Köhler  Illumination

Köhler Illumination for Brightfield 

 Köhler illumination is a method for evenly illuminating the sample with white light. This method is very important for brightfield imaging in widefield microscopy, and for using the T-PMT for confocal imaging. Brightfield illumination from light source focuses onto the sample via condenser, as a result extra steps are necessary to ensure the light-path is focused on the sample. 

 Bring the sample into focus 

 Close the field diaphragm ( 2 ) until you can see at least one edge 

 Adjust condenser height ( 3 ) until the edges of the diaphragm are crisp. You’ll see an octagon. 

 Center the diaphragm ( 4 ) mage using the two centering screws 

 Open the field diaphragm (2) just until the image fills the field of view (FOV) 

 

 Link for setting up Köhler illumination 

 ZEISS Microscopy How-to: Set up Köhler Illumination on your ZEISS Axio Imager (Upright): 

 https://www.youtube.com/watch?v=Qk8dbT-N1xw  

 MicroscopyU has a great interactive on guide for Köhler illumination:  https://www.microscopyu.com/tutorials/kohler