Living Image 3.1

Living Image ® Software User’s Manual
6.8 Subtracting Tissue Autofluorescence Using Background Filters
High levels of tissue autofluorescence can limit the sensitivity of detection of exogenous
fluorophores, particularly in the visible wavelength range from 400 to 700 nm. Even in the
near infrared range, there is still a low level of autofluorescence. Therefore, it is desirable
to be able to subtract the tissue autofluorescence from a fluorescent measurement.
The IVIS ® Imaging Systems implement a subtraction method based on the use of blueshifted
background filters that emit light at a shorter wavelength (see Table 6.2, page 101).
The objective of the background filters is to excite the tissue autofluorescence without
exciting the fluorophore. The background filter image is subtracted from the primary
excitation filter image using the Image Math tool and the appropriate scale factor, thus
reducing the autofluorescence signal in the primary image data. (For more details, see
Chapter 6, page 101.) The assumption here is that the tissue excitation spectrum is much
broader than the excitation spectrum of the fluorophore of interest and that the spatial
distribution of autofluorescence does not vary much with small shifts in the excitation
wavelength.
Figure F.17 shows an example of this technique using a fluorescent marker. In this
example, 1× 10 6 HeLa-luc/PKH26 cells were subcutaneously implanted into the left flank
of a 6-8 week old female Nu/nu mouse. Figure F.18 shows the spectrum for HeLa-luc/
PKH26 cells and the autofluorescent excitation spectrum of mouse tissue. It also shows
the passbands for the background filter (DsRed Bkg), the primary excitation filter
(DsRed), and the emission filter (DsRed). Figure F.17 shows the IVIS ® images using the
primary excitation filter, the background excitation filer, as well as the autofluorescentcorrected
image.
The corrected image was obtained using a background scale factor of 1.4, determined by
taking the ratio of the autofluorescent signals on the scruff of the animal. The numbers
shown in the figures are the peak radiance of the animal background within the region of
interest. In the corrected image, the RMS error is used to quantify the background. The
signal-to-background ratio of the original fluorescent image (DsRed filter) is 6.5. The
ratio increases to 150 in the corrected image, an improvement factor of 23. This
improvement reduces the minimum number of cells necessary for detection from 1.5× 10 5
to 6.7× 10 3.
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