Microscopy Workshop Summer 2009 Objectives
The objective of this day long workshop is two-fold. 1) Learn how the microscope works. Participants
will be able to trace light paths of bright-field and fluorescent microscopes, identify major parts of the
bright-field microscope, and compare microscopy technologies: light, fluorescent and electron
microscopy. 2) Explore Microscopy Applications. Participants will perform simple stain to observe
microorganisms, use the laser scanning confocal microscope to answer a scientific question, and gain
experience with the Nikon Microscopy U. website, a microscopy education website.
Activity 1
Introduction to the Microscope and Simple Stains
In this activity we will be familiarizing ourselves with the light microscope and performing a
simple stain.
Procedure:
I. The Microscope
1. In pairs, look at prepared slides using the microscope. To focus the microscope: Begin
with the lowest magnification and with the stage in the lowermost position. Keep the
condenser low and a medium setting in the light intensity. Slowly bring the stage up using
the coarse focus until you see some color. With the fine adjustment, clarify your image
until you can see the details. Have your partner watch to ensure you do not hit the slide
with the objective.
2. To increase the magnification, rotate the next objective into place, ensure that you do not
scratch the objective on the cover slip. When ready for the 100X add a drop of oil onto
the slide. Do not use the 100X without oil as it is an oil immersion lens. Do not go back
to the 10X or 40X after applying oil to the slide as these are air objectives and the oil will
destroy the lens.
a. TIPS: The objectives are parfocal meaning when you change magnification the
specimen should still be in focus with very little adjustment needed. This also
means that once you are in focus at 100X a new slide should also be pretty much
in focus i.e. you do not have to start at the lowest magnification again.
b. To go to a lower magnification after using oil or when finished, remove the slide
(use a new one for a lower objective) and use lens paper to clean the 100X oil
immersion lens.
c. Very little oil is actually needed on the slide, so use sparingly.
d. When looking at microorganisms you must use the 100X, eventually you will be
able to start on the 100X objective.
3. Observe as many prepared slides are your group feels necessary to become comfortable
with the microscope. Do not spend too much time here as the real fun comes with the
next procedure.
II. Simple Stain
The simple stain, also known as basic or acid stain depending on the dye used, relies only on
one dye and does not confer any differentiality to the procedure. This stain simply allows us to
see under the microscope something that otherwise will not be recognizable as anything. The
advantage of this procedure is that any stain will work. You can choose from variety of stains
that are available in any common lab (methylene blue, congo red, safarin, or crystal violet).
1. In pairs, one student will use Bacillus subtilis, while the other will use Micrococcus luteus.
Make a smear; by aseptically placing a small amount of growth in a drop of sterile water.
2. Place these on the slide, making a circle in the middle of the slide. Do not extend it to the
ends of the slide or you will not find the smear. See Figure 1.
Figure 1
3. Let the smear air dry.
4. When smear is completely dry, heat fix it by quickly running the slide through the Bunsen
burner a few times. You must be careful not to burn your fingers. Use a clothes pin to
hold the slide.
5. Place the slide on the staining rack and flood with the selected stain (safarin or crystal
violet are the easiest to work with). Leave the stain on the slide for 30 sec to 1 minute
and then wash off the stain with distilled water. You can air dry or blot dry the stained
smear.
6. Observe under the scope. Have your partner watch to make sure you do not hit your
slide with the objective. Also remember not to switch to a lower objective after using the
100X oil.
7. Repeat with any additional organisms
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Observations:
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Worksheet 1
Name:
Due Date:
1. Label the microscope below:
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2
3
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2. What are the observed differences between the two microbial broth samples on which
you used the simple stain?
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3. Draw the Light path of the microscope starting from the light source to your eye. Include
all objects the light must pass through and the function of each.
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Activity 2
Fluorescent Microscopy
In this activity we will explore the Nikon Microscopy University website and perform an actual
experiment using the Laser Confocal Scanning Microscope.
