Compared with localization schemes solely based on evaluating patterns of molecular emission, the recently introduced single-molecule localization concept called MINFLUX and the fluorescence nanoscopies derived from it require up to orders of magnitude fewer emissions to attain single-digit nanometer resolution. Here, we demonstrate that the lower number of required fluorescence photons enables MINFLUX to detect molecular movements of a few nanometers at a temporal sampling of well below 1 millisecond. Using fluorophores attached to thermally fluctuating DNA strands as model systems, we demonstrate that measurement times as short as 400 microseconds suffice to localize fluorescent molecules with ∼2-nm precision. Such performance is out of reach for popular camera-based localization by centroid calculation of emission diffraction patterns. Since theoretical limits have not been reached, our results show that emerging MINFLUX nanoscopy bears great potential for dissecting the motions of individual (macro)molecules at hitherto-unattained combinations of spatial and temporal resolution.

与仅基于评估分子发射模式的定位方案相比，最近引入的称为 MINFLUX 的单分子定位概念以及由此衍生的荧光纳米显微镜需要减少几个数量级的发射才能获得个位数的纳米分辨率。在这里，我们证明所需的荧光光子数量较少，使得 MINFLUX 能够以远低于 1 毫秒的时间采样检测几纳米的分子运动。使用附着在热波动 DNA 链上的荧光团作为模型系统，我们证明短至 400 微秒的测量时间足以以~2 nm 的精度定位荧光分子。对于流行的基于相机的通过发射衍射图案的质心计算进行的定位来说，这种性能是无法达到的。由于尚未达到理论极限，我们的结果表明，新兴的 MINFLUX 纳米显微镜具有以迄今为止未达到的空间和时间分辨率组合来剖析单个（宏观）分子运动的巨大潜力。