Download biology 163 laboratory use of the compound light microscope

BIOLOGY 163 LABORATORY
USE OF THE COMPOUND LIGHT MICROSCOPE
(Revised Fall 2012)
Microscopes in various forms are important tools for biologists. Two types of microscopes that you will
use frequently are the compound and dissecting light microscopes. Of these two, the compound
microscope is more difficult to use well. Successful use of the compound light microscope depends on a
variety of factors including quality of slide preparation, proper focusing, and adjustment for optimal
illumination.
Compound light microscopes employ a series of lenses to enlarge (i.e., magnify) the image of the object
being studied. Total magnification is the product of the magnifying power of the objective lens times the
magnifying power of the ocular lens. Typical microscopes have four objective lenses (4X, 10X, 40X, and
100X) that can be rotated into position. Combined with a 10X ocular lens (standard on our microscopes),
this gives the user the option of 40X, 100X, 400X and 1000X magnification.
Magnification, however, is only part of the equation. The real purpose of a microscope lies in its ability to
allow the user to see small details (i.e., resolving power). Resolving power is expressed as the limit of
resolution (lr). This is the smallest distance by which two neighboring points can be separated and still
be seen as separate entities. The limit of resolution of your naked eye is about 0.2 mm. That of a good
quality light microscope is about 0.2 µm (1 µm = 0.001 mm), a 500-1000 fold improvement over your eye.
Good illumination is critical to realizing the full potential of the microscope. In 1893, August Kohler
described a method to achieve the most uniform illumination from a non-uniform light source such as a
light bulb. The result has become known as Kohler illumination and is obtained (1) by focusing the bulb
filaments at the plane of the condenser diaphragm and (2) by focusing an image of the lamp diaphragm at
the plane of the object. In microscopes with built-in illuminators the microscope manufacturer has done
Step (1). You will need to do step (2) after some initial set-up procedures with the microscope.
You will use microscopes to study both "wet" and "dry" preparations. The latter are prepared for you and
consist of thin sections (about 0.01 mm thick) permanently set in some mounting medium. You make
"wet" preparations to examine fresh material. Information on preparing wet-mount slides is provided for
your reference in Appendix B.
USING A LIGHT MICROSCOPE WITH KOHLER ILLUMINATION
Please respect the equipment by observing the following precautions!
•
Carry the microscope two hands: one supporting the arm and the other under the base. Keep the
microscope UPRIGHT or the ocular lenses may fall out.
•
If the lenses are dirty, clean them ONLY WITH LENS PAPER. Other substances may scratch the
precision lenses. If you dirty a lens, clean it IMMEDIATELY with lens paper.
•
ALWAYS focus by moving the stage/specimen AWAY from the lens. Failure to do so may allow the
slide to contact the lens, potentially damaging both.
•
ALWAYS focus on your specimen with a lower power before moving to a higher power. If you canʼt
find your specimen at 100X, you will definitely not find it at 400X (and you may risk damaging the
specimen or the microscope if you try).
•
ALWAYS use a cover slip when observing a “wet mount” specimen.
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Use the following procedure to properly set up the microscope for viewing specimens. Refer to
Appendix F if you are unfamiliar with the parts of the microscope!
1. Using the condenser height adjustment knob, place the condenser in its uppermost position. Open
the condenser diaphragm fully by sliding the lever on the front of the condenser all the way to the left.
2. Turn on the lamp using the power switch located on the front of the microscope base. Open the lamp
diaphragm fully by rotating the ring on the top of the lamp clockwise until it stops. You can adjust the
intensity of the light by rotating the numbered ring on the right-hand side of the base.
3. Rotate the objective lenses until the 10X objective locks into place over the condenser.
4. Open the spring-loaded clip on the stage and insert your slide. Use the stage controls to position your
specimen in the center of the beam of light. Do this looking from the side, with your naked eye; donʼt
attempt to “hunt” for the specimen while looking through the ocular lenses!
5. Raise the stage so that the specimen is close to the objective lens. Look through the oculars and
slowly lower the stage with the course focus adjustment knob until the specimen become visible.
