Background

When we use brightfield, either on one of the widefield microscopes, or on the confocals its important to set it up properly in order to achieve the best images.

Fluorescence light is focused onto the sample from the objective and is viewed back through the same objective (Reflected light), which means, if you can see the sample in focus, the light (fluorescence) must be focused onto the sample. For brightfield (transmitted light) this is not always the case. Brightfield illumination comes from a separate light-source and focused onto the sample via the condenser. Therefore extra steps are required to ensure this alternate light-path is focused onto your sample.

The method of aligning the transmitted light (brightfield) is called Kohler illumination, and utilises conjugate focal planes in the field-stop and condenser to help you align (focus) the light onto your sample. The method utilises the same process for either Upright or Inverted microscopes.

Laser transmitted light alignment

 

Setting up Koehler illumination

 

Microscope alignment for optimal image quality: Koehler Illumination

 

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

 

ZEISS Microscopy How-to: Set up Köhler Illumination on your ZEISS Axio Observer (Inverted)