To Set Up A Microscope Properly For Transmitted Light
Illumination: Köhler Illumination
order to get the best image possible from
brightfield, phase contrast, differential
interference contrast, or polarization optical
setups with the light microscope, it is
crucial that the light path be set up properly.
method for doing this is called Köhler
illumination after August Köhler, the
man who invented it. It is also know as double
diaphragm illumination because it employs
both a field and an aperture iris diaphragm
to set up the illumination. If the light path
is set up properly, you will have the advantages
of an evenly illuminated field, a bright image
without glare and minimum heating of the specimen.
following instructions apply to any microscope,
upright or inverted which is equipped for
transmitted light bright field illumination.
Focusing of the field diaphragm as discussed
here should be done for phase and differential
interference optics as wel
set up Köhler illumination:
on the light source and make sure that
light is coming through the field diaphragm
at the base (upright microscope) or the
top (inverted microscope) of the microscope
stand. It may help to place a piece of
paper over the field stop to see the light.
Place your specimen on the stage and turn
the nosepiece (which holds the objective
lenses) to the 10X or 20X lens. Open the
field diaphragm as far as it will go.
whether or not your specimen is illuminated.
It will help to place a piece of paper
over the top of the specimen to see if
light is getting through to it. If you
are using the brightfield condenser stop,
open the iris diaphragm (or aperture diaphragm)
on the condenser turret (which contains
the stops for brightfield and phase, etc)
wide open to give the maximum illumination.
If there is a swing-in front lens for
the condenser (directly above (inverted)
or below (upright) the specimen), you
may need to swing it into the light path.
The front lens should be about 1-3 mm above
or below the specimen. There are condenser
focusing knobs to do this.
bring your specimen into focus with the
coarse and fine focusing knobs. the best
way to do this is to rack the lens as
close possible to the specimen watching
the objective lens all the time (and NOT
looking into the oculars) to make sure
that the lens does not run into the slide.
Then rack the lens away from the stage
(or vice versa) while looking through
the oculars to bring the specimen into
focus (details are as sharp as they can
be). If the light is too bright, reduce
it with the rheostat on the light source.
the specimen is in focus, start to close
the field diaphragm and also begin to
carefully move the condenser up and down
with the condenser focusing knobs. Look
for a sharp image of the edge of the field
diaphragm. This may be a little with a
long working distance condenser. Also,
if the iris diaphragm in the condenser
turret is open wide, the glare may obscure
the edge of the field diaphragm silhouette
somewhat. Furthermore, you may find that
this edge is not centered.
the edge of the field diaphragm silhouette
is sharply defined, center it with the
two knobs (usually knurled knobs) coming
out diagonally from the condenser. Close
down the field diaphragm most or all the
way to get it centered properly. When
it is centered, open the field diaphragm
until its edge is outside the field. If
you are doing brightfield or differential
interference microscopy, do not yet open
the field diaphragm.
stated before, you may notice some glare
around the edge of the field diaphragm,
that the edge area outside the edge is
not completely dark like the outer part
of the whole field as you should see it
now. This glare comes from light bouncing
around in the light path and going in
and illuminating the specimen in such
a way as to obscure detail in the specimen.
To reduce this glare, close down the iris
aperture in the condenser turret until
all of the dark area outside of the field
stop silhouette is evenly dark. Now open
up the field diaphragm until the edge
of the diaphragm silhouette is outside
the field of view. You should also now
be able to turn up the light at the power
specimen should be properly illuminated
and should give you a great image. If
it does not, check to make sure your lenses
and other optical components are clean.
Then recheck to see that you have followed
each step properly. If you still cannot
get a good image and you are at UCLA,
give me (Matt Schibler) a call at X59783
and I'll be glad to try to help you.