White Balance Calibration
The color balance of a digital image produced by an optical microscope is dependent not only upon the spectrum of wavelengths transmitted through or emitted by the specimen, but also on the spectral content of the light source. In color digital cameras that utilize charge-coupled device (CCD) imaging detectors, a range of balance adjustments is often necessary in order to produce acceptable color quality in digital images.
The human eye can readily adapt to identify a white object as white even when the surrounding light intensity changes. On the other hand, digital cameras require careful adjustment of the red, green, and blue signal settings by software control in order to achieve the same end. In most digital cameras, illumination intensity and color temperature must be measured and adjusted to ensure that a white object is recorded as white. This process is often referred to as adjusting the white balance, and is a software or hardware option on many digital cameras.
Modern scientific CCD digital cameras incorporate reference circuitry that contains a digital signal processing (DSP) integrated circuit for adjusting the color balance. This circuit provides a series of look-up tables (LUT), which are utilized by the software to adjust the red, green, and blue (RGB) signal amplitudes to arrive at the proper color balance for a specific illumination intensity and color temperature. Often, the look-up tables will contain information about tungsten-halide (at a variety of color temperatures), mercury arc, and xenon arc lamps, among others. The RGB system is one of the primary color models utilized to specify and represent colors in computer-controlled cameras and software. White is produced by combining equal parts of all three colors (red, green, and blue) at levels of 100 percent.
A typical adjustment of white balance in a culture of adherent Indian Muntjac deer skin fibroblast cells is illustrated in Figure 1. On the left (Figure 1(a)) is a digital image of the cells before white balance adjustment. Note that the image exhibits an overall bluish color cast and fine details are relatively indistinct. After white balancing (Figure 1(b)), the image appears more natural with a greater tonal range, and reveals minute image details more clearly.
The Digital Sight camera system is equipped with a white balance feature that allows setting the image white balance based on the contents of the currently displayed image. If the camera head is mounted on a microscope, then the field of view should be adjusted so as not to include any specimen details before white balancing is activated. Alternatively, the specimen can be removed so that the entire viewfield is white before setting the white balance. Setting the image white balance while the display window is not empty may cause the image to take on a color cast, which can seriously impair image contrast and result in colors that deviate from those of the specimen.
There are a number of ways to activate the white balance adjustment feature of the Digital Sight CCU. Information on the Digital Sight's white balance controls can be found in the following sections:
CAM Menu Features - This important menu contains a number of features useful for measuring and calibrating image focus, controlling exposure mode and time, and adjusting camera sensitivity.
Room 1 Network Client Interface - This virtual room provides network-based remote access to the Digital Sight's camera control unit, allowing a remote user to download live microscope images from the camera control unit. Other features include an image annotation function and an electronic zoom function. An option is also available to allow the remote user to take limited control of the camera control unit.
Contributing Authors
Matthew J. Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
