Living Image 3.1

F. Fluorescent Imaging
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level of autofluorescence. Autofluorescence may be generated by the system optics,
plastic materials such as microplates, and by animal tissue. Filter leakage, which may also
occur, is another source of background light.
The optical components of the IVIS ® Imaging Systems are carefully chosen to minimize
autofluorescence. Pure fused silica is used for all transmissive optics and fiber optics to
reduce autofluorescence. However, trace background emissions exist and set a lower limit
for fluorescence detection.
To distinguish real signals from background emission, it is important to recognize the
different types of autofluorescence. The following examples illustrate sources of
autofluorescence, including microplates, other materials, and animal tissue.
Microplate Autofluorescence
When imaging cultured cells marked with a fluorophore, be aware that there is
autofluorescence from the microplate as well as native autofluorescence of the cell.
Figure F.9 shows autofluorescence originating from four different plastic microplates. The
images were taken using a GFP filter set (excitation 445-490nm, emission 515-575nm).
White polystyrene Clear polypropylene
Clear polystyrene Black polystyrene
Figure F.9 Examples of microplate autofluorescence emission
The black polystyrene plate emits the smallest signal while the white polystyrene plate emits
the largest signal. (Imaging parameters: GFP filter set, Fluorescence level Low, Binning=8,
FOV=15, f/1, Exp=4sec.)
Two types of autofluorescent effects may occur:
Overall glow of the material - Usually indicates the presence of autofluorescence.
Hot spots - Indicates a specular reflection of the illumination source (Figure F.10).
The specular reflection is an optical illumination autofluorescence signal reflecting
from the microplate surface and is not dependent on the microplate material.