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MLAB 2360 Clinical I Microscopy Training 2
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.
MLAB 2360 Clinical I Microscopy Training 4
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 5
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.
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.
http://www.austincc.edu/biocr/1406/labv/microscope/index.html
http://www.austincc.edu/biocr/1406/laba/microscope/index.html
http://www.udel.edu/biology/ketcham/microscope/scope.html
MLAB 2360 Clinical I Microscopy Training 6