Fig 2: Evaluation of pre-QC cell clustering results from Datasets 6 (large intestine, CODEX) and 2 (CRC, CyCIF).a, UMAP embedding of Dataset 6 showing ~3.8 × 104 cells colored by HDBSCAN cluster. Black scatter points represent ambiguous cells (10.5% of total). b, Silhouette scores for CODEX clusters in (a) revealing cluster 29 as an under-clustered population. c, Mean signal intensities of clustering cells from Dataset 6 normalized across clusters (row-wise). d, Correlated, non-specific signals in a region of Dataset 6 as seen in channels MUC6 (red), CD154 (green), and NKG2D (blue). Yellow points highlight cluster 0 cells formed due to this artifact. e, Tissue fold in a region of Dataset 6 as seen in channels GATA3 (red), CD68 (green), and CD66 (blue). Yellow points highlight cluster 9 cells formed due to this artifact. f, Image blur in a region of Dataset 6 as seen in channels HLADR (red), CD206 (green), and CD38 (blue). Yellow points highlight cluster 13 cells formed due to this artifact. g, Location of CRC cluster 3 cells in (g) revealing no spatial bias in the distribution of these cells. h, Top three most highly expressed markers (1: green, 2: red, 3: blue) for the 25 members of CRC cluster 3 (memory helper T) cells represented by the rugplots of Fig. 2n. White asterisks highlight cells shown in enlarged format in Fig. 2m. A single white pixel at the center of each image patch highlights the reference cell. Nuclear segmentation outlines (translucent white outlines) and Hoechst (gray) shown for reference. i, Regression plots showing correlation among CD4, CD45, and CD45RO marker expression by 1.9 × 103 CRC cluster 3 cells (two-sided, Pearson R, p < 0.05). j, CRC cluster 3 cells in (h) after signal intensity cutoffs have been adjusted per image to improve their homogeneity of appearance. White asterisks highlight cells shown in enlarged format in (Fig. 2o). k, CRC cluster 3 cells with channels shown separately for clarity. Top panels show cells before contrast adjustment (h), bottom panels show cells after contrast adjustment (j). l, Top three most highly expressed markers (1: green, 2: red, 3: blue) for 25 CRC cluster 7 (TReg) cells. A single white pixel at the center of each image patch highlights the reference cell. Nuclear segmentation outlines (translucent white outlines) and Hoechst (gray) shown for reference. m, Regression plots showing strong correlation among CD4, CD45, and CD45RO marker expression of 1.9 × 103 CRC cluster 7 cells (two-sided, Pearson R, p < 0.05). n, Regression plots showing weak correlation between FOXP3 and CD4, CD45, and CD45RO marker expression of 1.9 × 103 CRC cluster 7 cells (two-sided, Pearson R, p < 0.05).