Abstract Single molecule localization microscopy (SMLM) is irreplaceable among super-resolution microscopies in revealing biological ultra-structures, given its unmatched high resolution. However, its sub-optimal quantitative capability, which is critical for characterizing true biomolecular organization of ultra-structures in cells, has hindered its widest application in biomedical research. Here, in SMLM imaging of cellular structures such as lipid rafts and microtubules with saturation labelling, we identified ultra-bright localizations, each of which is contributed by simultaneous emission of multiple molecules within a diffraction-limit region and has been regarded before as a regular localization from single molecule. Consistently, ultra-bright localizations are also observed in simulated SMLM imaging of endoplasmic reticulum or microtubules from public resource. Furthermore, after calibrating each ultrabright localization into multiple single-molecule localizations using the photon-number-based models, the density of total localizations shows linear correlation with the true molecule density, presenting SMLM with new reconstruction method as a quantitative analysis approach. Therefore, identification and dissection of ultra-bright localizations in SMLM enable the close and quantitative estimate of the true biomolecular organization.

Identification and calibration of ultrabright localizations to eliminate quantification error in SMLM