Single-molecule localization microscopy (SMLM) relies on the blinking behavior of a fluorophore, which is the stochastic switching between fluorescent and dark states. Blinking creates multiple localizations belonging to the same fluorophore, confounding quantitative analyses and interpretations. Here we present a method, termed distance distribution correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on obtaining the true pairwise distance distribution of different fluorophores naturally from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that, using the true pairwise distribution, we can define and maximize the likelihood, obtaining a set of localizations void of blinking artifacts. DDC results in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications, enabling accurate reconstruction and quantification of SMLM images.

单分子定位显微镜 (SMLM) 依赖于荧光团的闪烁行为，即荧光和暗态之间的随机切换。闪烁会产生属于同一荧光团的多个定位，混淆定量分析和解释。在这里，我们提出了一种称为距离分布校正 (DDC) 的方法，无需任何额外校准即可消除眨眼引起的重复定位。该方法依赖于通过使用比平均荧光生存时间长得多的时间分隔的定位之间的距离，从成像序列中自然地获得不同荧光团的真实成对距离分布。我们表明，使用真正的成对分布，我们可以定义并最大化可能性，获得一组没有闪烁伪影的定位。DDC 显着改善了在广泛应用中获得真实空间组织和荧光发射器数量的最接近估计，从而实现了 SMLM 图像的准确重建和量化。