Chemical Physics Letters FRONTIERS ARTICLE 457 (2008) 285–291

        single-molecule tracking spectroscopy 就是单分子轨迹追踪。单分子跟踪技术结合了录像显微镜和数字相机，以一定的频率连续拍摄标记的单分子，经过图像数据处理，描绘出单分子的运动轨迹。目标分子以绿色荧光蛋白、量子点(200 －500nm)或者胶体金(40 － 100 nm)标记，直径往往小于可见光波长范围(390 － 780 nm)，因此会散射光，形成同心圆的衍射图纹，中心处分辨率可精确到10nm。

        以前文献中已经发展出一些image analysis-based 3D tracking schemes，但是得到的images的信噪比很差不足以得到可靠的光谱。而利用confocal microscopy虽然有足够信噪比，但是目标分子经常会很快的跑出focal volume，使得可以tracking的时间和距离大大的缩短。实际上，因为溶液中的单分子不停地进行Brownian motion，这使得利用宏观的设备来追踪这些随机的纳米级的运动变得非常困难。不过近年来仍然有些革新的设计来克服这些困难，下面就对它们进行一些简单的介绍。

1. Circularly scanning laser tracking

          Enderlein[26]提出了一个思路，利用一个feedback system控制激光focus去有效地在二维平面中track单分子。他的idea很简单， 激光束腰(beam waist)大概在微米量级（远远大于单分子的纳米量级），所以可以利用激光束腰的强度梯度来map out目标分子到激光束腰中心的距离。但是单次测量是作不到这一点的，所以Enderlein设计让激光绕着目标分子转一圈进行扫描，从而可以确定单分子在二维空间中的位置。这个idea虽然简单，但是第一个实验却用了两年才由Gratton group[27]实现，他们发现实际上不需要记录激光束的坐标，而是利用了fast Fourier transform (FFT)分析。后来Mabuchi group又利用了lock-in amplifier[28]来改进这个设计，并且他们不再移动激光束位置，而是利用把sample放在一个3D piezoelectric translation stage上来移动sample，这就克服了当移动激光位置进行扫描时需要移动检测器的困难。

         通过利用两次扫描，一次在目标分子上方，一次在目标分子下方，Gratton group[27,33]发展出了三维空间中tracking的方法。接着Mabuchi group[34]发展出了双光束的方法从而避免了z方向的sacn。

2. Confocal 3D tracking

        本文作者Yang group发展出了confocal tracking method [35–37]，他们把一个pinhole放在共轭焦点处稍微在z方向off一点的位置。这样当目标分子移动的时候，它的image就会在pinhole附近移动，从而导致通过pinhole的能量改变，然后利用avalanched photo diode (APD)或photon multiplier tube (PMT)来获得目标分子在z方向上移动的信息。在xy方向上，他们利用一对正交放置prism mirrors和四个APDs来检查目标分子在xy平面内的移动。该方法的好处首先是激发激光可以聚焦在单分子粒子上，从而提高信噪比；并且不需要scan。

3. Trapping

        In the above two implementations, the actuator's mechanical movement establishes the ultimate limit of the tracking bandwidth: Both the laser-scanning scheme (which equires the detector center to be moved accordingly) and the sample-moving scheme are limited by the 1-kHz bandwidth of the currently availably piezoelectric stages. Cohen and Moerner [41–45] have devised a creative alternative, an electrophoretic trap, termed as the Anti-Brownian-Motion-Electrophoretic (ABEL) trap. As shown in Fig. 5, four orthogonal gold electrodes are fabricated on a cover glass. The movement of the target of interest is restricted to 2D by confining it within a 800-nm thick sample chamber. As soon as a charge-coupled device camera detects the movement of the molecule, voltages are applied to the four electrodes to steer the molecule back to the center. If the particle is charged, it will be moved directly by the electric force. For neutral particles, the electric field will induce a hydrodynamic drag from the flow of the fluid. Since the electrodes are very close to each other, the force induced by the two mechanisms can be very strong. Using the electrophoretic trap, they have successfully demonstrated trapping fluorescence spheres, quantum dots, fluorescence stained DNA, and fluorescent proteins. From the applied voltages, Cohen and Moerner were able to deduce the trajectory of the moving target. In principle, the electrophoretic trap can also be extended to 3D by fabricating a 3D structure of electrodes.
        One unique advantage of the trapping approach is that it is capable of manipulating individual molecules in solution, which could be exploited to create molecule-scale patterns. Though expected to be challenging, in principle, it might be possible to simultaneously manipulate many nanoscale objects using methods that have been demonstrated for steering micron-sized particles [46].

Applications

One immediate application of tracking nanoscale targets in 3D is to follow the transport dynamics in cells. Levi and others studied the phagocytosis process in fibroblasts [33].

The phagocytosis experiment and others [56–58] are examples of how direct visualization of probe trajectories enables the study of problems in intracellular trafficking.

One may envision that the capability to follow individual nonfluorescent nanoparticles could allow the real-time observation of the self-assembly process from nanoscale building blocks [60], where the nanoscale hydrodynamic interactions are expected to play a significant role.

Holding a single molecule at a fixed position in space also allows a critical test for the theory of polymer dynamics, e.g., the Zimm model [62,63].

该实验技术可以通过记录和分析单个分子或粒子在时间和空间中的动力学轨迹来研究单分子的振动和转动动力学。

Reference: 

1. Chem Phys Lett 457, 285  (2008) Progress in single-molecule tracking spectroscopy

2. 生命科学 20, 29 (2008) 细胞膜表面单分子事件的实时观察

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