Fig. 1: Highly multiplexed immunohistochemistry reveals scale-crossing features of developing retinal organoids and primary human retina.

a, 4i was performed on tissues of a timecourse of retinal organoid development (6, 12, 18, 24 and 39 weeks) and adult retina tissue. Schematic shows retinal cell type organization. b, Schematic of the 4i methodology and analyses. FFPE tissue sections were placed on a 96-well format coverslip that was subsequently attached to a 96-well superstructure allowing immunohistological treatment, followed by imaging and antibody elution. Here, we performed 21 immunohistochemical staining cycles. Images were acquired at ×40 magnification with a high-NA silicone oil objective, tiled across the tissue. c, Representative 4i dataset image showing overview of a Hoechst stain of a 39-week organoid section and progressive magnifications from millimeter to nanometer scales. d–f, Example of pixel clustering of a 39-week organoid section. d, Thirty-two global MTUs resolve the tissue structure with image quality and label biological structures in individual samples. e,f, Biological structures identified by unique MTUs include HCs, BC cytoplasm and nuclei, ACs and structural elements such as collagen-rich areas (e) and peripherally located structures such as mitochondria, the OLM, PR cytoplasm and nuclei, long-wave (LW) cones and the plexiform layers (f). g, MTU 6 (top) is enriched for EPHB2 protein expression (bottom) and non-uniformly distributed in the organoid section (outlined by red line). Insets show regions with high (i) and low (ii) detection of EPHB2 fluorescence immunohistochemical signal. Patterned EPHB2 staining was observed in all 39-week replicates (11 sections and three organoids). h, Heterogeneity analysis of nuclei with UMAP projection based on protein features and colored by labels transferred from sequenced cells. All major types are identified, including RGCs, HCs, cones, ACs, rods, BCs and MG. i, Feature plots highlighting median signal level per nucleus of HES1, identifying nuclei located in the INL; PRKCA, enriched in BCs; and RHO, identifying cells located in the ONL. j–n, Primary adult human retina section (j) and retinal organoid sections from different timepoints (k–n) with pixels labeled by MTUs derived from communal clustering and comparable across samples.