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)
ZEISS Microscopy How-to: Set up Köhler Illumination on your ZEISS Axio Imager (Upright):
MicroscopyU has a great interactive on guide for Köhler illumination: https://www.microscopyu.com/tutorials/kohler