MLAB 2360 Clinical I
Microscopy Training
Activity 1: Parts of the Compound Microscope
Aperture
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MLAB 2360 Clinical I
Microscopy Training
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Parts of the Microscope Defined:
1. Main Switch: On/Off
2. Rheostat: Light intensity knob
3. Coarse adjustment knob: This knob is used to raise and lower the stage of the
microscope. The coarse adjustment is used to first bring the object into APPROXIMATE
focus starting first with the stage as close to the objective lens without touching. Then
move the coarse adjustment so that the stage moves away from the lens until the object
is in relative focus. **Note: The coarse objective will ONLY be used when focusing with
the 4X or 10 X objectives to avoid damage to the other objectives.
4. Fine adjustment knob: This knob is used to focus the image into a sharp, critical focus
after achieving relative focus with the coarse adjustment knob. **Note: These
microscopes are PARFOCAL microscopes, meaning that if you are properly focused on a
lower objective (10X), you will be able to switch to a higher objective and maintain
relative focus. Do NOT use the coarse adjustment when on 40X, 50X or 100X.
5. Field Iris Diaphragm: Using the field iris diaphragm ring, the diameter of the field iris
can be adjusted to circumscribe (encircle) the field of view to exclude extraneous light
and improve image contrast. We will use this “field diaphragm” when setting the
microscope for Kohler illumination.
6. Condenser Lens: Below the stage is the condenser lens. This focuses light onto the
object and is NOT involved in the magnification. The focusing adjustment is a rack and
pinion movement to permit vertical movement of the condenser. Clear images are
obtained only when the condenser lens is in proper focus (Kohler illumination). **Note:
In Urinalysis and when using hemacytometers, it is often necessary to LOWER the
condenser to achieve greater contrast. This reduces resolution, but it is helpful when
searching for transparent elements in the specimen.
 Aperture Iris diaphragm: The recommended setting of this aperture iris
diaphragm is between 75-85% of the NA of the objective in use. When using the
100X objective, the aperture iris diaphragm should be set to 0.9 on the scale.
 Condenser centering screws: These screws are used during the Kohler
illumination procedure to ensure that the condenser is centered in the Field Iris
diaphragm.
7. X-axis : Moves the stage left to right
8. Y-axis: Moves the stage forward and back
9. Objectives: These are lenses mounted on the revolving nosepiece. Each lens is marked
with its Magnification/numerical aperture (NA) and Focal Distance. Most of our
microscopes are equipped with 10x and 40X DRY objectives as well as 50X and 100X OIL
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Microscopy Training
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objectives. The DRY objectives use only air between the lens and the slide, while the OIL
objectives utilize a SMALL drop of oil between the lens and the slide. The objective
lenses must be kept clean. Use lens tissue or KIMWIPES to clean the outer, exposed
surfaces of the lenses. Although the OIL immersion lens is designed to work in oil, the
oil must be removed after used and before you put the microscope away at the end of
each class period. **NOTE: Once you have used the 50x or 100x oil immersion objective
to examine a slide, DO NOT “go back” to the 40x objective. Doing so will cause oil
contamination of the 40x objective, rendering it useless until it can be cleaned. The oil
will cause the gaskets in the objective to erode and cause permanent and EXPENSIVE
damage to the objective. Oil is ONLY used on the 50X and I00x oil objectives.
10. Oculars: The ocular lenses usually magnify 10X. Thus the total magnification observed
is the multiplication of the power magnification of the ocular times the objective. The
oculars can be moved back and forth to adjust to the interpupillary distance of the
student.
 When first using the microscope, adjust the ocular lenses back and forth until a
circular field is viewed with both eyes open. The index dot indicates the
interpupillary distance on the Interpupillary Scale.
 Adjust the Diopter: Looking through the right eyepiece with your right eye,
rotate the coarse and fine adjustment knobs to bring the specimen into focus.
