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High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging



Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand–receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at

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Fig. 1: CosMx SMI chemistry and workflow and 3D mapping of RNA at single-cell and subcellular resolution using morphology-based cell segmentation.
Fig. 2: Single-cell distribution and detection sensitivity of low, medium and high expressers and comparison of SMI data with RNA-seq and RNAscope data.
Fig. 3: Spatial RNA detection to identify cell types and cell–cell interactions in FFPE human NSCLC tissue.
Fig. 4: Paired LR expression between interacting tumor cells and T cells varies across tumors.
Fig. 5: Concordance between serial FFPE lung sections over a spatial grid.
Fig. 6: Spatial subcellular protein analysis on SMI.

Data availability

The full RNA NSCLC dataset used in this study is available at

Code availability

Data from this publication (the full RNA NSCLC dataset) has been placed in the public domain in a format that can be analyzed and visualized using a variety of open-source packages, such as Seurat ( and Giotto ( Nearly all of the analyses performed in this paper can be accomplished using these open-source packages. For any of the specialized code/analyses performed in this manuscript that are not available through open-source packages, requests can be made through email to the corresponding author.


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Research and development reported in this publication was supported in part through a strategic development collaboration between NanoString Technologies and Lam Research (Fremont, CA). The authors thank B. Birditt, B. Filanoski, J. Jenkins and E. Zhao from NanoString Technologies for provision of technical support.

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Authors and Affiliations



S.H. undertook conception and design of the work, supervised data collection, analysis and interpretation and drafted the manuscript. R.B. developed protein data processing algorithms. C.B. was responsible for instrument control software, SMI automation and integration of all instrument subsystems software. E.A.B. reviewed and validated protein data (Fig. 6) and performed the nested protein multiplex validation experiment (Supplementary Fig. 17). D.L.B. developed manual and automated processes for inventorying, quantitation, normalization, pooling, purification and quality control of oligonucleotides in 980-plex RNA panel and SMI reporters. K.C. performed oligo conjugations of antibodies used in SMI protein assays. P.D. designed the 980-plex RNA panel with E.P., performed comparison with RNA-seq in Fig. 2, performed NSCLC analyses in Fig. 3, performed reproducibility analysis in Fig. 5 and contributed to manuscript writing/editing. D.D. led the team that developed the system, instrumentation and software. R.G.G. developed reporter chemistry, assembly, quality control and manufacturing, and contributed to the section SMI reporter design and assembly and Supplementary Fig. 18. G.G. led development of protein-based SMI assays and design and interpretation of protein experiments. M.T.G. analyzed profiling data of human lung cells. M.L.H. contributed to the development of reporters and analysis of FFPE RNA quality and created Supplementary Fig. 6. R.K. designed and developed reporter structure. E.E.K. contributed to SMI methods. D.K. developed the overall concept, designed and guided experiments and analyzed data. T.K.K. supervised data collection and interpretation of human lung samples. Y.K. supervised data collection, analysis and interpretation of human lung samples. A. Klock developed the SMI ISH probe design pipeline and designed SMI ISH probes used in the 980-plex RNA panel. M.K. developed the primary data analysis pipeline for RNA and protein targets. A. Kutchma designed SMI ISH probes and wrote the section SMI ISH probe design. Z.R.L. designed and developed the protein assay, executed protein analyses and performed manuscript writing and editing. Y.L. conducted a pathological review to identify the correct staining pattern for all antibodies used. J.S.N.’s team was responsible for all chemistry process development efforts and supply chain management pertaining to the outsourcing of oligonucleotide synthesis, process improvements, price negotiation and supply agreement, key component scale-up and validation of all custom reagents and DNA components in R&D required for SMI—including contract manufacturing operation and validation of the PC spacer, synthesis and quality control of the three large-scale component sets needed for SMI reporter manufacturing plus high-throughput synthesis, outsourcing, quality control and processing of the thousands of required ISH probes. G.T.O. developed morphology and segmentation markers. E.P.P. developed the SMI instrument optical subsystem, instrument validation and support. J.C.P. developed the encoding scheme, screened reporter sequences and developed readout sequences. T.P.-E. optimized protein assay and undertook data collection, protein analyses and manuscript writing/editing. E.P. designed the 980-plex RNA panel with P.D. and contributed to manuscript writing/editing. T.R. developed the secondary analysis and target decoding pipeline for RNA targets, and the SMI instrument fluidic subsystem and workflow software. Z.R. developed the LR interaction analysis method, analyzed LR interactions across all tumors and contributed to manuscript writing/editing. M.R. performed initial chemistry development, supervised data release and contributed to writing the manuscript. A.R. was responsible for SMI protein assay content design and reagent validation. D.R. created plots of transcript positions overlaid on morphology and segmentation images for the figures. H.S. carried out manuscript development, writing and editing and created figures and tables. A.W.W. designed and developed the cell segmentation pipeline and contributed to manuscript writing/editing. C.A.W.-W. performed lung RNA isolation, processing and quality measurement and NGS library preparation and sequencing. L.W. established the cell segmentation pipeline, optimized the on-instrument SMI readout workflow and contributed to manuscript writing/editing. J.M.B. conceived the project, helped with experimental design and analysis and contributed to manuscript writing/editing.

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Correspondence to Joseph M. Beechem.

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All authors are employees of NanoString Technologies and hold NanoString stock or stock options. D.K. is an employee of Dxome Co.

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Nature Biotechnology thanks Sanjay Tyagi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Figs. 1–18.

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Supplementary Tables 1–11; combined tables separated by tab.

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He, S., Bhatt, R., Brown, C. et al. High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging. Nat Biotechnol 40, 1794–1806 (2022).

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