BIO 7505: Biology: A Molecular Approach, Laboratory
Microscope Theory and Operation
I. What Microscopy Is:
As your instructor lectures them, write-in the following definitions:
1. Microscopic anatomy
2. Compound light microscope
3. Magnification
4. Resolution
5. Contrast
6. Brightness
II. Proper Care of a Compound Light Microscope:
Read the following instructions for how to care and transport a compound light microscope. After you have
read the instructions, go to the microscope cabinet, and safely transport a microscope to your lab bench.
Uncover the scope but please do not plug it in or turn it on yet.
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Always pick-up the microscope by the arm AND the base simultaneously.
Always be sure the cored is secured before moving the scope anywhere or using it. Microscopes are
literally worth thousands of dollars. The cord needs to be secured to prevent accidents. If someone trips
over a cord or catches a cord when they walk by a bench and the microscope falls on the floor it will
break.
Never touch the lens (eyepieces or objectives) with your hands. To change lenses, turn the rotating
nosepiece (also called a turret) but NEVER touch the lenses themselves. They unscrew and are
extremely expensive to replace should one fall on the floor and crack.
Start with the lowest power and move to high power objectives to focus your microscope. ALWAYS
carefully watch the lenses move in place above the specimen. You can scratch the lens by rubbing it
against the slide!
Never leave slides on the stage of the microscope.
Before you put away your microscope, clean it as described below.
When you put away your microscope, rotate the 4x objective into place, lower the stage to its lowest
point, secure the cord, and cover the microscope.
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III. Identifcation and Function of Microscope Parts:
Use the diagam on the next page to identify the following microscope parts. Then, as the instructor lectures
them, write-in their functions.
1. Ocular lens (eyepiece)
2. Interpupillary distance
3. Revolving nosepiece
4. Objective lenses
5. Specimen holder (mechanical stage)
6. Stage
7. Knobs controlling movement of mechanical stage
8. Condenser
9. Iris diaphragm level
10. Illuminator condenser
11. Light intensity control knob
12. Base
13. Arm
14. Coarse focus knob
15. Fine focus knob
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Figure 1- Compound Microscope
(http://www.digitalsmicroscope.com/wp-content/uploads/2011/04/Compound-Light-MicroscopeParts.jpg)
IV. Cleaning Microscope Lenses:
Read the information below. Then, obtain lens paper and isopropyl alcohol, and clean your microscopes
eyepieces, objective lenses, condenser, and illuminator condenser. Also, clean those parts before you put your
microscope away today.
Microscope lenses, condensers, and illuminators get dusty and smudged and need to be cleaned. To prevent
scratching and damage, lenses can be cleaned with only two things: lens paper and/or alcohol. Lens paper is grit
free and will not scratch the lenses. Alcohol will dissolve and clean away smudges from fingers and eyes. Do
not use any other kind of paper! Do not use any other liquid!
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V. Total Magnification:
Read the information below. Then, calculate total magnification when viewing specimens for each objective
lens.
Most compound microscopes have four objective lenses to give four different levels of magnification. The four
different levels of magnification from lowest to highest are scanning, low power, high power, oil immersion.
For our microscopes, the scanning is 4x, low power is 10x, high power is 40x and oil is 100x.
The eyepieces also magnify. Our eyepieces are 10x.
The total number of times an object is magnified is called total magnification and is equal to the magnification
of the eyepieces times the magnification of the objective lens used.
1. Total Magnification for Scanning Objective
2. Total Magnification for Low Power Objective
3. Total Magnification for High Power Objective
4. Total Magnification for Oil Immersion Objective
VI. Viewing a Specimen, Part 1:
Follow the instructions below:
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Obtain a letter e slide.
Adjust your seat height so can look into the oculars comfortably.
If you wear glasses you do not need to wear them when using the microscope unless they correct a
severe astigmatism. Near- and far-sightedness can be corrected by adjusting the microscope focuses.
However, if you are comfortable wearing your glasses while looking through the eyepieces, you may
leave them on.
Use the coarse focus knob to move the stage down.
Use the revolving nosepiece, not the objectives, to rotate the 4x objective into place. You should feel a
click to indicate it is properly positioned.
Use the condenser knob (your instructor will show you where it is) to adjust the condenser to its highest
position.
Move the iris diaphragm lever all the way to the left.
Use the clip to insert the e slide into the mechanical stage.
Set the light intensity to its middle setting.
Plug in and turn on the microscope.
Use the mechanical stage knobs to center the e over the light.
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Look through both eyepieces at the same time. Then, adjust the interpupillary distance. While doing so,
gently push apart or pull together the eyepieces to see only one image.
Use the coarse focus knob to move the stage up but do not ram the stage into the objective. Then, bring
the e into coarse focus.
Use the fine focus knob to bring the e into clear, sharp focus.
Using the mechanical stage knobs, center the e in the middle of the viewing field.
Use the revolving nosepiece to rotate the 10x objective into place.
Using ONLY the fine focus, bring the e into clear, sharp focus.
Using the mechanical stage knobs, center the e in the middle of the viewing field.
Use the revolving nosepiece to rotate the 40x objective into place.
Using ONLY the fine focus, bring the e into clear, sharp focus.
Change the light intensity setting and observe how that affects the image.
Move the iris diaphragm lever to the right and observe how that affects the image.
VII. Image Orientation, the Iris Diaphragm Lever, and the Effects of Magnification on Image Quality:
As your instructor lectures them, write-in the following definitions:
1. Image orientation
2. Iris diaphragm lever
3. Centering
4. Brightness
5. Field
6. Depth of field
7. Working distance
8. Parfocal
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VII. Viewing a Specimen, Part 2:
Follow the instructions below to prepare and view a wet mount of your cheek cells:
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Obtain a slide, cover slip, toothpick, and bottle of methylene blue.
