Living Image 3.1

Living Image ® Software User’s Manual
Table 6.1 Image Math window
Item Description
6.2 Subtracting Tissue Autofluorescence
Display Result for Measuring Opens the image generated by image math in an image
window.)
To remove tissue autofluorescence from image data, the IVIS ® Imaging System
implements a subtraction method using blue-shifted background filters that emit light at
a shorter wavelength (Table 6.2).
Table 6.2 Emission, excitation, and background filters used to acquire data that can be corrected
for tissue autofluorescence
Emission Filter Excitation Filter (Primary
Image)
Fluorophore Passband (nm)
The objective of using a background filter is to excite the tissue autofluorescence
without exciting the fluorophore. To reduce autofluorescence signal in the primary
image data, use the image math tool to subtract the background filter image from the
primary excitation filter image. For more details on tissue autofluorescence, see
Appendix F, page 223
The software computes:
Background-corrected signal = (A - B) × k, where:
A = primary image (acquired using the excitation filter)
B = background image (acquired using the background filter)
k = (primary signal/background signal)
Background Filter
(Background Image)
GFP 515-575 445-490 410-440
DsRed 575-650 500-550 460-490
Cy5.5 695-770 615-665 580-610
ICG 810-875 710-760 665-695
The background signal is obtained from a measurement ROI that is
located in an area where no fluorophore signal is present. The scale factor
k accounts for different levels of tissue autofluorescence due to different
excitation wavelengths and filter transmission characteristics.
After you acquire an image sequence that includes a primary and background image, use
the image math tool to subtract tissue autofluorescence. (For more details on acquiring
an image sequence, see Chapter 3, page 30.)
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