Living Image 3.1

Living Image ® 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.
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