Download 2006S Bio153: Lab Exercise 1 Basic Microscopy Microscopes are

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2006S Bio153: Lab Exercise 1
Basic Microscopy
(adapted from a document by Dr. D. Dilkes, UTM Biology)
Objectives: To learn the principles of light microscopy; to learn the proper handling
and use of compound and dissecting microscopes; to learn the function of parts of the
compound microscope; to learn how to calculate magnification and to measure objects
viewed under the microscope.
Microscopes are precision instruments. Please handle carefully at all times! Each
student will use the same compound microscope throughout the course and should
report any problems to either the technician or the T.A.
Introduction: Principles of Microscopy
The human eye can distinguish two objects that are separated by as little as 0.1mm.
Typical prokaryotic and eukaryotic cells range from 0.001 mm to 0.05 mm in diameter,
with only the very largest cells (0.1 mm) entering into the range discernible by the
human eye. Thus, microscopes are needed for detecting the existence of most cells and
for visualizing the internal structure of all cells.
For a compound, bright field light microscope, the material being examined is
placed on a glass slide and viewed with visible light. The path of light from the light
source through the lenses and specimen to the eye is shown in Figure 1. After the light
waves have passed through the specimen, the image is magnified by passing the light
through two lenses, the objective lens and an eyepiece (ocular) lens.
Ocular lens
Light Beam
Objective lens
Condenser lens
Lamp
Figure 1. The path of light through a compound light microscope.
Note that the image you will see is both inverted and reversed. The crucial property
of the microscope is not magnification, but resolving power. The resolving power of a
lens system is the ability to distinguish two adjacent objects as distinct. For example,
there are prokaryotic cells on the surface of our skin, but we cannot take a picture of our
skin with a regular camera, enlarge the photograph and see those cells! This is because
the resolving power of the camera’s lens is not great enough. Modern microscopes
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increase both magnification and resolution by a careful matching of light source and
precision lenses. Student microscopes usually can resolve objects that are 0.5 µm or
more apart. The best research light microscopes can resolve objects that are 0.1 µm or
more apart. Magnification which enlarges an image beyond the resolving power of the
microscope is useless and is called empty magnification. The maximum useful
magnification for a bright field light microscope is 1,000X.
Contrast is the difference in the darkness or color of the structures being
examined. Living cells consist mainly of water, and thus are nearly transparent. A
reduction in the amount of light improves contrast when viewing unstained specimens,
but contrast is usually generated in biological specimens by applying a stain which
colors or otherwise alters the light transmitting property of some of the structures of a
cell. There are a few stains that are used with live cells, but usually cells are first
treated with fixatives that kill the cells while preserving their structural appearance.
After fixation, the cells typically are stained.
Light must pass through the specimen to create an image, thus the specimen
must be thin. Specimens to be viewed with a compound microscope are placed on a
microscope slide and are usually covered with a cover slip. Specimens are mounted
on slides in two ways. The specimen for a prepared slide (permanent slide) usually has
been fixed and stained; a cover slip has been attached permanently using a special
mounting media. A wet-mount slide (temporary slide) has the specimen mounted in a
liquid, usually water, and covered with a cover slip. Wet mounts have to be handled
carefully to ensure that the cover slip remains centered over the specimen. Always carry
slides by their edges to avoid getting dirt and grease on them. When given a prepared
slide to examine, inspect its surface for dirt and grease and if necessary carefully clean
with a cotton swab moistened with an ethanol solution. When finished with your
wet mount slides, place the used slides and cover slips in the beakers by the
sinks.
Table 1: Parts of the compound microscope
Name
Function of Part
Ocular
The eyepiece lens that magnifies the image by 10x
Nosepiece
Holds the objective lenses and rotates each one into place
Objectives
Magnifying lenses of various powers (3.2x, 10x, 40x, 100x)
Coarse focus knob
Used for initial focusing with the scanning lens (3.2x power) by
raising or lowering the stage
Used for fine adjustments of focus with higher powered
objective lenses
Supports the slide perpendicular to the path of light
Fine focus knob
Stage
Stage knobs
Condenser
Permits movement of the slide and specimen in the horizontal
plane
Focuses light from the lamp onto the specimen
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The compound microscope has two principal lens systems: an objective lens, which
magnifies and projects a "virtual image" into the body tube of the microscope, and the
ocular lens, which further magnifies the image (X10) and projects the enlarged image
into the eye. The compound microscope does NOT give a stereoscopic image, since
the objective lenses are not paired. Your microscope has three objectives. The shortest
is the scanning objective, which has a magnification of 3.2X. The low-power
objective with a 10X magnification is intermediate in length. The high-power (highdry) objective is the second longest and has a magnification of 40X. The longest
(100x) lens is called an oil-immersion objective.
