We introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

我们介绍了 MINSTED，这是一种基于受激发射耗尽 (STED) 的荧光团定位和超分辨率显微镜概念，可提供低至分子尺度的空间精度和分辨率。在 MINSTED 中，STED 甜甜圈的强度最小值以及因此的最小 STED 点用作荧光团定位的可移动参考坐标。由于与大多数其他 STED 显微镜和定位方法相比，STED 速率、背景和所需的荧光检测数量较低，因此 MINSTED 需要显着减少荧光团漂白。在我们的实施中，每个荧光团检测 200–1,000 次可提供 1–3 nm 标准偏差的定位精度，结合独立的单个荧光团切换，远场显微镜分辨率比衍射极限提高了约 100 倍。通过对人类细胞线粒体内膜中 Mic60 蛋白的分布进行成像，证明了 MINSTED 纳米镜的性能。