Living Image 3.1

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F. Fluorescent Imaging 216 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.
<strong>Living</strong> <strong>Image</strong> ® Software User’s Manual Figure F.10 Specular reflection. The four symmetric hot spots on this black polystyrene well plate illustrate the specular reflection of the illumination source. (Imaging parameters: GFP filter set, Fluorescence level Low, Binning=8, FOV=15, f/1, Exp=4sec.) Black polystyrene microplates are recommended for in vitro fluorescent measurements. Figure F.9 and Figure F.10 show that the black polystyrene microplate emits the smallest inherent fluorescent signal, while the white polystyrene microplate emits the largest signal. The clear polystyrene microplate has an autofluorescent signal that is slightly higher than that of the black microplate, but it is still low enough that this type of microplate may be used. Control cells are always recommended in any experiment to assess the autofluorescence of the native cell. Miscellaneous Material Autofluorescence It is recommended that you place a black Lexan ® sheet (Xenogen part no. 60104) on the imaging stage to prevent illumination reflections and to help keep the stage clean. NOTE The black paper recommended for bioluminescent imaging (Swathmore, Artagain, Black, 9"x12", Xenogen part no. 445-109) has a measurable autofluorescent signal, particularly with the Cy5.5 filter set. Figure F.11 shows a fluorescent image of a sheet of black Lexan on the sample stage, as seen through a GFP filter set. The image includes optical autofluorescence, light leakage, and low level autofluorescence from inside the IVIS ® System imaging chamber. The ringlike structure is a typical background autofluorescence/leakage pattern. The image represents the minimum background level that a fluorophore signal of interest must exceed in order to be detected. 217
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Living Image 3.1

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