Then look through the left eyepiece with your left eye (cover the right eye) and
focus the specimen using the adjustment ring on the left ocular. This allows for
both eyes to be focused individually.
 Using the Eye shades:
o When using the microscope with eyeglasses, the eye shades should be in
the folded back position.
o When using the microscope WITHOUT eyeglasses, the eye shades should
be extended toward you to prevent extraneous light from entering
between the eyepiece and the eye.

Reticles: (Eyepiece micrometers) They are clear circular glass inserts with a scale
inscribed on them. The reticle or eyepiece micrometer sits at the focal plane
inside the eyepiece lens of the microscope and allows the investigator to make
accurate measurements of specimens. In binocular scopes like the ones you are
using, there will be a reticle in ONE of the lenses. Reticles come in many
varieties and in different diameters.
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Microscopy Training
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The "actual" distance between any two marks on the reticle are a function of the objective
lenses only. The best way to calibrate your reticle is to use what is called a stage micrometer.
This is a slide that has tiny marks of a known dimensions inscribed on it. By making a
comparison of the marks on the stage micrometer to the marks on the reticle, one can establish
the actual value for each mark on the reticle.
When you look into your eyepiece lens, the markings will always be the same but the size of the
image superimposed under them will get larger with more magnification. So, as you change to
a higher power objective lens, the represented value between marks will change
proportionately.
MLAB 2360 Clinical I
Microscopy Training
Let's say each division of the metric stage micrometer above is 0.01mm or 10 µm or 10µ (10
micron). The eyepiece reticle is divided into 100 units.
**Note: 1 micron = 1 micrometer so 1 µ=1 µm


First determine how many divisions of the eyepiece reticle correspond to a certain
distance on the stage micrometer by finding the FIRST line of the eyepiece reticle that
perfectly lines up with the stage micrometer.
Calculate the length which corresponds to one division of the eyepiece reticle. In this
example, 30 divisions of the eyepiece reticle corresponds to 20 divisions of the stage
micrometer. Each division of the stage micrometer equals 10µ so 20 divisions of the
stage micrometer would equal 200 µ. To calculate the value of one division of the
eyepiece reticle we would divide 200µ by 30 and the result would be 6.67µ per reticle
division. The reticle value, in this case 6.67µ, would apply only to the objective for
which the calibration was made. Each microscope objective must be calibrated
independently.
**Note: Care should be taken when removing dust covers from microscopes in order to avoid
pulling an eyepiece off of the scope and having it drop to the floor or workbench. Dropping
eyepieces can damage them beyond repair.
**Note: Eyepieces should be kept clean. Fingerprints can blur images and mascara can
“appear” in the field of view. A fresh piece of lens paper should be used to clean each eyepiece.
Gently “fogging” the eyepiece with your breath before wiping can aid in removing fingerprints.
Rubbing mascara when it is on a lens can etch the glass; therefore, it is important to “blow off”
as much of the mascara as possible before wiping the lens.
Activity 2: The Compound Microscope Video
http://www.austincc.edu/biocr/1406/labv/microscope/index.html
Activity 3: Review the parts of the Microscope
http://www.austincc.edu/biocr/1406/laba/microscope/index.html
Activity 4: Watch Virtual Compound Microscope Demonstration and
Practice on Your Microscope
http://www.udel.edu/biology/ketcham/microscope/scope.html
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Microscopy Training
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1. Place microscope on the workbench as shown in the first video.
2. Select a specimen slide from the slides provided by the instructor.
3. As the instructor goes through the Virtual Microscope Demonstration, find the parts
of the microscope and perform the tasks in the checklist.