Put a drop of methylene blue on a slide.
Gently scrape the inside of your cheek with the flat side of a toothpick. Scrape lightly.
Stir the end of the toothpick into the water and throw the toothpick away into the orange bag in the
beaker.
Place a coverslip onto the slide. Put one edge of the coverslip on the slide and then gently drop the other
edge into place. See the Figure below. Your goal is to try and not produce air bubbles in the fluid as you
lower the coverslip into place.
Place your wet mount into the microscope, and locate cells under scanning or low power. Cells may be
hard to see, but they can be found. As you learned above, adjust coarse and fine focus, light intensity,
and the iris diaphragm lever.
Once you think you have located a cell, center it in the field, switch to high power, and using only the
fine focus knob, bring the cell into clear, sharp focus. You should be able to view the nucleus,
cytoplasm, and plasma membrane of the cell.
Coverslip
Methylene blue
Cheek cells
Slide
Wet Mount Preparation
VIII. Electron Microscopy:
Read the information below:
The compound microscope is a valuable tool to microscopic things in the lab, but it has its limitations. Most
notably, compound microscopes have a maximum magnification of 1000X , beyond which light itself fails to
properly resolve structures. To improve on this method several alternatives are possible now.
Electron Microscopy (EM)- Compound microscopes use light to illuminate an object. The light goes through
the sample and is magnified by the objective and eyepieces. But instead of light if you use electrons which are
tiny, it is possible to see even smaller objects in the specimen. Electron microscopes magnify specimen
1,000,000 times! So you can see ribosomes, proteasomes and many other structures in cells. Typically the
sample is coated with an electron dense material like gold. The sample is then bombarded with electrons by the
EM and imaged.
There are 2 types of EM:
Scanning EM (SEM)- Samples are kept whole, coated and bombarded with electrons in an electron
microscope. The electrons scan across the surface of the object so surface structures can be seen. See Figure 2
which shows pollen visualized by a SEM.
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Figure 2- SEM of pollen
(http://en.wikipedia.org/wiki/File:Misc_pollen.jpg)
Transmission EM (TEM)- Samples are sectioned, coated and bombarded with electrons in an electron
microscope. The electrons transmit through the specimen and the resulting image is magnified. TEM allows the
visualization of internal structures in cells (see the mitochondria in Figure 3).
Figure 3- TEM of Mitochondria. Note the scale is in nanometers. The arrows indicate internal membranes of
the mitochondria.
(http://www.bing.com/images/search?q=Transmission+Electron+Microscopy++and+pic+and+mitochondria&vi
ew=detail&id=2E733536D3BAAD4541859FC0CB12E930FF6B14AD)
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VIII. Fluorescence Microscopy:
Read the information below. Then,if there is time at the end of lab, your instructor will take you to see some
flourescent microscopic images.
Fluorescence is a highly sensitive method of tagging things with fluorescent molecules which can be stimulated
with one wavelength of light and emit a different wavelength which has an associated color. For example, the
molecule Fluorescein Isothiocynate (FITC) is stimulated with 494 nm light and emits 521 nm light which
appears green (Figure 4).
Figure 4- Molecular Structure of Fluorescein Isothiocynate (FITC)
(http://en.wikipedia.org/wiki/File:FITC-2D-skeletal.png)
So if you chemically attach FITC to a protein and treat it with 494nm light, the protein will glow green. One can
use this principle in a technique called Immunofluorescence by binding fluorescent molecules like FITC to
immunological proteins called antibodies.
Antibodies tag non-self for removal by the immune system. One finds antibodies in many places in an
organism, but most people think of antibodies as being in the blood. For example, when someone is infected
with bacteria, immune cells produce antibodies to mark the bacteria for removal. They work very well because
antibodies are highly specific for the target non-self protein (e.g. in this case bacteria). They will only bind to
the specific target to which they were made.
It is thus, possible to inject biomolecules you need to study into an organism. The biomolecules will be
recognized as non-self and the organism will induce antibodies against them. Typically the blood containing the
antibodies are harvested and used in biotechnology to detect proteins in a cell. In immunofluorescence, they are
chemically conjugated with a fluorescent molecule like FITC and used in experiments.
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Figure 5- FITC-Conjugated Antibodies Binding to One HUGE Cell on a Slide. The pink stars represent the
target proteins. The yellow arrows are antibodies and the green bursts are the FITC after it is stimulated with
494 nm light.
The sample is placed on the stage of the fluorescence microscope which is set to treat the samples with the
correct wavelength of light to excite the fluorescent molecule on the protein. It is also set to detect the colored
emission from the fluorescent molecule and produces a colored image. See Figures 6 and 7.
Figure 6- A Real Example of Immunofluorescence Used to Detect
the Cytoskeleton in Bovine Pulmonary Artery Endothelial (BPAE)
cells. FluoCells® prepared slide #2 contains BPAE cells, but stained
with redfluorescent Texas Red®-X conjugated actin antibodies, mouse
monoclonal anti‑α‑tubulin in conjunction with green-fluorescent
BODIPY® FL for labeling microtubules and blue-fluorescent DAPI for
labeling the DNA in the nuclei.
(www.invitrogen.com)
Figure 7- FluoCells® prepared slide #1 contains bovine pulmonary
artery endothelial (BPAE) cells stained with a combination of
fluorescent dyes. Mitochondria were labeled with red-fluorescent
MitoTracker® Red CMXRos, actin was stained using green-fluorescent
Alexa Fluor® 488 conjugated to actin antibodies, and blue-fluorescent
DAPI was used to label the DNA in the nuclei.
(www.invitrogen.com)
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