Excitation spectroscopy in multispectral optical fluorescence tomography: methodology, feasibility and computer simulation studies

Molecular probes used for in vivo optical fluorescence tomography (OFT) studies in small animals are typically chosen such that their emission spectra lie in the 680–850 nm wavelength range. This is because tissue attenuation in this spectral band is relatively low, allowing optical photons even from deep sites in tissue to reach the animal surface and consequently be detected by a CCD camera. The wavelength dependence of tissue optical properties within the 680–850 nm band can be exploited for emitted light by measuring fluorescent data via multispectral approaches and incorporating the spectral dependence of these optical properties into the OFT inverse problem—that of reconstructing underlying 3D fluorescent probe distributions from optical data collected on the animal surface. However, in the aforementioned spectral band, due to only small variations in the tissue optical properties, multispectral emission data, though superior for image reconstruction compared to achromatic data, tend to be somewhat redundant. A different spectral approach for OFT is to capitalize on the larger variations in the optical properties of tissue for excitation photons than for the emission photons by using excitation at multiple wavelengths as a means of decoding source depth in tissue. The full potential of spectral approaches in OFT can be realized by a synergistic combination of these two approaches, that is, exciting the underlying fluorescent probe at multiple wavelengths and measuring emission data multispectrally. In this paper, we describe a method that incorporates both excitation and emission spectral information into the OFT inverse problem. We describe a linear algebraic formulation of the multiple wavelength illumination-multispectral detection forward model for OFT and compare it to models that use only excitation at multiple wavelengths or those that use only multispectral detection techniques. This study is carried out in a realistic inhomogeneous mouse atlas using singular value decomposition and analysis of reconstructed spatial resolution versus noise. For simplicity, quantitative results have been shown for one representative fluorescent probe (Alexa 700®) and effects due to tissue autofluorescence have not been taken into account. We also demonstrate the performance of our method for 3D reconstruction of tumors in a simulated mouse model of metastatic human hepatocellular carcinoma.