Comparing_Microscopes_v2c.docx
ComparingMicroscopes
Students compare the characteristics of a stereo dissecting scope and an inverted microscope.
1
1.1
OBJECTIVES
EXPERIMENTAL GOAL
In this introduction to the characteristics and use of microscopes, students will measure the field of view
and depth of field at different magnifications on two different kinds of microscope.
1.2
PREREQUISITE SKILLS AND KNOWLEDGE
Students are expected to have completed Optics 1 and 2.
1.3
RESEARCH SKILLS
After this lab, students will have had practice in:
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1.4
following laboratory protocols
using a laboratory notebook
using a dissecting scope
using an inverted microscope
calculating magnification of a microscope
estimating the size of an object viewed through a microscope
measuring the field of view of a microscope
LEARNING OBJECTIVES
After this lab, students will be able to:
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describe the relationship between magnification and field of view for microscopes
choose the appropriate microscope for the subject and task
2
2.1
PRE-EXPERIMENT ASSIGNMENT
THE MICROSCOPES
You will compare the characteristics of two kinds of microscope used in the X-Lab: a stereo microscope
and the Olympus CKX41 Culture Microscope. You may already have used the stereo microscope to view
living subjects; in this exercise you will examine the stereo microscope itself and compare its
characteristics to those of the inverted culture microscope.
2.1.1
The Stereo Microscope
A stereo microscope such as the one you will be using is often called a dissecting microscope. Why? The
image below shows a stereo microscope similar to the one you are using. Make sure you can find each
part.
Often the subject on the stage of a stereo microscope is illuminated only from above, but the stereo
microscopes you will be using have a light source below the stage, as well as one above it. If the abovestage light source is used, a black or white opaque disc can be inserted into the stage instead of the
transparent disc.
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Figure 1: Stereo microscope
2.1.2
The Inverted Culture Microscope
Some components of a culture microscope are the same as those in the stereo microscope, while some are
very different. Take some time to familiarize yourself with the identification and location of the specific
components labeled in the diagram (Figure 2) and described in more detail below the diagram.
Figure 2: Components of the inverted microscope.
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Oculars: The lenses through which you observe the image from the microscope.
Camera: A digital, monochrome (“black and white”) camera that allows you to record the image
from the microscope. Note that its field of view is less than what you can observe through the
oculars.
Halogen lamp: Produces light for transmitted light microscopy.
Phase contrast slider: Contains ring slits that, when slid into the transmitted light path prior to the
sample, allow you to view the sample using phase contrast optics.
Stage: Flat plate onto which the sample is placed.
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Mercury lamp: A lamp that produces very intense light for fluorescence microscopy. It produces
broad spectrum light, including UV light, so it has the potential to damage your retina. Make sure
you know how to safely operate the microscope in fluorescence mode before turning on the
mercury lamp.
Objectives: Lenses that collect light from the sample and magnify the image produced.
Filter cubes: Sets of optical filters and mirrors that reflect and filter the excitation light. There are
three cubes: U (passes UV light), B (passes blue light), and G (passes green light).
Focus: Dial that changes the height of the objectives. The outer dial is coarse focus, and the inner
dial is fine focus.
Lamp switch: Toggle switch to turn the halogen lamp on and off.
Light intensity dial: Rotary dial to change the intensity of the tungsten lamp.
2.1.2.1 Transmitted Light Microscopy
In transmitted light microscopy, light from a tungsten or halogen lamp passes through the sample, into the
objective, and then to the oculars and camera (Figure 3). The simplest and most common form of
transmitted light microscopy is referred to as bright field or Köhler illumination microscopy.
Figure 3: Light path through the inverted microscope in transmitted light microscopy.
2.1.2.2 Amplitude and Phase Contrast Microscopy
In order to see structure in a specimen through the microscope, there must be contrast between different
structures in the specimen. The contrast in bright field microscopy is due to differences in the amplitude
(brightness) of the light transmitted by the structures. If an object appears colored under bright field
microscopy, it does so because it absorbs light of specific wavelengths and transmits the wavelengths that
result in the color observed. Areas where no light is transmitted appear black.
Unless they contain pigments, many cell organelles are undistinguishable under bright field microscopy,
because there is no difference in the amplitude of the light they transmit. Phase contrast microscopy takes
advantage of differences in refractive index to make these otherwise clear structures visible. These
organelles refract (retard the phase of) the light passing through them by a fraction of a wavelength, more
or less, depending on their composition.
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Unaided, our eyes cannot discern these phase differences,
but the phase contrast method, invented in the 1950’s by
Fritz Zernike, provides a mechanism for transforming
phase changes into changes in amplitude. A phase contrast
apparatus requires a phase plate that retards light by exactly
one-quarter wavelength and a phase annulus consisting of a
clear ring on a black field. By destructive and constructive
interference of the unrefracted illuminating light and the
light refracted by passing through the specimen, the phase
changes can be made apparent.
