A General Analytic Approach for Rapid Diagnostics by a Simple Algorithm for Fluorescence Single Molecule Counting

Accurate biomolecule quantification at ultralow concentrations remains a major challenge because conventional ensemble assays report population averaged signals and therefore lose sensitivity in low-abundance regimes. Single molecule fluorescence counting can overcome this limitation by converting emission into discrete digital events, but practical implementation is often hindered by manual inspection, limited reproducibility, and the complexity of machine learning based analysis. Here, we present a simple and general analytical framework for rapid single molecule detection based on a deterministic threshold algorithm that exploits the temporal signature of fluorescence blinking. The method operates directly on time resolved fluorescence image stacks, applies median filter-based noise suppression, and identifies candidate single molecule events from consecutive frame-to-frame intensity transitions without the need for training data or model fitting. Applied to Alexa Fluor 488, Alexa Fluor 647, and Rhodamine Red–X datasets, the approach reproduced the concentration dependent trends observed by manual counting, while providing more standardized detection under weak signal and high background conditions. Dye specific operating thresholds yielded robust counting behavior and preserved approximately linear concentration dependent response across the tested range. Compared with manual analysis, which required inspection of only selected grid regions, the automated workflow processed full movie stacks and reduced analysis time from ~3 h to ~20 min per concentration, corresponding to an approximately 9-fold gain in efficiency. These results establish an interpretable, computationally lightweight, and experimentally adaptable strategy for fluorescence single molecule counting that can support rapid diagnostics and provide a practical foundation for future extensions in automated localization, clustering, and real time molecular analysis.