Dual-color image analysis for quantifying fluorescence intensity in plasma membrane region of cells

Quantitative evaluation of fluorescence intensity in the plasma membrane region is essential for a wide range of cell-based imaging studies, including membrane protein analysis and cell surface interaction assays. However, accurate quantification remains challenging because membrane regions are often manually selected, leading to subjective variabilities and limited reproducibility. Here, we developed a dual-channel image analysis framework and its web-based tool, DualCellQuant, which enables the objective and reproducible quantification of fluorescence intensity in the plasma membrane region. This method integrates automated cell segmentation using Cellpose-SAM with Euclidean distance transform-based radial normalization to define membrane regions at a fixed normalized distance from the cell boundary. The membrane region was determined on a reference channel, allowing robust quantification even for low-intensity target signals. In addition, ratiometric analysis between channels can mitigate cell-to-cell variability. We applied this approach to quantify rapamycin-induced binding between FKBP12 displayed on the cell surface and fluorescein-labeled FRB, demonstrating that membrane-associated fluorescence signals can be consistently extracted on a per-cell basis and used for binding-equilibrium analysis. This accessible framework is expected to facilitate the reproducible interpretation of multicolor cellular imaging data across diverse experimental settings.