The early photon tomography (EPT) experimental approach. (A) Experimental setup. Early arriving photons are detected by using the gated intensified CCD system. The animal is placed in the custom built cylindrical carbon-fiber tube (B) for scanning. As photons propagate through the diffusive medium, they disperse temporally (C). The experimentally measured Green's functions describing the path of photons propagating between a source (S) and detector (D) pair, measured at an early-time gate (D) and the early-photon forward model used in the reconstructions (E). This model was calculated by using a normalized cumulant approximation to the Boltzmann transport equation and agreed well with the measured weight function. The weight function for diffuse (CW) photons for the same geometry is also shown for comparison (F). The radial spread of the early arriving photons is drastically reduced, thereby allowing the excellent resolution possible with the EPT.

EPT imaging of a Lewis Lung Carcinoma tumor model in mice. (A and B) Three-dimensional rendering of a mouse with an LLC tumor 8 days after inoculation and a wild-type control. Mice were injected with a cathepsin-activatable probe (Prosense-750) 24 h before scanning with the EPT system. (C–E) Selected axial CT slices from the 2 mice. The 2 white circles on either side of the mouse are from the carbon-fiber holder. (F–H) Axial EPT reconstructions corresponding to the CT slices (arbitrary units). (I–K) Overlay of the EPT reconstruction on the CT slice. Histology was performed on excised lung samples. (L–N) H&E stain of tumor, lung tissue that was contralateral to the tumor tissue, and normal lung tissue, respectively. (O–Q) Macrophage-3 stain and (R–T) cathepsin B stain of the same tissue. Near-infrared fluorescence (NIRF) microscopy was also performed on adjacent tissue sections (U–W). The increased macrophage, cathepsin B, and NIRF levels in the primary tumor and the contralateral side corroborates the increased fluorescence signal from the protease-activatable probe measured with the EPT system. As discussed in the text, the increased fluorescence in the contralateral side was primarily due to the host inflammatory response of the lung in response to tumoral challenge as well as microscopic growth of LLC cancer cells into normal tissue. The fluorescent streaks emanating from the side of the animal are reconstruction artifacts.

Tumor-bearing (LLC tumor, 8 days after inoculation) and control mice were injected with the cathepsin-activatable probe Prosense-750 24 h before EPT imaging. After tomographic imaging they were immediately killed and dissected for surface-reflectance imaging. White light reflectance images of excised right (A) and left (B) lobes of the lung of a mouse with an LLC tumor and from a control mouse (C) are shown, as well as normalized planar-fluorescence images of the same lung tissue (D–F). The fluorescence was most intense in the tumor, and other microfoci, but was globally present at elevated levels throughout the entire organ versus controls. The time required for epiluminescence imaging herein resulted in significant microscopic degradation of the lung tissue so that subsequent immunohistochemical analysis of the tissue was impossible.

Longitudinal EPT imaging study measuring growth of LLC tumors in individual animals at 4-day intervals (n = 6). (A–C) Example X-ray CT axial slices at days 4, 8, and 12, from a single animal; (D–F) EPT reconstructions corresponding the CT axial slices in the same animal. (G) An excellent correlation was observed between the tumor volume calculated with the EPT system versus CT imaging. Tumors could be resolved with EPT after 4 days, corresponding to a minimum detectable tumor size of ≈0.02 cm (3).

Fluorescence reconstructions obtained by using early arriving photons compared with ungated (continuous-wave) photons in a mouse with an LLC tumor, 10 days after injection. The forward model for early arriving photons and ungated photons are shown (A and B) as well as the fluorescence reconstructions for the axial slice (C and D), and the corresponding X-ray CT axial slice (E). The use of early photons enables fluorescence reconstructions with superior resolution and localization of the activated fluorophore in the right lobe of the lung, as well as on the contralateral side compared with the ungated reconstruction.