(A) Structure of the ¹⁸F boron trap/heptamethine cyanine (C7-Cy) PET/NIR probe, [¹⁸F]BOMB 1, activated as an NHS ester for general bioconjugation. The boron-based ¹⁸F trap is colored red, while the NIR fluorophore is colored green. (B) Structure of PET/NIR dual probe Lymphoseek (DTPA-mannosyl-dextran) 2 and the aqueous ¹⁸F labeling scheme used to generate ¹⁸F-labeled [¹⁸F]BOMB Lymphoseek 3. (C) Excitation (blue) and emission (red) spectra of an aqueous solution of [¹⁸F]PET/NIR-Lymphoseek 3 (φ_f = 0.027, excitation maximum = 755 nm, emission maximum = 772 nm, 1.5 μM Lymphoseek solution in water). The amine (NN), mannose (NM), and DTPA (ND) densities on Lymphoseek are 5.8, 16.5, and 4.2 mol/dextran, respectively. The average preconjugation molecular weight of Lymphoseek is 16122.

Multimodality imaging of a mouse injected with a 10 μL, 1 nmol, 48.1 μCi dose of ¹⁸F-labeled Lymphoseek 3 (0.048 Ci/μmol). The red arrows indicates the location of the sentinel lymph node. (A) Pre-excision, live-mouse PET (red color table)/CT (blue color table) scan (left) and postoperative PET/CT scan (right) of the mouse with excised nodes (right and left lumbar and popliteal) that are placed below the excision site in the field of view. (B) Bright field images of the mouse with skin removed before (left) and after node excision (right). (C) NIRF images taken 90 min postexcision on a custom full field IR camera before (left) and after (right) lymph node excision. The injection site was covered with black cardboard to block intense signal from the foot and allow better visualization of the fluorescent lymph nodes. (D) H and E stain (top, color images) and NIRF (bottom, black and white images) histological verification of lymph node excision. A PET projection video of panel A is given as Supporting Information.

Nonradioactive NIRF imaging of [¹⁹F]Lymphoseek 3. (A) Typical NIRF imaging experiment of a mouse that had been injected with 1 nmol of [¹⁹F]3 diluted with 10 nmol of unconjugated Lymphoseek. The NIRF signal (colored red) is overlaid onto a bright field image (green) of a mouse. A red arrow indicates clear localization of [¹⁹F]Lymphoseek 3 to the sentinel (popliteal) lymph node, while the blue arrow indicates localization to the distal (lumbar) lymph node. (i) Post-mortem skin-on image in which tissue (skin) notably interferes with the sentinel lymph node visibility. (ii) Pre-excision image (skin removed) in which the sentinel node visibility is improved and lymph drainage tracks are visible. (iii) Postoperative image in which the excised lymph nodes have been placed in the field of view. (B) In vivo imaging of [¹⁹F]Lymphoseek 3’s mannose receptor specific activity. Fluorescent [¹⁹F]Lymphoseek 3 (1 nmol) was imaged in the presence of 2 (i), 29 (ii), or 300 nmol (iii) of unlabeled Lymphoseek, a competitor for lymph node mannose binding sites. Note that lymph tracks are clearly visible and that greater accumulation of fluorescent [¹⁹F]Lymphoseek 3 is seen in the sentinel lymph nodes with 3 and 30 nmol injections than with 300 nmol injections because of increased competition with unlabeled Lymphoseek for sentinel lymph node mannose receptors. (C) Ex vivo imaging of [¹⁹F]Lymphoseek 3’s mannose receptor specific activity. Sentinel lymph nodes were extracted from mice and placed on a bed for analysis. Greater accumulation of fluorescent [¹⁹F]Lymphoseek 3 is seen in the sentinel lymph nodes in 3 nmol injections than in 300 nmol injections (signal ratio of 3.5, n = 3 mice per dose, P = 0.015 for a two-tailed test, two-sample equal variance, quantization shown in the Supporting Information).

[¹⁸F]Lymphoseek 3 tissue biodistribution in mice that were sacrificed 1 h and 20 min following injection. Units of percent injected dose (ID, left) and percent injected dose per gram (right). Biodistribution data from 12 mice were used to compile this figure.