
also used to study membranes
Fluorescence
contrast depends on
absorption of light by
fluorophore and its quantum
yield
limited only by appropriate fluorescent
probes
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