Köhler illumination is a microscope alignment technique that ensures images are evenly illuminated and at maximum resolution. The microscope should be adjusted for Köhler illumination before transmitted light imaging, phase contrast and differential interference contrast (DIC).

Before adjusting for Köhler illumination you need to know where the relevant condenser components are on the microscope. This varies between manufacturers and models. The image below gives some examples of where to find the field diaphragm, iris aperture, etc. If in doubt, please ask a member of light microscopy staff or see the helpful links at the bottom of the page.

Condenser adjustment for Köhler illumination

The image above shows condensers of various upright and inverted microscopes. This selection is not exhaustive and components may be located elsewhere on other models

Condenser turret (Turret): Bright field position is H on Zeiss microscopes, A on Nikon microscopes and usually BF on Leica microscopes
Field diaphragm adjustment (FD): The DM2500 field diaphragm is on the other side of the stand. The Nikon Ti field diaphragm is near the top of the transmitted light pillar, labelled F
Condenser focus (Foc): This moves the condenser up and down but doesn't affect the focus of the specimen
Centring screws (CS): The Leica DM2500 and DMI6000 must be centred using hexagonal screwdrivers or Allen keys
Iris aperture adjustment (AP): The iris aperture on the DMI6000 is adjusted using the AP buttons on the left of the microscope stand

Alignment Procedure

Focus on a transmitted light image of the specimen using a 10x air lens
Rotate the condenser turret to the bright field position. Adjustment can either be manual or done through buttons or a touch pad, depending on the microscope model
Fully close the field diaphragm and adjust the condenser focus so the image of the field diaphragm is in sharp focus
Centre the image of the field diaphragm using the two centring screws
Open the field diaphragm until it is just outside the field of view
Remove an eyepiece and look into the empty opening
Adjust the iris aperture until it is about 70% open and then replace the eyepiece. The microscope is now adjusted for Köhler illumination

Links

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