Living Image 3.1

F. Fluorescent Imaging
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Unlike the method described in section F.6, Subtracting Instrument Fluorescent
Background, where you acquire an actual instrument fluorescent background image by
removing the fluorescent subject from the imaging chamber to correct the background, the
new method uses software correction. To perform adaptive background subtraction:
• Identify the fluorescent subject in the original image using the photo mask
• The software automatically fits the instrument background to the whole image
using the pixels outside of the subject
• The software subtracts the fitted instrument background from the original image
In most situations, such adaptive software correction works as effectively as the traditional
method except the following cases:
• The subject is dark, making it is difficult to mask the subject using the photo (for
example, experiments that use black well plates)
• The subject occupies most of the FOV (for example, high magnification or
multiple mice in the FOV). As a result, there is not enough information outside the
subject that can be used to help fit the background.