In 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.

The 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.

The following instructions apply to any microscope, upright or inverted which is equipped for transmitted light bright field illumination. Focussing of the field diaphragm as discussed here should be done for phase and differential interference optics as well.

    To set up Köhler illumination:
  1. Switch 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.

  2. 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.
  3. Notice 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.

  4. The front lens should be about 1-3 mm above or below the specimen. There are condenser focussing knobs to do this.
  5. Now bring your specimen into focus with the coarse and fine focussing 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.
  6. When 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 focussing 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.
  7. When 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.
  8. As 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 source.
  9. Your 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.

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