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Leica DME Microscope Use

Compound microscopes will be used in this lab. Microscopes are expensive pieces of precision equip-
ment, susceptible to damage by careless handling. Please use them with care. It is likely that you have
already used microscopes before. However, we ask that follow the procedures as written here, even if
they may vary from your past experience.
Setting up a Microscope for Use
Remove the dust cover, fold it and put 1.
it aside.
The compound microscope is usually 2.
stored in its storage position, with the
eyepieces pointed backwards toward
the arm. This is not the position with
which it is to be used. Gently rotate
the entire binocular head into its work-
ing position. This may require that you
temporarily loosen (and then retighten)
a screw holding the head onto the mi-
croscope.
Plug in the microscope. Make sure 3.
that the cord is positioned where it will
not get in the way.
Turn on the light source in the base. 4.
Adjust the illumination for your com-
fort.
Putting Away a Microscope
Remove your slide. 1.
Rack down the stage. 2.
Turn off the light and allow it to cool. 3.
Clean the lenses (eyepieces and ob- 4.
jectives) with lens paper ONLY. It is es-
pecially important to remove all oil, if
the 100X oil immersion lens was used.
Do not use any other paper, a hand-
kerchief or your fngers as they could
damage the lenses.
The lowest power objective should be 5.
in place for microscope storage.
Make sure that the stage is clean. 6.
Return the stage mechanism to its 7.
central position.
Wrap the electrical cord. 8.
Replace the dust cover. 9.
A. Components
Compound microscopes magnify thin speci-
mens mounted on microscope slides. They are
ideal for observing unicellular or very small or-
ganisms, cells, and cell structures. Below is a
list of parts of a compound microscope, and the
defnition or function of each.
Base: the bottom of the microscope, which sup-
ports the entire instrument.
Illuminator or light source: the light source is
usually built into the base of the microscope,
and directs light through the condenser to the
specimen. Alternatively, the light source may be
separate, and be directed toward the condens-
er with a mirror. The intensity of the light can
be adjusted using the rheostat (light) control
knob on the left side of the base.
Arm: the frame that supports all components
above the base.
Revolving nosepiece or turret: a revolving
disc-shaped support or frame for the objective
lenses.
Objective lenses: the primary optical system
which produces a magnifed image of the spec-
imen. There are typically four objective lenses
attached to the nosepiece: a 4X scanning ob-
jective, a 10X low power objective, a 40X high
power (dry) objective and a 100X oil immersion
objective. The magnifcation of each objective
is engraved on its side.
Ocular lens or eyepiece: the secondary optical
system that you look through. The ocular lens
further magnifes (10X) the image and brings
the light rays to a focal point. A binocular micro-
scope has two ocular lenses and a monocular
microscope has one ocular lens. They sit on the
adjustable binocular body.
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Leica DME Microscope Use
Stage: the fat surface upon which the slide with
your specimen is placed. Most microscopes
have a stage fnger assembly to hold the slide
on the stage. The entire mechanism including
the slide moves horizontally across the station-
ary stage (left/right and forward/back) using
two stage adjustment knobs situated under
the stage (variably on the left or right side, in
front of the focusing knobs).

Condenser: the lens located below the stage,
which focuses light (from the illuminator)
through the specimen being observed. Most
microscopes have a moveable condenser al-
lowing its distance from the specimen to be
adjusted using the condenser knob and con-
denser alignment screw.
Iris diaphragm: a unit below the condenser
that controls the amount of light directed to the
specimen. The diameter of the diaphragm can
be adjusted by turning it to increase or decrease
the size of the hole that light passes through.
Coarse adjustment or coarse focusing knob:
the large knob towards the back of the instru-
ment that is used to signifcantly raise or lower
the stage, when you frst focus on a specimen
at low power. It is never used when high power
objectives are in place.
Fine adjustment or fne focusing knob: the
smaller knob towards the back of the instru-
ment that is used to make small adjustments
in the height of the stage for fnal focusing on a
specimen. It is the only focusing knob used with
high power objectives.
Note: If the microscope does not appear to be functioning
properly, do not attempt to fx it. Bring the problem to the
immediate attention of your instructor. It is never good to
force a part of the microscope to move.
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Leica DME Microscope Use
B. Khler Illumination
Khler illumination is the alignment of the im-
age-forming light path and the illumination light
path of the microscope. In this process the con-
denser is centered and focused, thereby pro-
viding an evenly illuminated feld of view and
more importantly maximum resolution of the
specimen.
Directions:

Position focusing eyepiece to the appropri-
ate number for your eyesight on the left eye-
piece.

Position a stained slide on the stage.

Focus the specimen using the 10x objective
and comfortable illumination intensity, adjusting
focusing eyepiece as needed.

Close the feld diaphragm (black lever below
the stage and condenser) until about of the
feld of view is illuminated.

Partially close the aperture diaphragm (on
condenser).

Use the condenser height adjustment knobs
to position the condenser so the diaphragm im-
age is in focus.

Use the two condenser centration screws to
position the condenser the diaphragm image in
the center of the feld of view.

Fully open the feld diaphragm.

Adjust the aperture diaphragm for appropri-
ate contrast (also dependent upon intensity of
stain and thinness of sample).

Your microscope is now set to maximize res-
olution of the specimen.
Note: The microscope is now set to maximize resolution
of the specimen. If you adjust the condenser height to
gain contrast or adjust light intensity you will sacrifce the
resolution capability. Use the aperture diaphragm and /or
the illumination intensity to adjust contrast.
C. Calibration
Microscopes must be calibrated so accurate
measurements can be made. To calibrate a mi-
croscope both an ocular and a stage microm-
eter are used.
Ocular micrometer (O) - a very small ruler en-
graved on a piece of glass and placed inside
one of the ocular lenses
The rulers magnifcation does not change
when switching between the objective lens-
es.
At each objective, the value (in m) of an
ocular tick mark (division) must be deter-
mined.
Stage micrometer (S) - a very small ruler
engraved on a piece of glass then mounted
on a slide
The rulers magnifcation does change when
switching between the objective lenses.
The ruler is usually 1 to 2 mm (1000 to
2000 m) across.
Each tick mark (divisions) on the ruler is 10
m (0.01mm) from the next tick mark.
Directions (complete the steps below for 4X,
then 10X, and fnally 40X):

Focus on the stage micrometer (S).

Rotate the ocular lens until the ocular mi-
crometer (O) is exactly superimposed on S.

Adjust S so that its frst tick mark lines up
precisely with Os frst tick mark.

Look for another tick mark (as far from the
beginning as possible) where S and O line up
precisely.

Count the number of tick marks between
these two tick marks for both O and S.
See examples 1 and 2 on following page.
0.01 MM 1000 m
=
10 m
TICK MARK 1 MM TICK MARK
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Leica DME Microscope Use
70 TICK MARKS FOR S 10 UM
= 23 UM FOR EACH O TICK MARK (OCULAR DIVISION)
30 TICK MARKS FOR O S
Example 1:
Example 2: