Fig 2: Schematic representation of live small animal NIR wide-field time-resolved macroscopic fluorescence lifetime FRET imaging (MFLI-FRET) for preclinical studies. (A) AU565 tumor xenograft production; (B) Intravenous tail-vein injection of AF700–TZM and/or AF750–TZM; (C) Live small animal imaging using wide-field time-resolved FLI-FRET macroscopy (MFLI-FRET) imager; (D) Ex vivo validation using immunohistochemical (IHC) and H&E staining. Inset top left: illustration of TZM–AF700 and TZM–AF750 ligands. Inset bottom right: illustration of TZM–HER2 FLI-FRET events upon binding of donor- and acceptor-labeled antibodies to HER2 dimer at the surface of cancer cells. DMD: digital micromirror device; CCD: charge-coupled device.

Fig 3: Comparison of intracellular uptake of TZM–AF700 into HER2-positive and HER2-negative cell lines. (A) To test the specificity of HER2-mediated TZM–AF700 binding and internalization, competition experiments were performed. AU565 cells were loaded with 5 µg/mL TZM–AF700 (red) and 5 µg/mL Tf-AF568 (green) in the presence of increasing amounts of unlabeled TZM. Single slice confocal microscopy displays TZM–AF700 (red) and Tf–AF568 (green) binding and internalization into AU565 cells in the presence of increasing 0×, 10×, 50×, and 100× unlabeled TZM. (B) HER2-positive AU565 and HER2-negative MDA-MB-231 and MCF10A cells were subjected to 24 h uptake of 20 µg/mL TZM–AF700 (red) and then processed for anti-HER2 immunostaining (green). Nuclei were visualized with DAPI (blue). Maximum intensity projections of z-stacks consisting of 6–8 optical slices are shown. Confocal images were collected using identical settings on an LSM880 confocal microscope. Scale bar = 20 µm.

Fig 4: IHC ex vivo validation confirms TZM accumulation in the cancer cells both positive for HER2 and HER3. Consecutive sections of tumors M1–M3 and tumor from untreated mouse were processed for H&E staining, anti-HER2, anti-HER3, and anti-TZM immunohistochemical staining. NovaRED was used as peroxidase substrate (brown stain), tissue was counterstained with methyl green. Scale bar = 100 µm.

Fig 5: Confocal microscopy of Human EGF Receptor 2 (HER2) and trastuzumab (TZM)–AF700 distribution in AU565 cells. (A) Immunofluorescence analysis of HER2 (green; arrows), early endosomal antigen 1 (EEA1), or transferrin receptor (TfR) (red; arrows). Middle slices of z-stacks consisting of 6–8 optical slices are shown. (B) AU565 cells were loaded with 5 µg/mL TZM–AF700 (red) and 5 µg/mL Tf–AF568 (magenta) for 24 h and then processed for immunostaining of HER2 (green) and endosomal markers EEA1 or TfR (magenta). Maximum intensity projections of z-stacks consisting of 6–8 optical slices are shown. Nuclei are visualized with DAPI. Scale bar = 20 µm.