Köhler illumination is a type of specimen illumination used in transmitted-light microscopy. It was designed by August Köhler in 1893, and overcame limitations of previous types of sample illumination techniques (ie: critical illumination). Prior to the advent of Köhler illumination, the filament of the bulb used to illuminate the sample could be visible in the sample plane. This created what is known as a filament image. Various techniques were used to overcome the filament image such as lowering the power of the light source, utilizing an opal bulb, or placing an opal glass diffuser in front of the light source. However, all these techniques, while being semi effective in reducing the filament image, had the effect of reducing the quality and uniformity of light reaching the sample. Reducing the power of the light source and utilizing an opal bulb both caused a narrowing of the spectrum of incident light. For transmitted-light microscopy wide spectrum white light is desired to realize the maximum amount of contrast. Further, adding an opal glass diffuser will cause the light reaching the sample to not be uniform. Uniformity of light is essential to avoid shadows, glare, and incorrect contrast when taking images. Köhler illumination overcomes these limitations by utilizing a collector lens in front of the light source. This lens focuses the light at the condenser diaphragm, and this has the effect of putting the condenser diaphram and the filament image in conjugate planes. Thus, the filament image is no longer conjugate to the image plane, and is no longer visible.
Condenser Optical Components
- High intensity bulb
- Collector lens
- Field diaphragm
- Condenser diaphragm
- Condenser lens
Setting Up Köhler Illumination
1. Focus on the specimen.
2. Close the field diaphragm to its most closed state such that you can see the edges of the diaphragm (may be blurry) in the field of view.
3. Use the condenser focus knobs to bring the edges of the field diaphragm into the best focus possible.
4. Use the condenser-centering screws to center the image of the closed field diaphragm in the field of view.
5. Open the field diaphragm just enough so that its edges are just beyond the field of view.
6. Adjust the condenser diaphragm to introduce the proper amount of contrast into your sample. The amount of contrast added will depend on the sample, however too much contrast can introduce artifacts into your images.
7. Adjust the light intensity as necessary. To adjust light intensity it is best to use a neutral density filter rather than rasing or lowering the lightsource power supply. Neutral density filters block all wavelengths of light equally, while changing the power to the light source can reduce the spectrum of incident light creating a yellow/brown light effect.
- Bright field microscopy
- Phase contrast microscopy
- Dark field microscopy
- Differential interference contrast microscopy
- Polarized light microscopy
- Hoffman modulation contrast microscopy
Köhler illumination setup instructions from berkeley.edu.
This article needs additional citations for verification. (May 2007) (Learn how and when to remove this template message)