An ideal FT system should not only allow visualization of the fluorophore distribution in tissue but also provide quantitatively accurate concentration values in a heterogeneous medium. Quantitative accuracy is a pivotal factor for many practical applications of FT. For instance, MMPsense, which is an activatible fluorescence probe, accumulates three times higher in malignant tumors than benign ones in vivo
]. To be able to differentiate malignant and benign lesions, the reconstructed fluorophore concentration value should be independent of the size and location of the lesion. A stand-alone FT system, unfortunately, would not correctly differentiate a small malignant lesion buried deep in tissue and a large benign lesion located at subsurface lesion.
In order to address the need for quantitative FT imaging, we built a fully integrated FT/DOT/XCT system. There are two main objectives for developing this system. First, the XCT structural a priori information is used to guide and constrain the FT inverse problem, thus fluorophore concentration map can be recovered more accurately. Additionally, DOT provides an effective way of correcting the effect of optical background heterogeneity, thus improves the FT accuracy further.
Recently, Kepshire et al
has particularly investigated the depth dependence of a subsurface FT technique with simulation and phantom studies [44
]. They concluded that the fluorescence object could be localized but recovered image sensitivity was nonlinear with depth. Our second phantom study also confirms that the recovered fluorescence concentration is highly dependent on not only the depth but also the size of the fluorescence inclusions. On the other hand, the fluorophore concentration in the inclusions can be recovered within 10% error using the XCT structural a priori
information independent of their size and location.
Another factor that affects the FT reconstruction results is the background optical property distribution. Without proper modeling of emission and excitation light propagation between boundary of the medium and fluorophores, it is difficult to obtain accurate results. As an alternative, Born normalization method has been commonly used for heterogeneity correction, and applied to localize the fluorescence object robustly in vivo
]. For instance, Graves et al
have recovered a subcutaneously implanted fluorescence inclusion in nude mice with less than 30% error using slab geometry and matching fluid [41
]. However, the quantitative accuracy of Born normalization method has not been discussed especially for inclusions deeply embedded in the turbid medium [33
]. Furthermore, Herve et al
particularly compared Born normalization and a heterogeneity correction method, which used reconstructed optical property for modeling purposes. The results showed that the later was more accurate [47
]. Our results also confirmed that significant improvement can be achieved when DOT functional a priori
information is utilized during the FT reconstruction process. However, neither functional nor structural a priori
by itself is enough to obtain accurate FT maps. In essence, the strength of the tri-modality system described here comes from its ability to offer both XCT structural and DOT functional a priori
information that can be utilized to reconstruct quantitatively accurate fluorophore concentration images.
In our study, only absorption heterogeneity was considered and homogeneous scattering coefficient distribution was assumed. However, the scattering coefficient can also be very heterogeneous in reality. Time-dependent DOT measurements, which can be achieved using frequency- or a time-domain DOT system, are required in order to effectively separate the absorption and scattering coefficients. Nevertheless, the effect of the scattering error was evaluated using simulation studies. For example, when the scattering coefficient was chosen to be +/− 25% of the actual value, the recovered absorption coefficient from DOT gives +/− 28% for the absorptive object. In turn, when this absorption map is fed into the fluorescence reconstruction, the fluorescence concentration gives +/− 30% error.
A perquisite for any such multimodality approach is that the region of interest should be detected by each individual modality. This may not be true all the time and even if it is, the boundary of the ROI detected by different modalities may vary. As a solution for the latter case, soft a priori approach is used [48
] to reduce the effect of erroneous a priori
information on the reconstruction results. For the former case, XCT structural information can at least improve the optical property of the background medium obtained by DOT, which in turn will improve the FT results further. All the XCT images shown in this study is to provide phantom structure for FT reconstruction and demonstrate the importance of such multi-modality imaging system using proof-of-principle experiments. However, the limitation and optimized contrast and geometry for acquiring XCT image need to be addressed and investigated using contrast-detail analysis method in the future. Furthermore, various strategies such as utilization of XCT contrast agents or dual energy XCT technique can be potentially used to improve the vascular or soft tissue contrast [31
]. The most favorable case for such multi-modality imaging system is to use dual modality contrast agent where the location of the agent can be seen from the structural imaging modality and the amount of the agent can be quantified by the fluorescence imaging. This is possible with the advances of dual contrast agent development, such as dual MRI-fluorescence, dual XCT-fluorescence contrast agent [49
]. Meanwhile, extensive effort is being spent to develop new x-ray detector technology to improve XCT soft tissue contrast further.