2.2
2.2.1
CHARACTERISTICS OF MICROSCOPES
Field of View (FOV)
The field of view is the diameter of the area you see when looking into a microscope. It is traditionally
measured in microns (micrometers, µm). For each microscope, you will measure the field of view at
several magnifications, in order to determine the relationship between magnification and field of view.
You will use a ruler to measure the diameter of your field of view. The kind of ruler will vary with the
microscope. To measure the field of view of the stereo microscope, you will use a transparent millimeter
ruler, while for the culture microscope you will use a slide with a grid inscribed onto the coverslip. The
coverslip grid pattern consists of 9 squares, with different grids inscribed into different squares. The four
corner squares are divided into 16 smaller squares, while the center square is divided into 100 small
squares. What is the width of just one small square from the center square?
Figure 6: Field of view measured with a transparent millimeter ruler (left). Grid inscribed onto the slide coverslip (right).
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Figure 7: You can often use the known diameter of your field of view
to estimate the size of the organism you are observing.
What is the approximate length of the organism in this field of view?
2.2.2
Depth of Field (DOF)
Depth of field is measured in the direction perpendicular to the plane of the field of view. It is the distance
from the nearest object plane in focus to the farthest object plane in focus.
Figure 8: Only the red thread is in focus in this image.
Measuring depth of field is less straightforward than measuring the field of view. To measure depth of
field you will need to measure the distance between the object in focus closest to the objective and the
object in focus farthest from the objective. If the depth of field is a few micrometers, it can be measured
by stacking several transparent rulers and counting the number of rulers in the stack that are in focus.
Multiplying that number by the thickness of the ruler will give a good estimate of the depth of field.
2.2.3
The Limits of Light Microscopy
Microscopes can be powerful tools for observing the structures within organisms, and even within cells.
A light microscope allows you to view plant and animal cells and some cell organelles, but an electron
microscope is required to render the detail in cell organelles. Examine the Chart on the next page.
In the culture microscope, the light path goes through the specimen. This arrangement can allow you to
see structures with great detail, but also places limits on the thickness and opacity of the specimen. The
specimen must be thin and/or transparent enough to transmit light. Reflected light, however, is often used
to view objects with the stereo microscope.
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2.3
PREPARE FOR THE EXPERIMENT
Read through the entire Laboratory Manual to prepare your lab notebook. Consider carefully how you
will answer the questions. When you feel ready, test your preparation with the Pre-Experiment Quiz.
Prepare a spreadsheet to record your data. Set up any necessary calculations. Mail a copy of the
spreadsheet to yourself for use during the lab.
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3
3.1
LABORATORY MANUAL
MATERIALS CHECK OFF LIST
Each small group of (1-2) students will have:
Vernier caliper
Several clear plastic millimeter rulers
Slide with grid-inscribed coverslips
specimen slides
Piece of paper with text
Each large group of 1-2 small groups will share:
stereo dissecting microscope
inverted epi-fluorescence culture microscope
Box of lab wipes
3.2
SAFETY AND WASTE DISPOSAL PROTOCOLS
A lab coat, leg coverings, and closed-toe shoes are required in the X-Lab. Eating, drinking, or applying
make-up is not allowed within the laboratory.
Save all slides and rulers at the bench.
3.3
EXPERIMENTAL PROCEDURE
Your group will spend a portion of the lab period with the stereo microscope and the same length of time
with the inverted culture microscopes. You will need to complete the assigned measurements using both
kinds of microscope.
For each microscope, you will measure the field of view (FOV) and depth of field (DOF) at several
magnifications, in order to determine the relationship between magnification and each characteristic.
3.3.1
The Stereo Microscope
Examine the stereo microscope and make sure you can identify each part.
3.3.1.1 Determine magnification
The total magnification of the stereo microscope is a product of the magnification of the oculars and the
magnification of the objectives.
Q1. What magnifications are possible for the stereo microscope?
3.3.1.2 How to adjust the stereo microscope
As with the culture microscope, you will need to adjust the stereo microscope to fit you.
1. Turn the magnification adjustment ring to the lowest magnification.
2. Place the piece of paper with text onto the stage and adjust the light so it is illuminating the paper
from above.
3. Examine the oculars. Determine if only one of them has a diopter, or if both do.
Q2. Do both oculars have diopters or does only one?
4. If only one ocular has a diopter, cover that ocular with a card or folded piece of paper. If they both do,
then pick one to cover.
5. Look through the open ocular and adjust the focus knob until the text is clear.
6. Now move the card to the other ocular and adjust the diopter until the text is clear.
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7. Look through both oculars and adjust the distance between them until you see one image.
8. Turn the magnification adjustment ring to the highest magnification.
9. Adjust the fine focus until the text is clear.
Q3. What happens to the focus when you turn the magnification back to the low end?
3.3.1.3 Measure field of view at both magnifications
To measure the field of view of the stereo microscope, you will use a transparent millimeter ruler.