Figure 2. The compound microscope.
The body tube is supported by the arm and has 2 ocular lenses at the upper end and
a revolving nosepiece with the attached objective lenses at the lower end. The
nosepiece is rotated to bring different objectives into viewing position. The objectives
usually click into viewing position. The condenser lens(es) lie beneath the object
being viewed. This lens directs the light into a cone-shape, so that the light rays
converge exactly at the level of the object on the stage. There are actually two lenses
in the condenser assembly: the supplementary or front lens is used with all the
objective lenses except the 3.2x scanning lens. The front lens should be swung out of
the way when the scanning lens is in use. Usually, the condenser should be raised to its
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highest position. An iris diaphragm is built into the base of the condenser. The iris
diaphragm control lever varies the amount of light entering the condenser and the
lens system. The coarse-focusing knob has the larger diameter and is used to bring
objects into rough focus when using the 3.2X and 10X objectives. The fine-focusing
knob has a smaller diameter and is used to bring objects into fine focus. It is the only
focusing knob used with the high-power and immersion objectives.
Determining magnification: The magnification of each lens is inscribed on the lens.
The ocular usually has a 10X magnification. The powers of the objectives are 3.2X, 10X,
40X, and 100X. The total magnification is calculated by multiplying the power of the
ocular by the power of the objective. For example, if the objective lens has a
magnification of 40x and the ocular 10x, then the image produced by these two lenses is
400 times larger than the specimen
Switching Objectives: Your microscope is parcentric and parfocal. This means
that if an object is centered and in sharp focus with one objective, it will be centered and
in focus when another objective is rotated into the viewing position. However, slight
adjustments to re-center and refocus (with the fine-focusing knob) may be necessary. As
you switch objectives from 3.2X to 10X to 40X to increase magnification, the (1) working
distance, (2) diameter of the field, and (3) light intensity are reduced as magnification
increases. Note this relationship in Figure 3.
4x
10x
working distance
8.3 mm
25 mm
40x
0.5 mm
Figure 3. Relationship of objective power to working distance, diameter of
field and light intensity.
Depth of Field: The objective lens of a microscope has only a limited depth of focus,
so that only a part of a thick specimen is in focus at any one setting (just like a camera
lens). The higher the magnification, the shallower the depth of focus. The compound
microscope is therefore most frequently used to view very small whole organisms or thin
slices (sections) of tissue. When viewing objects with a greater depth (thickness) than
the depth of field, you see only a two-dimensional plane "optically cut" through the
object. To discern an object's three-dimensional shape, a series of these images must be
"stacked up" in your mind as you focus through the depth of the object. When the high
power (40X) objective is in use, constant fine adjustments of the control knob need to
be made when examining a specimen.
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Using the compound microscope: step by step
1. Cleaning the lenses: Use one or two drops of lens cleaning fluid on a piece of
lens tissue. (Note: paper towels or other tissues will leave permanent scratches on a
lens). This should be done whenever you start to use a microscope, and before you
put it away.
2. Initial Focusing:
a) Turn illumination on.
b) Adjust the distance between the two oculars to suit your own eyes, and to give a
single field of view.
c) Place slide on stage with specimen centered:
Figure 4. Placing the slide in the mechanical stage.
d) Turn nosepiece so that the Scanning Lens (3.2X) clicks into place.
e) Raise or lower the stage using the Coarse adjustment, until the image of the
specimen appears sharp.
f) Turn nosepiece, watching to see that the objective clears the slide, until the Low
Power lens (10X) clicks into place.
g) Swing Condenser Front Lens into place.
h) Looking at the side of the microscope, raise stage by turning Coarse adjustment
until objective is almost 2mm above the coverslip.
i) Looking into the oculars, slowly lower stage by turning the Coarse adjustment
towards you until the image is fairly clear.
j) Focus sharply by turning the Fine adjustment knob.