Once the specimen is visible, use the fine focus adjustment knob to make the specimen as clear as
possible. Remember to ALWAYS focus by moving the stage/specimen AWAY from objective lens!
6. Adjust the distance between the ocular lenses. WITH BOTH EYES OPEN, you should see a single,
fully illuminated, circular field of view. (TIP: If you are having difficulty with this step, your eyes may be
too close to the ocular lenses!)
7. Adjust the focus to accommodate for differences between your two eyes.
• Close your left eye. Looking with your RIGHT EYE through the RIGHT OCULAR, focus the image
using the FINE FOCUS ADJUSTMENT KNOB.
• Close your right eye. Looking with your LEFT EYE through the LEFT OCULAR, focus the image
using the DIOPTER ADJUSTMENT RING on the base of the left ocular (do NOT touch the fine
focus adjustment knob). Open both eyes.
8. Adjust the condenser to focus the light on the specimen.
• Rotate the lamp diaphragm ring counterclockwise until the diaphragm is at its minimum opening.
A bright spot with blurry edges should be visible in the center of the field of view. (If the spot is not
centered, ask your instructor how to adjust it.)
• Using the condenser height adjustment knob, slowly lower the condenser until the edge of the
spot comes into focus. (TIP: You shouldnʼt need to move the condenser very far from its
uppermost position.)
• Rotate the lamp diaphragm ring clockwise until the light just fully illuminates the filed of view.
9. Close the condenser diaphragm by sliding the lever on the front of the condenser toward the right.
Adjust this to obtain optimal contrast for your specimen. Always adjust contrast by using the
condenser diaphragm or the lamp intensity control. DO NOT adjust the height of the condenser, as
this changes the focus of the light on the specimen!
10. If you wish to increase the magnification of the image, carefully rotate the 40X objective into place
WITHOUT lowering the stage. You should now be able to bring the image into focus using ONLY the
fine focus adjustment knob. In order to optimize the light, repeat steps 8 and 9 with the new objective.
Avoid rotating the course focus adjustment knob, as this may cause the slide to impact the lens,
potentially damaging both!
NOTE: SEE APPENDIX C BEFORE ATTEMPTING TO USE THE 100X OBJECTIVE LENS!
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APPENDIX A
Use of the Phase-Contrast Microscope
Phase-contrast microscopy allows the viewing of faint or lightly colored specimens without the use of
stains. The phase ring, located just beneath the stage of the microscope, adjusts the light passing
through specimen such that contrast between the specimen and the background is enhanced.
Use of the phase-contrast microscope is very similar to use of the standard compound light microscope.
However, in order for the phase-contrast properties of the microscope to be realized, the number on the
phase ring must match the power of the objective being used (10X and 40X in most cases). Be sure to
rotate the phase ring to the proper setting each time you change the objective. When using the phase
ring, you do not need to make adjustments to the condenser diaphragm.
Do not touch any knobs or controls with which you are unfamiliar--doing so could put the phase ring out of
adjustment and the phase-contrast properties of the microscope will be reduced. If you are not certain of
how to use the microscope properly, please ask your instructor for assistance.
APPENDIX B
Preparation of Wet-Mount Slides
Wet-mount slides are made when you wish to view fresh specimens (as opposed to those that have been
stained, fixed, and sectioned, as is the case with prepared slides). Make wet preparations by placing a
small drop of water or other fluid in the center of a clean slide. Center the object of interest in the drop of
liquid. Lower a clean cover slip over the drop gently to avoid trapping air bubbles. If done properly, the
moisture of the drop will completely fill the space under the cover slip, with no excess outside that might
run onto the microscope stage. If the coverslip appears to be "floating" on top the slide, you have used
too much liquid! You will either need to make a new slide or wick some of the liquid away with a KimWipe or paper towel.
Studying wet preparations should show you that a microscopic preparation, while thin, does have
appreciable thickness when observed with the microscope. A sense of the depth of the field is an
important feature of study with the microscope. You can see depth of view by using the fine adjustment.
As you focus up and down, the two-dimensional images at various levels become integrated in your mind
to form a notion of the third dimension of the preparation.
