Multiplexed imaging and automated signal quantification in formalin-fixed paraffin-embedded tissues by ChipCytometry

Deciphering the spatial composition of cells in tissues is essential for detailed understanding of biological processes in health and disease. Recent technological advances enabled the assessment of the enormous complexity of tissue-derived parameters by highly multiplexed tissue imaging (HMTI), but elaborate machinery and data analyses are required. This severely limits broad applicability of HMTI. Here we demonstrate for the first time the application of ChipCytometry technology, which has unique features for widespread use, on formalin-fixed paraffin-embedded samples, the most commonly used storage technique of clinically relevant patient specimens worldwide. The excellent staining quality permits workflows for automated quantification of signal intensities, which we further optimized to compensate signal spillover from neighboring cells. In combination with the high number of validated markers, the reported platform can be used from unbiased analyses of tissue composition to detection of phenotypically complex rare cells, and can be easily implemented in both routine research and clinical pathology. [Display omitted] •ChipCytometry is an optical imaging method based on staining/bleaching cycles•ChipCytometry allows multiplexed imaging and is now applicable to FFPE sections•A fine-tuned analysis pipeline leads to accurate automatic signal quantifications•High data quality enables deep investigation of spatial and phenotypic parameters Simultaneous in situ analyses of a multitude of cells and markers is necessary for gaining insights into tissue biology and pathology. Here we describe the application of ChipCytometry, a user-friendly optical imaging-based technology for multiplexed staining, to widespread used formalin-fixed paraffin-embedded tissues. The multiplexing of up to 30 markers in combination with a developed open-source workflow for signal quantification facilitated high-dimensional tissue analyses. Understanding of tissue biology requires in situ analyses of cell phenotypes. Jarosch et al. describe the application of the multiplexed imaging method ChipCytometry to formalin-fixed and paraffin-embedded tissue sections. The high staining quality allowed them to develop a pipeline for automated signal quantifications facilitating reliable and reproducible analyses of high-parameter imaging.