Abstract
Standard immunofluorescence imaging captures just ~4 molecular markers (‘4-plex’) per cell, limiting dissection of complex biology. Inspired by multimodal omics-based data integration approaches, we propose an Extensible Immunofluorescence (ExIF) framework that transforms carefully designed but easily produced panels of 4-plex immunofluorescence into a unified dataset with theoretically unlimited marker plexity, using generative deep learning-based virtual labelling. ExIF enables integrated analyses of complex cell biology, exemplified here through interrogation of the epithelial-mesenchymal transition (EMT), driving significant improvements in downstream quantitative analyses usually reserved for omics data, including: classification of cell phenotypes; manifold learning of cell phenotype heterogeneity, and; pseudotemporal inference of molecular marker dynamics. Introducing data integration concepts from omics to microscopy, ExIF provides a blueprint empowering life scientists to use routine 4-plex immunofluorescence methods to achieve previously inaccessible high-plex imaging-based quantitative single-cell analyses.