Quantification and analysis of multiplexed fluorescence in situ hybridization data using open-source tools

Here, we present a protocol to quantify and analyze multiplexed fluorescence in situ hybridization (mFISH) data using two open-source tools, FijiFISH and RUHi. FijiFISH, an ImageJ-based plugin, enables image registration, cell segmentation, and gene expression quantification. RUHi, an R-based package, supports dimensionality reduction, clustering, and visualization through both code and a Shiny app. The protocol also accommodates experimentally induced exogenous fluorophores, providing multimodal, single-cell resolution insights into spatial gene expression. For complete details on the use and execution of this protocol, please refer to Sullivan et al.¹.