Figure 2.

Highly multiplexed imaging data collection and analysis pipelines. A selection of multiplexed imaging approaches is illustrated in brief (A). In cyclic IHC, primary antibodies for a protein of interest are detected by a secondary antibody conjugated to horseradish peroxidase, which catalyzes a reaction depositing a chromogenic dye which can then be visualized through light microscopy in rounds of bleaching, staining, and imaging. In Cyclic IF, secondary antibodies are detected using fluorophores, followed by bleaching, staining, and imaging. In IMC or MIBI, primary antibodies are directly conjugated to unique mass tags, and data are collected simultaneously. In CODEX, primary antibodies conjugated to unique nucleotide barcodes are stained simultaneously and are detected cyclically using complementary barcodes conjugated to fluorophores. After data collection, imaging data must be processed to quantify features of interest (B–E). Preprocessing steps include illumination correction (B), background subtraction, bleedthrough correction, and autofluorescence correction. Image registration often begins at the level of the gross tissue and ideally is followed by a cell–cell registration (C). Nuclear segmentation identifies single cells (D). Expression of proteins of interest and their localization can then be quantified per detected cell (E). Once an expression dataset has been produced, specific techniques are necessary to analyze high-dimensional imaging data (F–I). Dimensionality reduction tools such as t-SNE and UMAP can make high-dimensional relationships between cells easier to visualize (F). Clustering tools such as FlowSOM and SPADE identify populations within the dataset (G). Phenotypic characteristics of identified clusters can be determined using marker enrichment modeling (MEM) or expert-guided median expression analysis (H). Local niche characteristics can be discovered by identifying neighbor cells either by distance from the index cell or by selecting all first-order neighbors (I). Abbreviations: IHC, immunohistochemistry; IF, immunofluorescence; IMC, imaging mass cytometry; MIBI, multiplexed ion beam imaging.