Living Image 3.1

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E. Luminescent Background Sources & Corrections 206 If any part of the hemisphere exhibits what appears to be luminescent emission, it is actually the light reflected from a source illuminating the hemisphere. Observe the side of the hemisphere that is illuminated to help determine the source location. In Figure E.1 the pen appears very bright due to phosphorescent emission that is also illuminating the portion of the hemisphere next to the pen. If the pen had been outside the field of view, it would not have been imaged, and the source of the phosphorescence would be less obvious. However, the illumination of the hemisphere would still be very apparent and indicative of a light pollution problem. ! IMPORTANT E.3 Background Light From the Sample Figure E.1 Xenogen High Reflectance Hemisphere and a plastic marker pen Left: Photographic image. Right: Photograph with luminescent overlay. The hemisphere is illuminated by phosphorescence emitted from the pen. ALERT! Handle the Xenogen High Reflectance Hemisphere by its black base plate while wearing cotton gloves provided by Xenogen. Skin oils can phosphoresce and will contaminate the hemisphere. Latex gloves and the powder on them may also phosphoresce. If the hemisphere becomes contaminated, contact Xenogen technical support for a replacement. There are no known agents that can clean the hemisphere. To check the hemisphere for contamination, take several images of the hemisphere, rotating it slightly between images. A glowing fingerprint, for example, will rotate with the hemisphere, while a glowing spot due to external illumination most likely will not. Another source of background is the natural light emitted from a sample that is not due to emission from the source of interest in the sample. This type of background may be due to a material associated with the experimental setup. For example, the cell culture medium may phosphoresce. Materials should be screened so you can identify and eliminate problematic materials. If a background source is phosphorescent and the phosphorescent lifetime is relatively short, you can try keeping the sample in the dark for a long period before imaging to reduce background light emission. Occasionally there is no way to eliminate the natural light emission of the sample. The natural light emission associated with living animals (autoluminescence) is a major area of interest in in vivo bioluminescent imaging. Most animals exhibit a low level of autoluminescence. Usually this is only a problem when looking for very low signals at the highest levels of sensitivity.
<strong>Living</strong> <strong>Image</strong> ® Software User’s Manual Xenogen Corporation has conducted tests to try to minimize the source of the background light emission in mice. Test Description Observation Test 1: Subject animals were housed in the dark 12 hours prior to imaging. Test 2: White-furred animals were shaved prior to imaging Test 3: Alfalfa (known to be phosphorescent) was eliminated from the animal diet. Background emission levels were not reduced. A phosphorescent component in mouse fur or skin is not the source of light emission. No increase or decrease in background emission levels. An alfalfa-free diet reduced background emission slightly, but not significantly. The sources of autoluminescence are not yet fully understood. No external sources have been proven to cause natural light emissions, so it is possible that a chemiluminescent process associated with metabolic activity in living animals is the source of animal background. This is supported by the observation that the level of background light drops significantly in euthanized animals. In Figure E.2 the background light emission is clearly visible in the images of a control white-furred mouse and a nude mouse. The images are five minute, high-sensitivity (high binning) exposures. The average emission from a white-furred mouse and a nude mouse is approximately 1600 photons/s/cm 2/sr and 1000 photons/s/cm 2/sr, respectively. Since these values are well above the lower limit of detection of the IVIS ® Imaging System (~100 photons/s/cm 2/sr), the background light emission from the mouse determines the limit of detection. An approximation of this background (determined by making similar measurements on either control animals or regions of the subject animal that do not contain the primary signal) can be subtracted from ROI measurements. (For more information on ROI measurements, see Chapter 5, page 69.) Note that the background light emission is not uniform over the entire animal. In Figure E.2 images of control animals (mice) show a somewhat higher background component originating from the abdominal and thoracic regions. Therefore, care must be taken when selecting a representative background area. 207
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Living Image 3.1

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