What and why Köhler illumination

Köhler illumination is uniform alignment of the incident or illuminating light for microscopy.

Every time you use the microscope for transmitted light work, whether brightfield, phase or DIC, you must align the condenser lens to assure Koehler illumination is optimal.  If you fail to do this, you will have poor resolution, wacky contrast artifacts, and unevenly lit pictures.

When you put your first sample on the microscope, you need to focus on the cells and then align for Koehler illumination.

The first 6 minutes of this video shows it step-by-step:  https://www.youtube.com/watch?v=P1Tb_sllWII

For more information, please also see the first 17 minutes of https://www.youtube.com/watch?v=H5-CfX3XLf0

 

Brief Instructions for Köhler illumination

STEP 1
Focus your sample in brightfield. 
(Note the dark shadow in the upper right)
Kohler_start.jpg (9093 bytes)

STEP 2
Close the field diaphragm so it looks something like this.

The lower power the objective lens, the smaller the spot in the center.

kohler_close_field_dia.jpg (4677 bytes)
STEP 3
Focus the edge of the diaphragm by adjusting the condenser height, so it looks like this:   


(if the image moves out of your field of view, skip to step 4, then come back to step 3)
kohler_focus_field_dia.jpg (5403 bytes)
STEP 4
center the image using the two centering screws, so it looks like this:    

(Note centered, crisp edge)
kohlercenter.jpg (5764 bytes)
STEP 5
Open the field diaphragm until it is at the edge of the field of view.    
(Note that the shadow in step 1 is gone.)
kohler_open_field_dia.jpg (9487 bytes)

Contrast can be adjusted using the CONDENSER diaphragm.

However, be careful when adjusting the condenser diaphragm.  Closing the condenser diaphram reduces resolution.  To maximize both contrast and resolution, close the diaphragm just to the point where the image begins to get dark and no further.   This position is especially important when using Nomarski optics.

 

 

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last revised 05 Oct 2015 by mc; previous authoring by cbm in 2000