Achieving accurate color reproduction for brightfield microscopy using a color camer.

Technical Note: 7
Reads: 6808
Creation Date: 28/09/1999
Modification Date: 25/07/2005

The illumination of a sample is critical for high quality color imaging.

Color temperature

CCDs respond to light of different wavelengths or colors completely differently from the human eye and thus require completely different illumination to produce what a human describes as correct color balance in an image. For optimum color representation in an image, the illumination source should have a color temperature of 3200K. Some microscopes have a function whereby the intensity of the transmitted light source can be set to 3200K or a photography setting, otherwise it can be approximated by setting the voltage of the lamp to max.

This will be too bright to view comfortably therefore it will be necessary to insert neutral density filters in front of the lamp to cut down the intensity of the light without altering the color temperature.

Infra red compensation

The transmitted light has a high proportion of infrared (IR) compared to daylight and the CCD camera is more sensitive to red than the eye. IR in an image reduces the contrast so it may be necessary to redress the balance by using a BG 38 filter (or daylight filter) along with the neutral density filters

Koehler illumination

Use Koehler to provide a field with uniform illumination, maximum resolution and optimum contrast.

Setting up Koehler illumination: The procedure is broadly similar for all scopes, however see your microscope documentation for specific instructions and optimisations.

  1. Focus the specimen. Start with a 10x or 16x objective.
  2. Close the field diaphragm (FD) until the edge or part of the edge is visible.
  3. Move the condenser up and down (being careful not to let it make contact with the slide) to bring the edge of the FD into focus.
  4. Use the centering screws on the condenser to move the image of the FD into the center.
  5. Open the FD to almost fill the field of view (FOV), make final centering adjustments, and then open the FD so that the edge is just out of the FOV.
  6. Adjust the aperture diaphragm for contrast. Contrast is achieved at the expense of resolution. Ideally the aperture is between 60 and 80% open. Remove an eyepiece to see how much of the aperture is filled with light.

This is Koehler illumination set up for the current lens. The diaphragms will have to be adjusted again when a different lens is used.