Quantitative MINFLUX Imaging via Sequential Labeling Localization

MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy localizes individual switchable fluorophores using a probing donut-shaped excitation beam, achieving resolutions ranging from 1 to 3 nm for structures in both fixed and living cells. However, traditional MINFLUX data sets obtained through fluorophore multiple blinking and readout methods present untapped opportunities for quantitative investigations within its resolution limits. Here, we introduce a quantitative MINFLUX imaging method (q-MINFLUX) with nanobody point accumulation in nanoscale topography, followed by the sequential localization of target subsets. Notably, sequential localization events related to the same molecule share a common trace ID (exported parameter TID), which can be used for accurately calculating labeled targets, going beyond the mere binning of molecule localizations for spatial visualization. We use our method in a proof-of-principle demonstration to map the number of proteins in one cluster of molecular arrangement of the microtubules and the mitochondria in situ in 2D and 3D. Our methods provide a direct and adaptable approach for quantifying proteins in dense clusters using MINFLUX nanoscopy imaging.