Köhler illumination 1
Köhler illumination
Köhler illumination is a method of specimen illumination used in transmitted- or reflected-light microscopy[1] . It
was designed by August Köhler in 1893, and overcame the limitations of previous techniques of sample illumination
(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 remove the filament image, for example lowering the power of the light source, using an opal bulb, or
placing an opal glass diffuser in front of the light source. However, all these techniques, although effective in
reducing the filament image to a certain degree, had the effect of reducing the quality and uniformity of light
reaching the sample. Reducing the power of the light source and introducing an opal bulb both caused a reduction in
the spectrum of incident light. For transmitted-light microscopy wide spectrum white light is desirable in order to
realize the maximum amount of contrast. Further, adding an opal glass diffuser will cause the light reaching the
sample to be uneven. Uniformity of light is essential to avoid shadows, glare, and inadequate contrast when taking
photomicrographs. Köhler illumination overcomes these limitations by creating parallel light rays to pass through the
specimen. Because the light rays that pass through the specimen are parallel they will not be in focus when creating
the image of the specimen thus eliminating the image of the filament.
Principles
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 so 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 increasing or reducing the supply of power to the lightsource. Neutral density filters block all
wavelengths of light equally, while changing the power to the light source will alter the balance in the
spectrum of incident light giving a yellow/brown appearance to the image.
Köhler illumination 2
Used In
• Bright field microscopy
• Phase contrast microscopy
• Dark field microscopy
• Differential interference contrast microscopy
• Polarized light microscopy
• Hoffman modulation contrast microscopy
See also
• Optical microscopy
• August Köhler
External links
Köhler illumination setup instructions [2] from berkeley.edu.
References
[1] Olympus Microscopy Resource Center - Köhler Illumination (http:/ / www. olympusmicro. com/ primer/ anatomy/ kohler. html)
[2] http:/ / microscopy. berkeley. edu/ courses/ TLM/ condenser/ kohler. html
Article Sources and Contributors 3
Article Sources and Contributors
Köhler illumination Source: http://en.wikipedia.org/w/index.php?oldid=340027185 Contributors: ChrisHodgesUK, Coolbrdr, Jkbjob, LilHelpa, Peter G Werner, Rjwilmsi, Sole Soul,
TenOfAllTrades, 14 anonymous edits
License
Creative Commons Attribution-Share Alike 3.0 Unported
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