Department of Chemistry, Duke University, Durham, NC, USA.
Nat Commun. 2020 Jul 17;11(1):3607. doi: 10.1038/s41467-020-17444-6.
To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647 N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385 bp) of single DNA molecules at rates up to 10 µm/s. In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.
迄今为止，单分子研究一直依赖于系链或限制来实现长时间和高时间分辨率的测量。在这里，我们提出了一种 3D 单分子主动实时跟踪方法（3D-SMART），它能够在每次锁定溶液中的单个荧光团几分钟，具有光子限制的时间分辨率。作为演示，3D-SMART 被应用于主动跟踪 90%甘油溶液中的单个 Atto 647N 荧光团，在计数率约为 10kHz 时，平均持续时间约为 16 秒。主动反馈跟踪进一步应用于单个蛋白质和核酸，直接测量各种长度（99 至 1385bp）的单 DNA 分子的扩散速率高达 10μm/s。此外，3D-SMART 能够定量单个 Spinach2 RNA 适体的占有率，并捕获单个自由扩散 DNA 上的活性转录。3D-SMART 代表了朝着单分子光谱学解链迈出的关键一步。
