smICA: Open-Source Software for Quantitative, Lifetime-Resolved Mapping of Absolute Fluorophore Concentrations in Living Cells

Advanced microscopy techniques are essential in biomedical research for visualising and tracking biomolecules within living cells and their compartments. Conventional fluorescence microscopy methods, however, often struggle with accurately measuring the absolute concentrations of fluorescent probes in living cells. To overcome these limitations, we introduce an open-source analysis tool, smICA (Single-Molecule Image to Concentration Analyser). The smICA method offers quantitative mapping of absolute fluorophore concentrations, lifetime-resolved filtering methods of the signal, intensity-based cell segmentation, and requires only a few photons per pixel. Our approach also reduces the time required for the determination of the mean concentration per cell, compared to the standard FCS measurement performed in multiple posts. To highlight the robustness of the method, we validated it against standard fluorescence correlation spectroscopy (FCS) measurements by performing in vitro (aqueous solutions of polymers) and in vivo (polymers and EGFP in living cells) experiments. The presented methodology, along with the software, is a promising tool for quantitative single-cell studies, including, but not limited to, protein expression, degradation of biomolecules (such as proteins and mRNA), and monitoring of enzymatic reactions.