White Balance Calibration

The color 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 charged 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 most digital cameras, the 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 white balancing, 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 for white balance in digital photomicrography is illustrated in Figure 1, where the specimen is a multiply-stained thin section of a Dutchman's Pipe dicotic woody vine stem. On the left (Figure 1(a)) is a digital image of the thin section before white balance adjustment. Note that the overall color tones are dark and "muddy" and the background is a light gray in color. After selection of an appropriate area (the clear channel in the right central portion of the image) and application of white balance adjustment algorithms (Figure 1(b)), the image appears more natural with a greater range of tonal values and a white background.

The DXM 1200 is equipped with a software feature termed the Live settings panel (Figure 2), which is utilized to specify the conditions for determining white balance for digital images. Either a single point or a rectangular marquee-selected area on the image can be specified for measuring the white balance.

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To activate the white balance adjustment within the DXM 1200 software, click on the Live tab in the ACT-1 window to display the features available in this settings panel. Before attempting to set the white balance, carefully examine the image to determine the appropriate area that should be selected for the adjustment. In stained thin sections, an area devoid of stained tissue should be utilized. Some biological stains can bleed through the mounting medium and contaminate the areas between tissue sections, so avoid these parts of the specimen. Images viewed in polarized light, differential interference contrast, fluorescence, or other contrast enhancing techniques can present more difficulty in white balance determination.

When the appropriate white balance area has been chosen, select either the Point (eyedropper icon; Figure 2) or marquee selection (Rectangle Area; Figure 2) radio buttons in the White Balance section of the Live settings panel to activate the measurement mode. If the area to be measured is very small (limited to only a few pixels), then the Point (eyedropper) tool should be chosen for more accuracy in the exact location of the selection. To activate the Point sampling tool, click on the eyedropper button in the settings panel. This will initialize the Start software routine, which deactivates the exposure frame and focus mark overlays and allows selection of a point in the image. When a specific point in the image is chosen, the measurement position can be specified when the tip of the eyedropper icon is located directly on top of the point and the mouse button is clicked once.

A more accurate method of selecting an area for white balance adjustment is to utilize the marquee selection (Rectangle Area) tool. After clicking on the appropriate radio button, move the cursor to the desired position within the live image window and drag the mouse to specify a rectangular selection. When the Start button is depressed, the software will proceed with a white balance calculation and the Measuring White Balance dialog box appears (Figure 3). At this point, the calculation can be halted by clicking on the Stop button in the dialog box.

The white balance is measured by automatically toggling through sensitivity (gain) values through the range Normal, High, and Max. Either insufficient or excess light intensity may confuse the software and make it difficult or impossible to correctly measure white balance. Always adjust the Sensitivity radio buttons and the exposure slider to optimize image brightness prior to making a white balance calibration.

The specimen area should always be carefully selected for white balance measurement, because artifacts in the specimen can often lead to errors in the determination of white balance. Differences in refractive index for the specimen or the presence or absence of a cover glass can lead to spherical aberration, which can affect the measurement (although in a minimal manner). As mentioned above, bleeding of biological stains can also induce color shifts in the mounting medium, leading to errors in white balance calibration. Wherever possible, use the marquee-selection tool to choose the largest area available for white balance determination. This allows the software to average white balance calculations over a greater number of pixels and will generate more acceptable results. In some cases, especially when only a single pixel is selected, the overall white balance determination can be compromised leading to images that are not adjusted properly.

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