Calibration-free molecular counting from a single DNA-PAINT intensity trace using cumulants
Single-molecule localization microscopy achieves nanometer-scale resolution but fails to count molecular targets when multiple targets are in close proximity. DNA-PAINT uses reversible binding of fluorescently labeled probes to image molecular targets, but existing time-series based counting methods require prior knowledge of binding kinetics, calibration, or extensive data post-processing, which limits applicability across heterogeneous biological samples. Here, we present mCOAST, a calibration-free method that extracts molecular counts and kinetic parameters directly from a single DNA-PAINT intensity trace using cumulants. Unlike existing approaches, mCOAST requires no adjustable parameters, data normalization, or denoising. We demonstrate accurate counting for diffraction-limited clusters with up to 48 targets at high imager concentrations, with precision that improves, rather than decreases, as concentration increases. Critically, mCOAST counts targets in individual clusters despite kinetic heterogeneity across samples or between experiments. This paves the way towards quantitative imaging and counting in uncalibrated biological systems, such as living cells.