Living Image 3.1

Living Image ® Software User’s Manual
Appendix G Planar Spectral Imaging
G.1 Planar Spectral Imaging Theory
Planar Spectral Imaging Theory . . . . . . . . . . . . . . . . . . . . . . . . . 225
Optical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Luciferase Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
An Example of Planar Spectral Imaging . . . . . . . . . . . . . . . . . . . . . 227
Optimizing the Precision of Planar Spectral Analysis . . . . . . . . . . . . . . 231
The unique spectral signatures of the luciferase emission spectrum and the optical
properties of tissue enable the Living Image software to determine the depth and intensity
of light sources inside a living animal. The planar spectral imaging algorithm relies on a
diffusion model of light propagation in tissue and assumes a point source of light
embedded in a flat surface approximation of the mouse. The algorithm is designed to
provide a fast and robust method to approximate source location and brightness. The
analysis requires two or more single-view images at wavelengths between 560 and 660 nm.
The Diffuse Tomography (DLIT ) algorithm is a more complete and accurate model. It
analyzes images of surface light emission to produce a three-dimensional (3D)
reconstruction of the bioluminescent light sources in a subject. For more details on DLIT
analysis, see Chapter 11, page 137 and Appendix H, page 233.
An image acquired on an IVIS ® Imaging System is a diffuse projection on the surface of
the animal from the bioluminescent sources located deeper inside. Information about the
depth of the bioluminescent cells can help quantify the source brightness and provide
information on the location of the cells.
The Living Image software uses spectroscopic information from a single-view image to
estimate the depth of the bioluminescent cells. The method takes advantage of the fact that
firefly luciferase bioluminescence is emitted from 500 to 700 nm, a region of the spectrum
where there are major contrasts in tissue optical properties (Figure G.1).
In this portion of the spectrum, tissue absorption drops off dramatically between 500-580
nm (green/yellow wavelengths) and 600-750 nm (red wavelengths), due mainly to the
presence hemoglobin. As a result, the bioluminescent signal observed on the surface of the
animal is dependent on both the wavelength and the thickness of the tissue through which
it travels.
The depth and absolute photon flux of a single point source can be determined from two or
more images acquired at different wavelengths using relatively simple analytical
expressions derived from the diffusion model of the propagation of light through tissue.
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