Fig. 2. Qualifying the 16-plex single-shot Orion antibody panel.
a, Panels of images from FFPE tonsil sections showing single-antibody IHC for pan-cytokeratin, Ki-67, CD8α, CD163 and the matching channels extracted from 16-plex Orion IF images (the H&E stain was performed on the same section as the Orion imaging). Each image is from one representative specimen. For IHC/H&E, four serial sections were used from the same tonsil tissue; one additional section from the same sample was used for Orion. b, Orion IF images and CyCIF images from neighboring sections of an FFPE colorectal adenocarcinoma. The CyCIF images were collected using 2 × 2 binning, while Orion images were obtained with no binning. c, Plots of the fraction of cells positive for the indicated markers from whole-slide Orion IF and CyCIF images acquired from neighboring sections. Pearson correlation coefficients are indicated. d, t-SNE plots of cells segmented from an Orion IF image of an FFPE CRC specimen (C01) with inferred cells types (left) and the fluorescence intensities of selected markers (CD45, pan-cytokeratin, CD8α and α-SMA; right) overlaid on the plots as heat maps. The plots show a random sample of 50,000 cells. e, Orion images showing antibodies imaged across two cycles. Twenty-three of 29 antibodies are displayed across four marker groups from four different regions of interest (labeled ROI 1–4). Markers from cycle 2 are underlined. The locations of the four ROIs in the whole-slide image are shown in Extended Data Fig. 5a. Images are from one FFPE tonsil specimen/section; VIM, vimentin; Gr-B, granzyme B.