A lack of proper color temperature balance between the microscope illumination source and the film emulsion or electronic image sensor calibration is the most common reason for unexpected color shifts in photomicrography and digital imaging. If the color temperature of the light source is too low, images will have an overall yellowish or reddish cast, and if the color temperature is too high, images will have a blue cast. This interactive tutorial explores how the white and black balance settings on a digital camera system can be utilized to adjust color balance in digital images.
The tutorial initializes with a randomly selected specimen image, captured in the microscope, appearing in the left-hand window entitled Specimen Image. Each specimen name includes, in parentheses, an abbreviation designating the contrast mechanism employed in obtaining the image. The following nomenclature is used: (FL), fluorescence; (BF), brightfield; (DF), darkfield; (PC), phase contrast; (DIC), differential interference contrast; (HMC) Hoffman modulation contrast, and (POL), polarized light. Visitors will note that specimens captured using the various techniques available in optical microscopy behave differently during image processing in the tutorial.
Positioned adjacent to the Specimen Image window is the Balanced Image window, which displays the image after white or black balance correction algorithms have been applied to the specimen image. In order to operate the tutorial, select either the White Balance or Black Balance radio button from the Balance Mode listing at the bottom of the tutorial window. Next, choose Point Selection or Rectangular Selection from the Area Selection Method list. If Point Selection is chosen, position the mouse cursor over an area that should appear either white or black in the image and click on the button to apply the appropriate algorithm to the specimen image. The Balanced Image window will automatically display the processed image with either the white or black correction applied. Choosing the Rectangular Selection radio button will enable the marquee selection of a specific area in the image over which the correction algorithm will be applied.
When digital capture devices are utilized to image color specimens in optical microscopy, obtaining correct color balance to provide a true representation of the specimen is usually the primary goal. Intentional deviations from this strategy are usually made only to correct a problem with the specimen preparation that produces an undesirable color cast. Most scientific grade digital cameras, including those specifically designed for microscopy, rely on adjusting white balance by reference to a selected color value. In transmitted illumination, an appropriate region (usually white or a neutral gray) is chosen from the specimen field or the adjustment is performed on the illuminated field alone, with the specimen removed from the light path. In order to perform white balance adjustment in a microscope utilizing reflected illumination, a white or neutral-gray card (or section of paper) can be positioned on the microscope stage in place of the specimen. The white balance setting is subsequently acquired by measuring the light reflected from the surface of the white card.
The majority of digital cameras designed for microscopy are controlled through software residing on a host computer, such as Nikon's NIS-Elements microscopy software. When the white balance adjustment window is activated in the user interface, options are made available for selecting an area in the viewfield for white balance evaluation by the camera system. The live image on the display monitor should be carefully evaluated for an appropriate white or neutral gray area to serve as a reference point for the image sensor. If the image displayed on the monitor has a color cast that differs from the color balance observed in the microscope eyepieces, the camera system must be adjusted for white balance in order to render an accurate image of the specimen. Ideally, the displayed color cast will be removed by the color balance circuitry of the camera when the proper specimen area is selected for white balance adjustment.
Several typical examples of specimen areas that can be employed to set digital camera white balance algorithms are presented in Figure 1. The specimens are a living culture of fibroblasts imaged with differential interference contrast (Figure 1(a)), a quadruple-stained thin section of starch granules in potato tissue under brightfield illumination (Figure 1(b)), and human red blood cells in phase contrast (Figure 1(c)). The regions on each image that are suitable for white balance adjustment using the area selection technique are outlined in red, while the yellow arrows indicate specific points on the images that may produce satisfactory white balance calibration when selecting a single pixel.
The region selected as a white balance reference should be as large as possible and free from the coloration effects of specimen stains that have bled into the mounting medium. The white balance adjustment software in many systems enables the selection of either a single point (pixel) in the image, or a larger area that may be designated by marquee selection with the mouse cursor. Better results are generally obtained by selecting the largest possible region. A much wider variation in results can occur if a single point is chosen for adjustment, because fluctuating localized combinations of red, green, and blue pixel intensities can contribute to the overall visual effect of white. By selection of a larger area, an average is obtained over a larger number of pixels in the sensor array, with improved probability of accomplishing acceptable color balance. Following reference area selection, the white balance adjustment is initiated, and the camera system utilizes either an algorithm or look-up table (LUT) to set appropriate electronic values (such as sensor gain for each of the component colors) that produce a neutral or white color value.
Matthew J. Parry-Hill, Thomas J. Fellers, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.