Living Image 3.1

Living Image ® Software User’s Manual
G.2 Optical Properties
G.3 Luciferase Spectrum
The results of the fit are the total flux of the bioluminescence source S and the source depth
d.
Planar spectral image analysis requires prior knowledge of the tissue optical properties at
the wavelength used at image acquisition. The two main optical parameters are the:
• Absorption coefficient (µ a ) that defines the inverse of the mean path before photons
are absorbed by the tissue.
• Reduced scattering coefficient (µ' s ) that defines the inverse of the mean path before
photons are scattered isotropically in the tissue.
The effective attenuation coefficient (µ eff ) is a function of the absorption and reduced
scattering coefficients:
µ eff = (3µ a (µ' s + µ a )) 1/2 (2)
Calculation of the function K in Equation 1 requires all three coefficients (µ a , µ' s , and µ eff )
as input. The function K includes a term called the effective reflection coefficient to account
for the reflection of light at the air-tissue boundary due to a mismatch in the index of
refraction. The tissue index of refraction is generally assumed to be close to 1.4.
The model assumes that the tissues are optically homogeneous and the Living Image
software provides several factory set tissue optical property values to choose from.
Analyzing spectrally filtered images requires knowledge of the spectral dependence of
bioluminescent light emission. The luciferase bioluminescence spectrum was measured in
vitro at 37° C and pH≈ 7 in various cell lines. This spectrum is used to normalize the photon
flux values that the software measures at each wavelength.
Source spectra for several reporters are included in the database, including firefly, click
beetle, renilla, and bacteria (Figure G.1).
NOTE
G.4 An Example of Planar Spectral Imaging
The firefly luciferase spectrum is temperature and pH dependent. The luciferase
spectra included in the software are only valid for measurements performed at 37° C
and pH 7.0-7.5. If you use other temperature or pH conditions for an experiment, the
associated luciferase spectral curve is required for planar spectral image analysis. For
more information on the pH and temperature dependence of the luciferase spectrum,
please contact Xenogen Corporation.
Melanoma cells were injected intravenously into the tail vein of nude mice. After 13 days,
metastases developed in the lungs, kidney, and hind limb bone. An image sequence was
acquired on the IVIS ® Imaging System 200 Series using filters at six wavelengths from 560
to 660 nm, in 20 nm intervals.
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