Single-molecule fluorescence resonance energy transfer (smFRET) is widely used to investigate dynamic (bio)molecular interactions occurring over distances of up to 10 nm. Recent advances in super-resolution methods have brought their spatiotemporal resolution closer towards the smFRET regime. Although these methods do not suffer from the spatial restrictions of FRET, they only visualize one emitter at a time, thus making it difficult to capture fast dynamics of the interactions. Here we describe two approaches to overcome this limitation in pulsed-interleaved MINFLUX (pMINFLUX) microscopy by using its intrinsic fluorescence lifetime information. First we combine pMINFLUX with smFRET, which enables tracking a FRET donor with nanometre precision while simultaneously determining its distance to a FRET acceptor, yielding the acceptor position by multilateration. Second, we developed pMINFLUX lifetime multiplexing—a method that simultaneously tracks two fluorophores with similar spectral properties but distinct fluorescence lifetimes—to extend co-localized tracking beyond the FRET range. We demonstrate applications on DNA origami systems as well as by imaging the paratopes of an antibody with precision better than 2 nm, paving the way for nanometre precise co-localized tracking for inter-dye distances between 4 nm and 100 nm, and closing the resolution gap between smFRET and co-tracking.

单分子荧光共振能量转移 (smFRET) 广泛用于研究在长达 10 nm 的距离内发生的动态（生物）分子相互作用。超分辨率方法的最新进展使其时空分辨率更接近 smFRET 体系。尽管这些方法不受 FRET 的空间限制，但它们一次只能可视化一个发射器，因此很难捕获相互作用的快速动态。在这里，我们描述了两种方法，通过利用其固有的荧光寿命信息来克服脉冲交错 MINFLUX (pMINFLUX) 显微镜中的这一限制。首先，我们将 pMINFLUX 与 smFRET 结合起来，从而能够以纳米精度跟踪 FRET 供体，同时确定其与 FRET 受体的距离，从而通过多边测量得出受体位置。其次，我们开发了 pMINFLUX 寿命多重分析（一种同时跟踪具有相似光谱特性但不同荧光寿命的两种荧光团的方法），以将共定位跟踪扩展到 FRET 范围之外。我们演示了在 DNA 折纸系统上的应用，以及以优于 2 nm 的精度对抗体互补位进行成像，为 4 nm 至 100 nm 之间染料间距离的纳米精确共定位跟踪铺平了道路，并缩小了分辨率smFRET 和协同追踪之间的差距。