KNOW YOUR MICROSCOPE
Basic terminology
Light source
The light source, or "illuminator", should have
adjustable brightness and may include an adjustable field
diaphragm to minimize scattered light.
Condenser (light-gathering lens)
The condenser is located beneath the stage and
includes an adjustable aperture, controlled by a diaphragm
within the condenser. Proper condenser adjustment, or is
essential for optimal resolution (see "Kohler illumination"
on the other side of this page).
Stage (specimen support platform)
The stage supports the microscope slide and allows
specimen movement in x-y plane. The stage may move up
and down for specimen focus.
Specimen
Note that the slide and coverslip are both parts of the
optical system. At high magnification, optimal optical
function requires that coverslip thickness be appropriate
for the objective lens.
Objective lens (image-forming lens)
Functionally, the objective lens is the most critical (and
most expensive) element of the microscope. Objectives
may move up and down for specimen focus. Never touch
the optical surface of the objective lens.
Eyepiece (enlarging lens)
If eyepieces are paired, then interpupillary distance is
adjustable. At least one eyepiece should be independently
focussable. Eyepieces are removeable; when held
upsidedown, an eyepiece can serve as a hand lens.
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Three rules for carrying a microscope:
1. Always carry the microscope upright
(otherwise the eyepieces can fall out).
2. Use both hands.
3. Give the job your full attention, just as if you
were carrying a baby.
Three rules for cleaning a microscope:
1. Don't! It easy to cause permanent damage
by injudiciously cleaning a microscope.
 Clean eyepieces and other optical surfaces only
when absolutely necessary. It is better to ignore
dirt than to cause permanent damage to the
microscope.
 Never the touch the objective lens surface, unless
you own the microscope and have been properly
trained.
2. If you see dirt when you look through the
microscope, determine by logic and
experiment where the dirt is located.
 Dirt on a slide moves when you move the slide.
 Dirt on the condenser goes in and out of focus
when you focus the condenser.
 Dirt on an eyepiece will move when you rotate the
eyepiece.
 Dirt on an objective cannot be seen while viewing
through the microscope, but manifests itself as a
decrease in image quality.
3. If dirt is objectionable, use the least-
intrusive procedure to make it go away.
 Some dirt can blown away without touching the
surface. Use this procedure freely, but blow gently
and be careful not to spit.
 Dirt on slides may be wiped off at will, using the
same care you would use for eyeglasses.
 Dirt on an eyepiece is often accentuated by having
the condenser aperture too small; such dirt may
become much less conspicuous when the
condenser diaphragm is set correctly (see "Kohler
illumination" on the other side of this page).
 Dirt on the condenser may become inconspicuous
if you lower the condenser (i.e., move the dirt out
of focus).
 If dirt must be removed manually, use clean lens
paper and moisture from breath. Wipe gently in
one direction. Do not scrub with a circular
motion. Do not try to clean an objective lens.
Kohler Illumination
The following instructions apply to most research
microscopes, including the multi-viewer-
microscopes in Lindegren Room 206. Condenser
adjustment may be simplified on student
microscopes.
Initial set up: Turn on the lamp, place a slide on the
stage, focus on the specimen, adjust interocular distance to
match that of your own eyes.
1. Focus the condenser.
Close the field diaphragm located on the base of the
microscope. This will reduce the illuminated field of
view, as seen through the eyepieces, to a small circle of
light. Then adjust the substage condenser up or down
until the edge of this circle appears sharp.
2. Center the condenser.
Open the field diaphragm until the illuminated
circle almost fills the viewing area. Then adjust the
screws at either side of the condenser until this circle is
precisely centered in your field of view. Now open the
field diaphragm completely to restore illumination over
the entire field of view.
3. Adjust the condenser diaphragm.
Remove one eyepiece and look into the open tube.
You should see an illuminated field of view whose size
is controlled by the diaphragm within the condenser.
Adjust the condenser diaphragm until its edge just
barely encroaches on the illuminated field of view.
Then replace the eyepiece.
This adjustment should be repeated whenever the
objective lens is changed (i.e., whenever magnification
is changed).
4. Adjust illuminator brightness.
Use the voltage regulator on the base of the microscope
to adjust image brightness to suit your eyes.
5. Repeat steps 3 and 4 whenever you change
objectives.
Congratulations. Your microscope should now
be adjusted for proper Kohler illumination.
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Additional details may be found at http://www.siumed.edu/~dking2/intro/kohler.htm
For yet more information . . .
http://www.micrographia.com/tutoria/micbasic/micbpt01/micb0100.htm
http://www.microscopy.fsu.edu/primer/anatomy/anatomy.html
Magnification
 Magnification is determined in principle by
multiplying the power of the Objective Lens
by the power of the Eyepiece.
 Magnification is determined in practice by
calibration with a specimen of known size.
o For research purposes, calibration is done with a
reference standard, such as a "calibration slide",
engraved with a very finely-calibrated ruler.
o An "eyepiece micrometer" (a small, transparent ruler
mounted within the eyepiece) can be used to measure
specimens, after calibration with a specimen of
known size (see preceding item).
 In routine practice, magnification is estimated
with reference to familiar objects of known
size, such as red blood cells (the diameter of
human RBCs is approximately 6-8µm).
CALIBRATE YOUR OWN MICROSCOPE.
 A good calibration standard can be made by
using a size-reducing photocopier to make a
miniature copy of a millimeter ruler. Measure
the actual size of this miniature ruler, so you
know the length between marks.
 Put your calibration standard on a microscope
slide and view it through the microscope.
Measure edge-to-edge across measure the field
of view. Repeat for each magnification (i.e.,
each different objective lens). Or, measure
once with lowest power and use arithmetic to
compute the change when magnification is
increased. (For example, if magnification is
increased from 4x to 40x, which is a factor of
10, the real area visible in the field of view
will decrease by a factor of 10.)
 If your microscope has an eyepiece pointer,
you can also measure the thickness of the
pointer and use that number as a standard for
estimating the size of objects much smaller
than the field of view.