Measure the field of view at the lowest magnification first, and then move on to the higher magnification:
1. Place the transparent ruler onto the white disc in the stage, or place a piece of white paper on the stage
with the ruler on top of it.
2. Select the magnification and adjust the oculars, light level, and focus so that you can comfortably
view the ruler.
3. Place one edge of the ruler at one edge of your field of view. Measure the distance to the other
opposite edge of your field of view.
4. Record the field of view in micrometers µm at each magnification. (Note: Both partners should
measure the field of view at lowest magnification and compare results and methods before continuing
to higher magnifications.)
Q4. Plot your results in a meaningful way and paste a snip of your plot here.
Q5. What relationship did you find between magnification and field of view?
Q6. Use the measured diameter of the field of view to estimate the size of one of the organisms on the
prepared slides. Report the name of the organism and your method for estimating the size.
3.3.1.4 Measure depth of field (DOF)
1. Starting at the highest magnification, stack the printed transparent rulers until you reach the limit of
the depth of field.
2. Measure and record, in micrometers, the thickness of the stack.
3. Repeat this measurement for each possible magnification.
Q7. At lower magnifications you may run out of transparent sheets. Suggest and test another way to
measure the depth of field.
Q8. Plot your results in a meaningful way and paste a snip of your plot here.
Q9. What relationship did you find between magnification and DOF?
3.3.2
The Inverted Culture Microscope
Examine the inverted microscope and make sure you can identify each part.
3.3.2.1 How to adjust the Olympus CKX41 culture microscope
3.3.2.1.1 Adjusttheoculars
1. For bright field and phase contrast microscopy, make sure the filter cube slider is pushed all the way
to the right. The letters U, B, and G should all be visible
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2. Push the phase contrast slider all the way to the left, to allow all the light through.
3. Turn the revolving nosepiece so that the 10x
objective is engaged beneath the stage center
plate.
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4. Place the specimen slide or slide with gridinscribed coverslips in the slide holder and
use the X-axis and Y-axis knobs to move the
specimen/grid into the light path.
5. Turn on the halogen lamp and adjust the brightness. Start at the lowest setting (all the way to the
bottom) and increase as needed.
6. Use the coarse and fine adjustment knobs to focus the specimen.
7. Adjust the interpupillary distance. Look through the oculars and adjust the distance between them
until the left and right fields of view coincide completely. The index dot indicates your interpupillary
distance; make a note of it for future reference.
The index dots show an interpupillary distance of
60.
8. Looking through just your right eye and the right ocular, adjust the focus using the fine focus knob.
9. Looking through just your left eye and the left ocular, turn the diopter adjustment ring until the
specimen is in focus. Make a note of your diopter adjustment for future reference.
This user’s diopter setting is about +0.5.
3.3.2.2 Using Phase Contrast
Move the phase contrast slider so the appropriate light annulus is centered under the light source. The
empty annulus is for bright field microscopy. The other two are labeled to coordinate with the
magnification (4, 10, 20, or 40) of the objective in use. Choose the correct annulus for your objective.
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View your specimen under both bright field and phase contrast.
Q10. What differences do you notice?
3.3.2.3 Determine magnification
The total magnification of the inverted microscope is a product of the magnification of the oculars and the
magnification of the objectives.
Q11. What magnifications are possible for the culture microscope?
3.3.2.3.1 Measurefieldofviewateachmagnification
Measure the field of view at the lowest magnification first, and then move on to higher magnifications:
1. Place the slide with grid-inscribed coverslips into the slide holder and adjust the position to place one
of the grids within the light path.
2. Select the magnification and adjust the oculars, light level, and focus so that you can comfortably
view the grid.
3. Place one edge of the grid at one edge of your field of view. Measure the distance to the other
opposite edge.
4. Record the field of view and magnification. (Note: Both partners should measure the field of view at
lowest magnification and compare results and methods before continuing to higher magnifications.)
5. Repeat 1-4 for the remaining magnifications.
Q12. Plot your results in a meaningful way and paste a snip of your plot here.
Q13. What relationship did you find between magnification and field of view?
3.3.2.3.2 Measuredepthoffield(DOF)
1. Starting at the highest magnification, stack the printed transparent sheets rulers until you reach the
limit of the depth of field.
2. Measure and record, in micrometers, the thickness of the stack.
3. Repeat this measurement at two different magnifications.
Q14. Plot your results in a meaningful way and paste a snip of your plot here.
Q15. What relationship did you find between magnification and DOF?
3.4
POST-LAB ASSIGNMENT
Q16. Can the field of view and depth of field data sets for both inverted and stereo microscope be
described by the same equation? Explain.
Q17. Use the measured diameter of the field of view to estimate the size of one of the organisms on the
prepared slides. Report the name of the organism and your method for estimating the size.
Compare the two kinds of microscopes. Think about what each kind would best be used for.
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Q18. Describe at least one limitation of each type of microscope.
Q19. Describe possible uses for each kind of microscope.
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