Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity. RASTMIN delivers ~1–2 nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures.

单个荧光发射器的定位是纳米级和超越整体平均的物理化学和生物物理测量的关键。例子包括单分子跟踪和单分子定位显微镜的超分辨率成像。在众多可用的定位方法中，MINFLUX 在分辨率方面比基于广角相机的方法提高了约 10 倍，在中等光子计数下达到分子尺度。设置的技术复杂性阻碍了 MINFLUX 和相关方法的广泛应用。在这里，我们提出了 RASTMIN，一种基于光栅扫描包含最小强度的光图案的单分子定位方法。RASTMIN 使用通常的荧光团提供约 1-2 nm 的定位精度，并且只需少量修改即可在标准共聚焦显微镜上轻松实现。我们展示了 RASTMIN 在单分子定位和 DNA 折纸结构的超分辨率成像方面的性能。