Living Image 3.1

Living Image ® Software User’s Manual
F.6 Subtracting Instrument Fluorescent Background
The fluorescence instrumentation on an IVIS ® Imaging System is carefully designed to
minimize autofluorescence and background caused by instrumentation. However a
residual background may be detected by the highly sensitive CCD camera.
Autofluorescence of the system optics or the experimental setup, or residual light leakage
through the filters can contribute to autofluorescence background. The Living Image
software can measure and subtract the background from a fluorescence image.
Fluorescent background subtraction is similar to the dark charge bias subtraction that is
implemented in luminescent mode. However, fluorescent background changes day-to-day,
depending on the experimental setup. Therefore, fluorescent background is not measured
during the night, like dark charge background is.
After you acquire a fluorescent image, inspect the signal to determine if a fluorescent
background should be subtracted (Figure F.16). If background subtraction is needed,
remove the fluorescent subject from the imaging chamber and measure the fluorescent
background (select Acquisition → Fluorescent Background → Measure Fluorescent
Background on the menu bar). In the Living Image ® software, the Sub Fluor Bkg check
box appears on the Control panel after a background has been acquired. You can toggle the
background subtraction on and off using this check box.
NOTE
The fluorescence background also contains the read bias and dark charge. Dark
charge subtraction is disabled if the Sub Fluor Bkg check box is checked.
Figure F.16 Comparison of dark charge bias subtraction (left) and fluorescent background subtraction (right).
The autofluorescence from the nose cone and filter leakage have been minimized in the image on the right by using Sub
Fluor Bkg option.
F.7 Adaptive Background Subtraction
Adaptive background subtraction is a simple way to reduce the "instrument fluorescent
background" by fitting and removing the background using the existing image (for
example, the left image in Figure F.16).
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