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    Setting Up Köhler Illumination:

     

    Purpose:

    Setting up Köhler illumination is a critical component of performing light microscopy. This illumination technique provides the optimum balance between contrast and resolution. Therefore, “Köhlering” should be the first thing to occur when one sits down at a microscope.

     

    *Throughout the process delineated below, be sure to avoid contacting the specimen (as always) with any lenses.*

     

    Steps –

    1.       1. Turn on the microscope power


    2.       2. Turn the nosepiece such that the 10x objective will be the active objective

    3.       3. Open the variable field diaphragm and the aperture diaphragm to the fullest extent possible.

     

    4.       4. Place sample on the microscope stage and focus the sample using the present settings on the microscope.

    5.       5. Close the variable field diaphragm as much as possible. You should see an outline (either crisp or rather fuzzy) of a polygon with greater than 6 sides (in truth, I believe it to be a dodecagon in our microscopes, though I have not counted).

     

    6.      6.  Use the condenser focusing knob (raising and lowering the condenser) to bring the edges of the polygon to optimal focus (with crisp, clean edges)

    7. The polygon should be located in the center of the field of view. If this is not the case, use the condenser centering screws to move the polygon to the location that you believe is the center of the field of view.

     

    7.       8. Open the variable field diaphragm until the edges of the polygon have almost left the field of view and center the enlarged polygon once again.

    a.       It is usually helpful to try to place the polygon such that all corners contact the edge of the field of view. In such a case, you are assured that the condenser is centered.

    8.       9. Open the variable field diaphragm such that the polygons edges are located just barely out of the field of view.

    9.      10. Remove one eyepiece and look down the opening you just created.

    10.    11. You should see a bright shape in the center of a darker circle. Open/close the aperture diaphragm such that this shape fills roughly 3/4 (75%) of the viewing field.

     

    11.    12. Replace the nosepiece and begin your work

    12.   REPEAT THIS ENTIRE PROCESS EACH TIME YOU SWITCH TO A NEW OBJECTIVE

    a.       Switching to different objectives changes the amount of light that enters through the objective, requiring “reKöhlering” of the illumination path.

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    For Step #9, once the eyepiece is removed, insert the "Centering Telescope" to better visualize the bright shape in the center of a darker circle. This is referenced in Step #12!