Appendix 1. Characteristics of CCD Color Cameras
CCD cameras come in three types:
Single chip monochrome
A single CCD detector, offers the highest resolution for fluorescence measurements. They are the easiest to apply and have the highest sensitivity because of the simple optical path through a single filter. The main problem with single chip cameras is the lack of color information, which can only be gotten by sequencing RGB filters over the detector, thus making them too slow for real time viewing. Microscopes with a single chip camera frequently have a color camera in addition.
Single chip color
The most popular color cameras have single chip CCD detectors that have a checkerboard pattern of different colored pixels across the surface. There is no location on the sensor that receives full color information. In the popular Bayer pattern, 50% of the pixels are green, 25% red, and 25% blue. Averaged together, a Bayer chip misses 67% of the color information in the image. Complex calculations from surrounding pixels are made to interpolate the missing color at each pixel. What is more difficult to construct is the image detail contained in a missing color, which can result in reduced sharpness and color artifacts in the image. Bayer pattern color cameras do not produce equivalent resolution to true RGB pixel cameras.
Triple chip color
The highest quality color images are obtained by dividing the image into three colors, each detected by a separate chip. The prism system with color coatings that separates the images is very expensive. In addition, a high-quality imaging lens that is matched to the prism system is required to produce three color images with exactly the same magnification. High precision registration of the pixels on the three chips is necessary for best performance. Because of the extensive optical system in front of the detectors, a 3 CCD camera is the least efficient optically and has the lowest sensitivity. Triple chip camera filters are fixed and cannot be exchanged for different wavelength bands as may be desired in a fluorescence system.
All CCD camera systems have a common set of difficulties.
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Sensitivity decreases with increased resolution. Smaller pixels are less sensitive, so frame times slow down or higher illumination power is required. Work arounds include binning 4 or more adjacent pixel charges to increase the signal. However, this reduces resolution by 4. Cooling the detector reduces thermal noise and allows higher video amplifier gain or longer integration times to be used. Very expensive cameras have electron multipliers in front of a single CCD detector for much higher sensitivity, but lower resolution.
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Blooming is caused by very bright parts of the image. Excess photoelectrons flow into the neighboring pixels causing expansion of the white areas in the image. There are three different types of blooming, which require special circuits to minimize.
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Smear can occur in interline transfer CCDs when photons enter the dark vertical shift registers during readout time. In frame transfer CCDs, smear occurs from the light hitting the sensor during the frame shift to memory. Light blocking shutters are required to eliminate smear.
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Aliases or Moiré effects can occur if the sample has a periodic structure whose spacing is close to the CCD detector array spacing. A beat frequency can be generated resulting in wavelike interference patterns, called aliases, or Moiré patterns, that are not real in the image (see Figure 5). The variable periodicities of the Bayer pattern can make this effect worse.
Figure 5. Moiré Pattern in Crossed Diatoms Image