Leica DME
Microscope Use
Compound microscopes will be used in this lab. Microscopes are expensive pieces of precision equipment, 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
Putting Away a Microscope
1. Remove the dust cover, fold it and put
it aside.
1. Remove your slide.
2. The compound microscope is usually
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 working position. This may require that you
temporarily loosen (and then retighten)
a screw holding the head onto the microscope.
3. Turn off the light and allow it to cool.
3. Plug in the microscope. Make sure
that the cord is positioned where it will
not get in the way.
6. Make sure that the stage is clean.
4. Turn on the light source in the base.
Adjust the illumination for your comfort.
8. Wrap the electrical cord.
2. Rack down the stage.
4. Clean the lenses (eyepieces and objectives) with lens paper ONLY. It is especially important to remove all oil, if
the 100X oil immersion lens was used.
Do not use any other paper, a handkerchief or your fingers as they could
damage the lenses.
5. The lowest power objective should be
in place for microscope storage.
7. Return the stage mechanism to its
central position.
9. Replace the dust cover.
A. Components
Compound microscopes magnify thin specimens mounted on microscope slides. They are
ideal for observing unicellular or very small organisms, cells, and cell structures. Below is a
list of parts of a compound microscope, and the
definition or function of each.
Base: the bottom of the microscope, which supports 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 condenser 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 magnified image of the specimen. There are typically four objective lenses
attached to the nosepiece: a 4X scanning objective, a 10X low power objective, a 40X high
power (dry) objective and a 100X oil immersion
objective. The magnification of each objective
is engraved on its side.
Ocular lens or eyepiece: the secondary optical
system that you look through. The ocular lens
further magnifies (10X) the image and brings
the light rays to a focal point. A binocular microscope 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 flat surface upon which the slide with
your specimen is placed. Most microscopes
have a stage finger assembly to hold the slide
on the stage. The entire mechanism including
the slide moves horizontally across the stationary 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).
be adjusted by turning it to increase or decrease
the size of the hole that light passes through.
Condenser: the lens located below the stage,
which focuses light (from the illuminator)
through the specimen being observed. Most
microscopes have a moveable condenser allowing its distance from the specimen to be
adjusted using the condenser knob and condenser alignment screw.
Fine adjustment or fine focusing knob: the
smaller knob towards the back of the instrument that is used to make small adjustments
in the height of the stage for final focusing on a
specimen. It is the only focusing knob used with
high power objectives.
Iris diaphragm: a unit below the condenser
that controls the amount of light directed to the
specimen. The diameter of the diaphragm can
Coarse adjustment or coarse focusing knob:
the large knob towards the back of the instrument that is used to significantly raise or lower
the stage, when you first focus on a specimen
at low power. It is never used when high power
objectives are in place.
Note: If the microscope does not appear to be functioning
properly, do not attempt to fix 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. Köhler Illumination
C. Calibration
Köhler illumination is the alignment of the image-forming light path and the illumination light
path of the microscope. In this process the condenser is centered and focused, thereby providing an evenly illuminated field of view and
more importantly maximum resolution of the
specimen.
Microscopes must be calibrated so accurate
measurements can be made. To calibrate a microscope both an ocular and a stage micrometer are used.
Directions:
Position focusing eyepiece to the appropriate number for your eyesight on the left eyepiece.
Ocular micrometer (O) - a very small ‘ruler’ engraved on a piece of glass and placed inside
one of the ocular lenses
•
The ruler’s magnification does not change
when switching between the objective lenses.
•
At each objective, the value (in µm) of an
ocular tick mark (division) must be determined.
•
Stage micrometer (S) - a very small ‘ruler’
engraved on a piece of glass then mounted
on a slide
•
The ruler’s magnification does change when
switching between the objective lenses.
•
The ‘ruler’ is usually 1 to 2 mm (1000 to
2000 µm) across.
•
Each tick mark (divisions) on the ruler is 10
µm (0.01mm) from the next tick mark.
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 field diaphragm (black lever below
the stage and condenser) until about ¼ of the
field of view is illuminated.
Partially close the aperture diaphragm (on
condenser).
Use the condenser height adjustment knobs
to position the condenser so the diaphragm image is in focus.
Use the two condenser centration screws to
position the condenser the diaphragm image in
the center of the field of view.
Fully open the field diaphragm.
0.01 mm
tick mark
1000 µm
1 mm
=
10 µm
tick mark
Directions (complete the steps below for 4X,
then 10X, and finally 40X):
Focus on the stage micrometer (S).
Rotate the ocular lens until the ocular micrometer (O) is exactly superimposed on S.
Adjust the aperture diaphragm for appropriate contrast (also dependent upon intensity of
stain and thinness of sample).
Adjust S so that its first tick mark lines up
precisely with O’s first tick mark.
Your microscope is now set to maximize resolution of the specimen.
Look for another tick mark (as far from the
beginning as possible) where S and O line up
precisely.
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 sacrifice the
resolution capability. Use the aperture diaphragm and /or
the illumination intensity to adjust contrast.
Count the number of tick marks between
these two tick marks for both O and S.
See examples 1 and 2 on following page.
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Leica DME
Microscope Use
Example 1:
Example 2:
70 tick marks for S
30 tick marks for O
10 um
S
=
23 um for each O tick mark (Ocular division)
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