APPENDIX C
Use of the Oil Immersion Lens
Since oil has a higher refractive index than air, viewing a specimen through a drop of can increase the
resolving power of the microscope at high magnification. The 4X, 10X and 40X objectives are “dry”
objectives—they are designed to be used in air. However, the 100X objective is a lens specially designed
to be used with oil. To use this lens:
1. Rotate the 40X objective out of the way. Place a drop of immersion oil directly over the area on the
slide you wish to observe. Rotate the oil immersion objective (usually 100x) into position. When doing
this, watch carefully from the side of the microscope so that the objective does not contact the slide.
2. Focus the specimen using the fine focus adjustment knob and adjust for proper Kohler illumination.
3. When finished, WIPE ALL OIL FROM THE OBJECTIVE WITH LENS PAPER. Clean up any oil that
may have found its way to other lenses or parts of the microscope!
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APPENDIX D
Use of the Ocular Micrometer
One ocular lens of your microscope should contain a glass disk with fine divisions that are numbered.
This is an ocular micrometer--it is used to measure objects in the field of view (Figure 1).
0
10
20
30
40
50
Figure 1. Scale of an ocular micrometer
It is important to note that the numbers on the ocular micrometer DO NOT represent any fixed unit of
distance! When you increase magnification, the field of view (i.e., the total area visible through the
microscope) decreases, and the ocular micrometer becomes superimposed over a smaller area. As a
result, the physical distance represented by each small division (or “ocular unit”) decreases with each
increase in magnification. For this reason, you need to calibrate the ocular micrometer for the specific
microscope you are using!
The ocular micrometer is calibrated with a stage micrometer, a precise scale prepared as a dry mount
slide. Since the units on the stage micrometer are constant, it may be used to “measure” the ocular
micrometer when used with the different objective lenses. This procedure has already been done for the
ocular micrometers you will be using, and the results are provided in Table 1. You will need to use this
information to be able to accurately measure the specimens that you view!
Table 1. Ocular unit distances of ocular micrometers used at Colby College. One ocular unit is
defined as the smallest division of the ocular micrometer.
At this magnification:
40X (4X objective)
100X (10X objective)
400X (40X objective)
1000X (100X objective)
Each ocular unit equals:
0.025mm (25um)
0.010mm (10µm)
0.0025mm (2.5µm)
0.0010mm (1.0µm)
The ocular micrometer is a useful
tool for measuring microscopic
specimens, but the micrometer
itself is not typically shown in
illustrations or micrographs. For
these applications, a scale bar
should be used (Figure 2). Itʼs
helpful to draw the scale bar
approximately the same length as
the specimen of interest, with the
actual distance being determined
using the ocular micrometer and
the information in Table 1.
Field of View
Specimen
Scale Bar
5µm
Figure 2. Illustration of a specimen with a scale bar included.
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APPENDIX E
Understanding the Limit of Resolution
The limit of resolution (lr) depends upon several factors; it is calculated using Abbe's equation:
lr =
where:
0.61 λ
n sin α
λ = the wavelength of illumination used (Fig. 1)
n = the refractive index of the medium used between the objective lens
and the specimen. n (air) = 1; n (oil) = 1.56
α = half the angle of light entering the objective lens (Fig.2); the
theoretical maximum value of sin α is 1.
Notice that the limit of resolution becomes smaller (resolving power increases) as the numerator
becomes smaller and/or the denominator becomes larger. With most compound light microscopes,
you can manipulate the size the angle α (by adjusting the condenser diaphragm) and the value of n
(by using an oil immersion lens). The wavelength of light is fixed unless you add a colored filter to the
light source.
UV
Violet
Blue
Gr een
Yellow
Or ange
Red
IR
4000 A
4100
4700
5200
5800
6000
6500
7000 A
Figure 1. The Visible Light Spectrum
Objective lens
Cone angle =
Slide
!
Stage
Condenser
lenses
2!
Condenser iris
diaphragm control
Figure 2. The angle of light entering the objective lens.
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2!
APPENDIX F
The Olympus CH-2 Compound Light Microscope
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