Precise immuno-fluorescence canceling enables highly multiplexed imaging [scRNA-seq]

Cell states are regulated by extrinsic signals from various external factors such as intercellular interactions, and intrinsic gene expression. Although comprehensive cell state profiling has been attempted, it remains simultaneous analysis of signal activation has still been challenging. Multiplexed imaging is a technique acquiring multiple protein information at a single cell level as traditional immunofluorescence. However, the method often compromises resolution, hindering the analysis of intracellular localization dynamics and post-translational modifications of proteins. To address these limitations, we developed an erasable fluorescence method using disulfide linkers to label antibodies. We term these antibodies ‘Precise Emission Canceling Antibodies (PECAbs)’. PECAb allows for high-resolution iterative imaging with minimal non-specific binding. Automation enables our system to achieve reproducible quantitative analysis using 206 antibodies. The resulting quantitative data allow reconstruction of the spatiotemporal dynamics of signaling pathways over both long and short timescales. Additionally, combining this approach with sequential RNA-FISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system serves as a comprehensive platform for analyzing complex cell processes, from signal transduction to gene expression.