Fig. 1. Same-section IF and H&E using the Orion platform.
a, Schematic of one-shot 16- to 20-channel multiplexed IF imaging with the Orion method followed by H&E staining of the same section using an automated slide stainer and scanning of the H&E-stained slide in transillumination (brightfield) mode. This method of discriminating the emission spectra of fluorophores is repeated using seven excitation lasers spaced across the spectrum (see Extended Data Fig. 1b and Methods). Using polychroic mirrors and tunable optical filters, emission spectra are extracted to discriminate up to 20 channels, including signal from fluorophore-labeled antibodies (15–19 in most experiments), the nuclear stain Hoechst 33342 and tissue-intrinsic autofluorescence (figure created with BioRender.com). b, Left, Orion multiplexed IF image showing CD31, α-SMA, Hoechst (DNA) and signal from the tissue autofluorescence channel; this image highlights an artery outside of the tumor region with red blood cells in the vessel lumen and elastic fibers in the internal and external elastic lamina of the vessel wall, numerous smaller vessels (arterioles) and stromal collagen fibers (the inset displays arterioles). Right, images of the H&E staining from the same tissue section (histologic landmarks are indicated). Images are from a single representative specimen (C18). c, Orion multiplexed IF image (showing CD45, pan-cytokeratin (PanCK), CD31 and α-SMA) from a whole-tissue FFPE section and matched H&E from the same section. Holes in the images are regions of tissue (‘cores’) removed in the construction of TMAs. Images are from a single representative specimen (C04). d, Zoom-in views of the regions indicated by arrowheads in c; marker combinations are indicated. The images are from a single representative specimen (C04). e, Intensities of fluorochromes (columns in heat maps) in each Orion channel (rows in heat maps) before (top) and after (bottom) spectral extraction. The extraction matrix was determined from control samples scanned using the same acquisition settings that were used for the full panel. The control samples included unstained lung tissue (for the autofluorescence channel), tonsil tissue stained with Hoechst and tonsil tissue stained in single plex with ArgoFluor conjugates used in the panel (for the biomarker channels). The values in each column were normalized to the maximum value in the column. Data were derived from a single pool (N = 1) of control beads.