Rules to remember:
1. Always use scanning lens first, to locate the specimen.
2. Always focus with Low Power (10X) before using High Power
3. When using High Power, use only the Fine adjustment knob.
4. Always watch from the side if you must raise the stage to find the specimen.
5. Use the condenser front lens with all objectives except the scanning lens.
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Once the lowest power objective has been focused, the other objectives should be
almost in focus, when swung into place. However, the image usually needs to be
sharpened slightly, using the Fine adjustment knob.
3. Focusing the Condenser Lens: By focusing the condenser lens, you will set the
distance between the condenser lens and the specimen, so that light rays now
converge on the specimen. (NOTE: The position of the condenser should remain
fixed during normal use).
a) Focus on a specimen on a slide, using low power (10X) objective.
b) Remove the slide and open the Iris Diaphragm.
c) Using your left hand, turn the Condenser Adjustment Knob until the condenser
is at the top of its travel. Keep hold of the knob.
d) With your right hand, hold the point of a pencil or mounted needle in the center of
the frosted glass covering the lamp, touching the glass.
e) Look down the eyepiece and slowly turn the condenser adjustment away from you,
until you see the pencil point in sharpest focus.
f) Remove the pencil. Defocus the image of the frosted glass by raising the condenser
slightly.
4. Adjusting Illumination:
A. Low Power Objective (10X):
a) Remove the eyepiece. Look down the body tube at the circle of light.
b) Close the Iris Diaphragm down until you can see its edge about 1/3 into the field,
like this:
c) Replace the eyepiece.
B. High Power Objective (40X):
a) Looking from the side, turn nosepiece until the 40X objective clicks into place.
Refocus, using fine adjustment only.
b) Readjust the Iris Diaphragm as in A, steps a-c. However, you will have to OPEN
the diaphragm slightly. By adjusting the diameter of the iris diaphragm, you have
optimized the angle, such that the cone of light entering the objective lens will not
cause internal reflections and distortions of the image.
5. Focusing the Ocular Lenses: Each time you begin to use a microscope, adjust
each ocular lens separately so that each eye receives a sharp image. Focus on a
specimen using either low or high power (10X or 40X).
a) Set both eyepiece rings at 65.
b) Close your left eye. Looking down the right ocular only, focus sharply on the
specimen using the fine adjustment.
c) Close your right eye. Looking down the left ocular only, sharpen the image by
turning the left eyepiece ring. Do not adjust the Fine focus knob.
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Measuring with the Microscope: The size of objects viewed under the microscope
can be determined by calibrating the microscope with a scale of known size. A stage
micrometer consists of a fine scale in 0.05 mm divisions, mounted permanently on a
slide. Using this instrument, the field diameters of the various objectives have been
measured for the compound light microscope, and are summarized in Table 2.
Table 2. Field Diameters
Objective
3.2 x
10 x
40 x
100 x
Description
Compound
Microscope
Scanning Lens
Low Power
High Power
Oil Immersion
Field
Diameter
4.85
1.55
0.40
0.16
mm
mm
mm
mm
As has been noted previously, the field of view decreases as the magnification is
increased. If you know the diameter of the field of view at each magnification, it is
possible to estimate the size of objects in the field of view. For example, when viewed
with the Low Power (10X) objective, an Amoeba stretches about 1/3rd across the field of
view in the microscope. Thus, the Amoeba occupies 1/3 of 1.55 mm. Thus, the Amoeba
is 1/3 x 1.55 = 0.52 mm long.
Calculating the Magnification of a Drawing: The magnifying power of a microscope
is the product of the powers of all the lenses in line with the object. Thus, if you
examine a specimen with the Low Power (10X) lens, you will see it magnified 10 x 10 =
100X (remember that the ocular lenses magnify 10X). Notice though, that a drawing of
the same specimen will have a different magnification. However, if you have calculated
the actual size of the specimen using the microscope and the table of Field Diameters
above, the magnification of a drawing is easily calculated:
Magnification of Drawing = drawn size
actual size
Putting the Microscope Away
1. Clean all the lenses. Put a drop or two of lens cleaning fluid on a piece of lens paper and
clean the oculars and objectives thoroughly.