I. Microscopy U.
Go to www.microscopyu.com.
This is the home page of Nikon microscopy education page.
1a. Under the Featured Sections (found on the left hand side of the page), click on
Fluorescent Microscopy.
1b. Scroll down to the Interactive Java Tutorials, click on Laser Scanning Confocal
Microscopy.
1c. This is a simulated version of what happens in a confocal microscope. On the left is a so
called wide-field image- obtained from a basic fluorescent microscope. The image on the
right is the confocal image. Select a specimen that you would like to view, then manipulate
the controls below the images. Focus and Z-axis controls will take you up and down (in the Z
plane) of the specimen. The PMT gain is determines how much light (either red or green) is
collected by the detector, in this case a PMT. The Scan Speed is how fast the pinhole scans
across the image, notice slower makes a better, cleaner, crisper image. Finally notice how
the larger you set the pinhole the more similar the image becomes to a wide field scope
image.
2. Back on the Fluorescent Microscopy page (see step 1a if you’re lost) scroll down to the
Digital Image Galleries. Click on Fluorescence Microscopy Digital Images. This shows a
variety of the types of fluorescent images that we can obtain. In each case different cellular
structures are fluorescently labeled and made visible. The images on the bottom half of this
page, are labeled (once you have clicked on them) as to what visible structure is. The
images in the galleries are quite beautiful but are not labeled as well.
3. In addition if you go back to the Fluorescent Microscopy page, in the Digital Image
Galleries, and click on Confocal Image Gallery. You can choose an image and scroll up
and down through the tissue. Check this out.
4a. Now go to the Live Cell Imaging Featured Section (from the Home page or the
Fluorescent Microscopy page) on the left hand side.
4b. Scroll down to the Digital Video Galleries, select Live Cell Imaging: Motility to observe
light microscopy images of cell motility. Quite impressive.
4c. Back on the Live Cell Imaging page, in the Digital Video Galleries, select Pond Life.
Here you can observe light microscopy images of organisms that live in our ponds.
5a. Finally take the time to explore Microscopy U. to see what else it has to offer.
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II. Laser Confocal Scanning Microscope.
In groups of 5 we will work together to use the confocal microscope to obtain images and
answer an experimental question.
Observation:
Question:
Hypothesis:
Experiment:
Results:
Conclusions:
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Observations/Notes:
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Worksheet 2
TakeHome
Go back to the Nikon Microscopy U. website, www.microscopyu.com.
Click on the Fluorescent Microscopy link under Featured Sections.
Under Review Articles, select Introduction to Fluorescent Proteins.
This is an article designed to give you exposure to what fluorescent proteins are, where we
find them, and how we use them.
After reading, answer the questions below.
1. What are fluorescent proteins (FPs) used for?
2. What was the first FP identified?
3. List some additional FP colors.
4. Now the hard part, how do you tag your protein of interest with an FP?
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Worksheet 2
Key
1. What are fluorescent proteins (FPs) used for?
To label/tag a protein/cellular structure of interest “to monitor cellular processes in living
systems using optical microscopy”
2. What was the first FP identified?
GFP (green fluorescent protein) in 1961 by Osamu Shimomura and Frank Johnson form the
Aequorea victoria jellyfish. The gene for GFP was not cloned until 1992.
3. List some additional FP colors.
See Table 1, the most common include blue (BFP), yellow (YFP), green (GFP), red
(RFP/dsREDFP), and cyan (CFP).
4. Now the hard part, how do you tag your protein of interest with an FP?
Using molecular cloning techniques you create a chimera protein. Using a vector/plasmid
containing the FP desired, one clones the gene of interest into the vector in frame with the
FP. This DNA construct is then introduced into the organism of interest, gene expression
occurs (transcription and translation) producing a chimera protein, half FP and half your
protein of interest, thus your protein now fluoresces when excited by the correct light
wavelength.
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