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However, the strengths of the commercially available ST platforms in studying spatial biology have not been systematically evaluated using rigorously controlled experiments. In this study, we used serial 5-m sections of formalin-fixed, paraffin-embedded surgically resected lung adenocarcinoma and pleural mesothelioma tumor samples in tissue microarrays to compare the performance of the single cell ST platforms CosMx, MERFISH, and Xenium (uni\/multi-modal) platforms in reference to bulk RNA sequencing, multiplex immunofluorescence, GeoMx Digital Spatial Profiler, and hematoxylin and eosin staining data for the same samples. In addition to objective assessment of automatic cell segmentation and phenotyping, we performed pixel-resolution manual evaluation of phenotyping to carry out pathologically meaningful comparison between ST platforms. Our study detailed the intricate differences between the ST platforms, revealed the importance of parameters such as tissue age and probe design in determining the data quality, and suggested reliable workflows for accurate spatial profiling and molecular discovery.<\/jats:p>","DOI":"10.1101\/2024.12.13.628390","type":"posted-content","created":{"date-parts":[[2024,12,21]],"date-time":"2024-12-21T12:20:11Z","timestamp":1734783611000},"source":"Crossref","is-referenced-by-count":1,"title":["Comparison of imaging-based single-cell resolution spatial transcriptomics profiling platforms using formalin-fixed, paraffin-embedded tumor samples"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6820-1456","authenticated-orcid":false,"given":"Nejla","family":"Ozirmak Lermi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Max","family":"Molina 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