Activity 5: Kohler Illumination
http://youtu.be/fNTNZ8nWPd4
Illumination of the specimen is the most important variable in achieving high-quality images in
microscopy and critical photomicrography or digital imaging. Köhler illumination was first
introduced in 1893 by August Köhler of the Carl Zeiss corporation as a method of providing the
optimum specimen illumination. The object of the process is to align the condenser to ensure
that the cone of light emitted from the illuminator (light source) is focused directly on the
specimen and the apertures are set to maximize contrast. The primary advantage of Kohler
illumination is even illumination free from glare; however, in urine samples and when using a
hemacytometers, the condenser is often lowered to increase contrast then raised to increase
resolution. Kohler illumination is not warranted in these circumstances, but IS recommended in
the hematology, microbiology, and parasitology areas of the lab.
Adjusting Contrast:
1. Place a specimen slide onto the microscope stage and secure between the specimen
holders.
2. Set the interpupillary distance to ensure comfort.
3. Place the 10X objective lens into position and focus the specimen using the coarse
adjustment knob.
a. Close the right eye. Use the fine adjustment knob to sharpen the image.
b. Close the left eye. Focus is obtained using the diopter ring shown below.
The diopter ring is normally located on the right eyepiece and moves both in
the counter clockwise and clockwise directions to achieve the necessary
focus.
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Microscopy Training
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4. As stated earlier, the aperture iris diaphragm knob should be set on the aperture iris
diaphragm scale to a setting between 75-85% of the NA of the objective. We will
perform the Kohler illumination on the 10X objective which has a NA of 0.25 (as seen
below); therefore, you are to set the aperture diaphragm knob to approximately 0.2
on the scale.
Objective
Magnification
Where video begins
NA
a. This can be done visually as shown in the video. Remove the removable
eyepiece and place it carefully on the table.
b. Look through the ocular.
c. When you move the aperture iris diaphragm knob from right to left you will
see a hexagon or octagon depending on the model expanding then becoming
a circle of light. You want to set the aperture iris at approximately 75-85% of
the capacity (gray circle) by sliding the aperture iris knob.
d. Replace the eyepiece carefully.
Centralizing Field Diaphragm
5. With the 10X objective in the viewing position, focus the specimen using the coarse
and fine adjustment knobs. This gets the stage in the appropriate position.
6. Once focused, close the diaphragm. This can be done by moving the field iris
diaphragm knob to the far left.
7. While looking through the oculars adjust the condenser knob until the
hexagon/octagon of light/specimen is at its sharpest.
8. Continue looking through the oculars and centralize the condenser by twisting the
condenser centering screws.
9. Open the field iris diaphragm by moving the field iris diaphragm to the far right.
10. You have now achieved Kohler illumination.
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Microscopy Training
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Activity 6: Focus with an OIL Objective
1. Select a blood smear that the instructor supplies.
2. Place the specimen slide onto the microscope stage and secure between the
specimen holders.
3. Set the interpupillary distance to ensure comfort.
4. Place the 10X objective lens into position and focus the specimen using the
coarse adjustment knob.
i. Close the right eye. Use the fine adjustment knob to sharpen the image.
ii. Close the left eye. Focus is obtained using the Diopter ring.
5. When the image is focused with the 10x objective, carefully hold the nosepiece
and move the 100X oil objective into viewing position. **Note: These slides
have had oil used on them before, therefore, be careful to go from 10X to 100X
WITHOUT passing the 40X over the slide.
6. Once the 100x oil objective is in place, apply a SMALL drop of oil to the space
between the objective and the slide.
7. Use ONLY the FINE adjustment knob to focus on the specimen. **Note: These
microscopes are PARFOCAL microscopes, meaning that if you are properly
focused on a lower objective (10X), you will be able to switch to a higher
objective and maintain relative focus.
8. Use the X and Y axis knobs to move through the slide.
9. When you have looked at a minimum of 5 fields (visible area of oculars), lower
the stage using the coarse adjustment knob, remove the slide, and GENTLY
remove the excess oil off the slide using a Kimwipe.
10. Using a clean Kimwipe and lens cleaner, clean the 100X oil objective.
11. Properly put away your microscope as shown in the first video.