Transcript Slide 1

1- bring sample into focus with objective focus
knob
image point for o1
and ω2
diaphragm open
image point for o2
and ω1
diaphragm closed
2- close field diaphragm to minimum size
back plane of objective lens
front of objective lens
3- focus condenser until you can see a clear
image of the field diaphragm (open and close
field diaphragm to check, center as needed)
sample (object)
condenser lens
4- open the field diaphragm until the field of
view is just completely illuminated
condenser diaphragm (focused at back
plane of objective)
field diaphragm (focused at object plane)
collector lens
Light source (filament)
adapted from Murphy 2001 and Nikon microscopy website
http://www.microscopyu.com/tutorials/java/kohler/index.html
Why do we use Kohler
illumination? For even
intensity at the object and
image, we want each small
part of our source to
contribute to all parts of the
image and we want each
small part of our image to be
made up of light from all areas
of the source. We don’t want
to see the shape of the light
source in our image! What
would we see if light rays
from A1 and A3 were focused
at the object/image planes?
5- now adjust light intensity and the condenser
diaphragm to optimize image; small aperture in
condenser diaphragm gives best contrast,
large aperture gives best resolution (default
starting point is for condenser aperture to fill ¾
of objective back plane, this can be viewed
with special telescope eyepiece).
Notes: Do not adjust light intensity with field or
condenser diaphragms, use the condenser
diaphragm to optimize contrast and resolution,
adjust lamp voltage to correct light intensity.