Approach. (A) A 30-nm nanoparticle with an iron oxide core and a dextran shell was used for multimodal imaging. The dextran shell carried ∼40 amines per particle, which were used to attach more ligands. The clinical PET isotope ¹⁸F was attached with click chemistry. A near-infrared fluorochrome was used for FMT, fluorescence histology, and flow cytometry. These underivatized nanoparticles are avidly taken up by myeloid cells (i.e., monocytes and macrophages). However, this platform also can be derivatized with affinity ligands, such as peptides (6) or small molecules (3). (B) (Top) An imaging cassette and an adapter specifically developed for use in the Inveon PET-CT platform. This adapter can be fixed to the gurney, allowing seamless positioning of the cassette in the CT and PET ring. (Middle) Surface-rendered CT image of a mouse inside the imaging cassette. The lid of the cassette was segmented out to show the fiducial wells that are used for FMT/PET-CT fusion (yellow arrows). (Bottom) A 3D reconstruction of a CT, FMT, and PET dataset after injection of ¹⁸F-CLIO-VT680 into a control mouse. Nanoparticle signal was predominantly seen in the liver and spleen. (C) The algorithm for multichannel fusion based on fiducials. The software plug-in is described in more detail in SI Methods. Raw image datasets are resampled and coregistered (horizontal arrow) to match image dimensions, and then fused (vertical arrow). Images on the right were acquired in a four-channel FMT-CT experiment (described in detail in SI Methods).

Signal correlation in phantoms. Two-dimensional images of tissue phantoms, acquired by PET and FMT imaging. ¹⁸F-CLIO-VT680 (A) and ⁶⁴Cu-CLIO-VT680 (B) were diluted serially, and the concentration was reported according to the iron content in the nanoparticle core. The correlation between FMT and PET signals is shown on the right. FMT signals are reported as fluorescence concentration (nM); PET signals, as counts per volume (Bq/mL).

Coregistration of FMT and PET in tissue phantoms. Three-dimensional, surface-rendered reconstruction of FMT-CT, PET-CT, and FMT/PET-CT images show good correlations in spatial signal distribution. Because of the greater sensitivity of PET, the positive area is slightly larger for this modality. The correlation of spatial FMT and PET signal distributions in coregistered data was quantitated with a CCF (the 3D mesh on the right) based on the PC (perfect coregistration, PC = 1; no coregistration, PC = 0). In high-colocalized conditions, the CCF is close to 1 at its maximum, which occurs for low relative translations, that is, Δx ≈ Δy ≈ 0. The CCF analysis shown here indicates very good concurrence between the PET and FMT reconstructions.

Comparison of modalities in vivo. (A) FMT-CT, PET-CT, and FMT/PET-CT reconstructions of a representative mouse with bilateral flank tumors. The signal intensity was color-coded and was found to correlate well in amplitude and location. (B) Correlations of in vivo FMT to in vivo PET signal (Upper), ex vivo tumoral activity (measured by scintillation counting) to in vivo PET (Middle), and in vivo FMT signal to ex vivo scintillation counting (Lower). (C) As shown in Fig. 3 for phantoms, signal distribution was compared using the CCF. A good correlation of modalities was found.

In vivo multichannel FMT/PET-CT. FMT/PET-CT of tumor-bearing mice, coinjected with a fluorochrome-derived RGD peptide targeting integrin (Integrisense), a protease sensor (Prosense), and the nanoparticle PET agent (⁶⁴Cu-CLIO-VT680). (A and B) In 2D images, FMT (A) and PET (B) signal intensities were color-coded, whereas 3D reconstructions show a surface-rendered signal. (C) Overall, the signals in all FMT channels and in PET colocalized in tumors; however, when assessed at higher magnification, distinct differences between probes due to their different target profiles became evident.

Ex vivo high-resolution imaging showing differential probe uptake in a tumor. (A–C) Ex vivo fluorescence reflectance imaging of the same tumor in respective wavelengths depicting probe distribution of coinjected molecular sensors. The box in C shows localization of the histology shown in E–J. (D) Autoradiography demonstrating a similar signal pattern as that of fluorescence imaging in C, because it reports on the same nanoparticle. (E and F) H&E histology. The box in E shows the locations of F–J. (G–J) Fluorescence microscopy of the same section in respective channels showing differential microscopic distribution of three coinjected probes. The integrin signal is accentuated in the tumor margin in vessel-like structures, whereas the nanoparticles accumulates in TAMs. (Original magnification 200×.)