Real-time 3D single molecule tracking
Real-time 3D single molecule tracking
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DOI：
10.1038/s41467-020-17444-6
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发表时间：
2020-07-17
影响因子：
16.6
通讯作者：
Welsher, Kevin
中科院分区：
文献类型：
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作者：
Hou, Shangguo;Exell, Jack;Welsher, Kevin
迄今为止，单分子研究一直依赖于束缚或限制，以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），该方法能够以光子有限的时间分辨率锁定溶液中的单个荧光团数分钟。作为示范，3D-SMART应用于主动跟踪90%甘油溶液中的单个Atto 647 N荧光团，平均持续时间类似于16秒，计数率类似于10 kHz。主动反馈跟踪进一步应用于单个蛋白质和核酸，以高达10 μ m（2）/s的速率直接测量单个DNA分子的各种长度（99至1385 bp）的扩散。此外，3D-SMART能够量化单个Spinach 2 RNA适体的占用率，并捕获单个自由扩散DNA上的活性转录。3D-SMART代表了单分子光谱学的关键一步。
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 647N fluorophores in 90% glycerol solution with an average duration of similar to 16s at count rates of similar to 10kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385bp) of single DNA molecules at rates up to 10 mu m(2)/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.