2. Rotate the nosepiece until the scanning lens (3.2X) is in place.
3. Check to see that the slide has been removed.
4. Wrap the cord around the base of the microscope.
5. Return microscope to its numbered place in the cabinet.
Although you are not required to submit biological drawings in this course, it is good
practice to learn how to make a proper drawing (good drawings make good study
notes!) Use a sharp, hard pencil. Draw clearly defined structures with smooth,
continuous lines. Maintain accurate proportions, but allow room for labels. Make note of
the name of the specimen and the type of view (e.g. dorsal, ventral, cross-section, etc.)
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Figure 5: Example of a good biological drawing.
A dissecting microscope is used to view objects that are too large or too opaque to
observe with a compound microscope. The two oculars enable stereoscopic
observations and usually are 10X in magnification. Most student models have two
objectives that provide 2X and 4X magnification so that total magnification is 20X and
40X respectively. Some models have a zoom feature that enables observations at
intermediate magnifications. Objects are
usually viewed with reflected light
instead of transmitted light, although
some dissecting microscopes provide
both types of light sources.
The dissecting microscope
enables you to observe objects that are
too large or too thick to be seen under
higher magnifications, and because of its
stereoscopic nature, allows you to
observe objects in three dimensions.
The large working distance between
the objective lenses and the object being
viewed allows for easier manipulation or
dissection of the object. Another
advantage of the dissecting microscope
is that it gives a true image of the
object (i.e., neither inverted nor
reversed).
Figure 6. The dissecting microscope.
The following exercise will give you some experience working with compound and
dissecting microscopes. You will not be asked specific questions about the specimens;
this is a chance to practice making observations and sketching what you see.
1. Make a wet mount of a sample of pond water (stir the pond water gently,
draw up a small amount in the pipette provided, place a drop on the slide and
cover with a cover slip). Examine using the compound microscope.
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2. Examine the various prepared slides using the compound microscope.
3. Look at a sample of brine shrimp under the dissecting scope (it can be a
challenge to observe living organisms!)
4. Examine some whole preserved insects under the dissecting scope.
Study questions:
1. Magnification
a) is the factor by which the image of a specimen is enlarged,
b) is how well details stand out against the background in the image,
c) is the degree to which two adjacent points in a specimen are seen as separate in the
image,
d) focuses the light rays from a specimen to produce an image.
2. Resolving power
a) is the factor by which the image of a specimen is enlarged,
b) is how well details stand out against the background in the image,
c) is the degree to which two adjacent points in a specimen are seen as separate in the
image,
d) focuses the light rays emanating from a specimen to produce an image of that
specimen.
3. A lens
a) is the factor by which the image of a specimen is enlarged,
b) is how well details stand out against the background in the image,
c) is the degree to which two adjacent points in a specimen are seen as separate in the
image,
d) focuses the light rays emanating from a specimen to produce an image of that
specimen.
4. Contrast
a) is the factor by which the image of a specimen is enlarged,
b) is how well details stand out against the background in the image,
c) is the degree to which two adjacent points in a specimen are seen as separate,
d) focuses the light rays emanating from a specimen to produce an image of that
specimen.
5. The maximum useful magnification for a light microscope is about
a) 100X
b) 1,000X
c) 10, 000X
d) 100,000X
6. The two image-forming lenses of a compound light microscope are the
a) condenser and objective
b) condenser and ocular
c) objective and ocular
d) condenser and eye
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7. Stains are usually added to sections of biological specimens to increase
a) depth of field
b) magnification
c) contrast
d) all of the above
8. If the magnification of the two image-forming lenses are each 10x, the total magnification
of the image will be
a) 1X
b) 10X
c) 100X
d) 1,000X
9. The higher the magnification, the _______ the depth of focus.
a) shallower
b) deeper
c) wider
d) narrower
10. The highest power objective lens has the:
a) longest working distance, smallest diameter of field and highest light intensity
b) longest working distance, largest diameter of field and highest light intensity
c) longest working distance, largest diameter of field and highest light intensity
d) shortest working distance, smallest diameter of field and lowest light intensity
Answers: 1a, 2c, 3d, 4b, 5b, 6c, 7c, 8c, 9a, 10d.