Fig. 3: MEZ-XRF enables molecular marker imaging of human FFPE tissue.

a–g, MEZ-XRF images of the indicated markers (rows) in HER2⁺, luminal A (LumA) and luminal B HER2⁻ (LumB HER2⁻) tissues (columns): Ho_Ka|HH3 (a); Lu_Ka|panCK (b); Er_Ka|CK19 (c); Tm_Ka|Vim (d); Gd_Ka|CD44 (e); Nd_Ka|HER2 (f) and Ce_Ka|ER (g). Overview scans were imaged with a 500 nm focused 69 keV X-ray beam with 2 µm raster steps at 20 Hz. ROI (indicated by red box and shown magnified to the right) were imaged with a 500 nm focused 69 keV X-ray beam with 0.5 µm raster steps at 5 Hz. XRF emissions were recorded with a prototype GeCMOS detector. Images are 0.5–99.5% gray levels of detector counts per element emission line matched across samples. h, Leiden clustering of single cells segmented from overview images colored by clusters as annotated by marker enrichment. i, Leiden cluster annotations (colors as in i) projected back onto single-cell segmentation masks of representative overview imagery for the indicated samples. j, Subcellular localization of molecular markers in each tumor sample. The relative nuclear to nonnuclear intensities were derived from nuclear and whole-cell segmentations of ROI scans. Violin plots (three dashed horizontal lines per violin show the lower quartile, median value and upper quartile values) show distribution of nuclear/nonnuclear marker ratios for all cells of a single field of view for one sample per diagnosis.