Living Image 3.1

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F. Fluorescent Imaging 218 Figure F.11 Light from black Lexan This image shows the typical ring-like structure of light from a sheet of black Lexan, a low autofluorescent material that may be placed on the imaging stage to prevent illumination reflections. (Imaging parameters: GFP filter set, Fluorescence level High, Binning=16, FOV=18.6, f/2, Exp=5sec.) Other laboratory accessories may exhibit non-negligible autofluorescence. The chart in Figure F.12 compares the autofluorescence of miscellaneous laboratory materials to that of black Lexan. For example, the autofluorescence of the agar plate with ampicillin is more than 180 times that of black Lexan. Such a significant difference in autofluorescence levels further supports the recommended use of black polystyrene well plates. NOTE It is recommended that you take control measurements to characterize all materials used in the IVIS ® Imaging System. Figure F.12 Comparison of autofluorescence of various laboratory materials to that of black Lexan
<strong>Living</strong> <strong>Image</strong> ® Software User’s Manual Despite the presence of various background sources, the signal from most fluorophores exceeds background emissions. Figure F.13 shows the fluorescent signal from a 96-well microplate fluorescent reference standard (TR 613 Red) obtained from Precision Dynamics Co. Because the fluorescent signal is significantly bright, the background autofluorescent sources are not apparent. Figure F.13 96 well plate fluorescent reference standard (TR 613 Red) The fluorescent signal is strong enough to exceed background emissions. (Imaging parameters: DsRed filter set, Fluorescence level Low, Binning=8, FOV=15, f/1, Exp=4sec.) Reference standard TR 613 Red is available through Precision Dynamics Co, http:// www.pdcorp.com/healthcare/frs.html. Animal Tissue Autofluorescence Animal tissue autofluorescence is generally much higher than any other background source discussed so far and is likely to be the most limiting factor in in vivo fluorescent imaging. Figure F.14 shows ventral images of animal tissue autofluorescence for the GFP, DsRed, Cy5.5, and ICG filter set in animals fed regular rodent food and alfalfa-free rodent food (Harlan Teklad, TD97184). Animals fed the regular rodent diet and imaged using the GFP and DsRed filter sets, show uniform autofluorescence, while images taken with the Cy5.5 and ICG filter sets show the autofluorescence is concentrated in the intestinal area. The chlorophyll in the regular rodent food causes the autofluorescence in the intestinal area. When the animal diet is changed to the alfalfa-free rodent food, the autofluorescence in the intestinal area is reduced to the levels comparable to the rest of the body. In this situation, the best way to minimize autofluorescence is to change the animal diet to alfalfafree rodent food when working with the Cy5.5 and ICG filter sets. Control animals should always be used to assess background autofluorescence. 219
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Living Image 3.1

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