STAMP: Single-Cell Transcriptomics Analysis and Multimodal Profiling through Imaging [Merscope]

Single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular diversity, but remains constrained by scalability, high costs, and the destruction of cells during analysis. To overcome these challenges, we developed STAMP (Single-Cell Transcriptomics Analysis and Multimodal Profiling), a highly scalable approach for the profiling of single cells. By leveraging transcriptomics and proteomics imaging platforms, STAMP eliminates sequencing costs, enabling single-cell genomics of hundreds to millions of cells at an unprecedented affordability. Immobilizing (‘stamping’) cells in suspension onto imaging slides, STAMP supports single-modal (RNA or protein) and multimodal (RNA, protein and H&E) profiling, while retaining cellular structure and morphology. Its flexible, ultra-high-throughput formats facilitate the analysis of single or multiple samples in the same experiment, enhancing experimental scalability and adaptability. We demonstrate STAMP's versatility across diverse experimental contexts, including the profiling of peripheral blood mononuclear cells (PBMCs), cell lines and stem cells. We also stamped cells and nuclei from dissociated tissues from mouse organs to simulate the generation of cell atlases. Accessibility was further enlarged by analyzing nuclei from archival formalin fixed and paraffin embedded (FFPE) tissue samples. Combining RNA and protein profiling, we applied STAMP for high-throughput immuno-phenotyping of millions of blood cells, providing multimodal insights into cellular heterogeneity. We highlight the capability of STAMP to identify ultra-rare cell populations, simulating clinical applications for detecting circulating tumor cells (CTCs). By capturing lineage dynamics during stem cell differentiation and subtle changes in in vitro activated PBMCs, we further showed its utility for large-scale perturbation studies. These results validate STAMP as a first-of-its-kind single-cell imaging analysis strategy. We present data for 10,962,092 high quality cells/nuclei and 6,030,429,954 high quality transcripts. By replacing sequencing with imaging, STAMP enables high-resolution cellular profiling that is more accessible, scalable, and cost-effective. STAMP has the potential to transform our ability to map biological diversity and dynamics, significantly advancing research and clinical applications. In this study, we transformed four advanced imaging platforms—Xenium Analyzer (10X Genomics), CosMx Spatial Molecular Imager (NanoString Technologies/Bruker), MERSCOPE (Vizgen), and PhenoCyler Fusion (Akoya Biosciences)—into scalable, flexible single-cell profiling tools. We termed this approach Single-Cell Transcriptomics Analysis and Multimodal Profiling (STAMP) through imaging. STAMP leverages single-molecule imaging to analyze hundreds to millions of individual cells with unprecedented flexibility and scalability. The platform supports both single-modal (RNA or protein) and multimodal (RNA, protein, and H&E) profiling in single- or multi-sample configurations. We validated its performance across diverse sample types and experimental conditions, comparing its quality control metrics to classical single-cell analysis methods. To assess scalability, STAMP was applied to cell populations ranging from hundreds to millions, demonstrating adaptability to varying sample sizes. It successfully profiled both whole cells and nuclei (from fresh-frozen and FFPE samples), enabling the analysis of archival tissues. Functional applications included: -Immunophenotyping millions of peripheral blood mononuclear cells (PBMCs), capturing subtle compositional and transcriptional shifts induced by in vitro perturbations. -Detection of ultra-rare cell populations, such as circulating tumor cells (CTCs), which are often undetectable using conventional scRNA-seq methods. -Cellular differentiation and lineage tracing, where STAMP was used to study human embryonic stem cell (hESC) differentiation induced by bone morphogenetic protein 4 (BMP4), effectively modeling early embryonic gastrulation. -Application to induced pluripotent stem cells (iPSCs), highlighting STAMP’s potential for large-scale perturbation and drug screening studies.