Fig 2: Western blotting analysis for TFRC proteins separated on a 12% reducing SDS gel. The proteins were transferred to a PVDF membrane and the size of the proteins seen 95 kDa and 37 kDa for GAPDH (House-keeping gene).

Fig 3: Basal macrophagic protein expression in mock-infected triple-cell co-cultures. Ferritin (a), FPN (b), TfR1 (c) and DMT1 (100 kDa and 65 kDa isoforms) (d) polypeptides were detected in THP-1 whole cell extracts from the same triple-cell co-cultures described in Supplementary Materials Figure S1. Isolated protein was used in immunoblotting analysis, and ß-actin was the loading control. The blots were subjected to densitometry, and the resulting expression values were plotted in expression histograms. Values were calculated as a percentage of the 24 h mock-infected control. All graphs depict the average expression from at least three individual experiments with representative accompanying blots.

Fig 4: Correlated magnetic and optical imaging in lung tumor tissues. (A) Correlated HE staining and IMM in the same tissue section. Hematoxylin and eosin stained the tissue’s cellular structure, simultaneously producing strong fluorescence signals (SI Appendix, Fig. S12), which significantly reduced the contrast of the NVs’ CW spectrum (SI Appendix, Fig. S13). Nevertheless, the robust IMM resisted the impact of fluorescence from hematoxylin and eosin. Although the magnetic image shows a slightly reduced signal-to-noise ratio, it still clearly resolves the distribution of PD-L1. The yellow ellipse and box in the merged images indicate squamous carcinoma cells and immune cells, respectively. (B) Double labeling of Ki67 and TfR by IHC and immunomagnetism in the same section, respectively. In IHC, DAB immunostained Ki67 proteins and hematoxylin stained cell nuclei. Again, the IMM resisted the impact of DAB and hematoxylin, and we obtained a high-quality magnetic image. (C) Double labeling of TfR and EGFR by IF and immunomagnetism in the same section, respectively. Alexa Fluor 488 in the green channel represents TfR, which was labeled via the routine immunofluorescence procedure. Shown are DAPI-stained cell nuclei. Both imaging methods work well without disturbing each other. The HE and DAB signals were recolored with the green color, and then the optical images were merged with the reconstructed IMM images. These results were confirmed in three (IMM and HE), four (IMM and IHC), and two (IMM and IFM) samples in independent experiments. (Scale bars, 100 µm and 20 µm [zoom].)

Fig 5: Magnetic images and quantification of a variety of cancer biomarkers. (A) Workflow of single-cell quantification from magnetic images. The magnetic image reconstruction is fulfilled by a deep-learning model, and the reconstructed image is segmented at the single-cell level in a single-cell segmentation software. (B–F) Magnetic images, corresponding reconstructed images, and single-cell expression distributions of five typical biomarkers in lung tumor tissues. Membrane proteins EGFR, TfR, EpCAM, and PD-L1 show obvious cell membrane distribution, while the nuclear localization protein Ki67 mainly locates in the cell nucleus. Color bars represent magnitudes of magnetic signals and MNP densities. Shown are DAPI-stained cell nuclei. The histograms display distinct single-cell expression distributions of different markers. These results were confirmed in five (EGFR), five (TfR), five (PD-L1), three (EpCAM), and three (Ki67) samples in independent experiments, respectively. (Scale bars, 100 μm.)