Detection of non-Brownian diffusion in the cell membrane in single molecule tracking
Detection of non-Brownian diffusion in the cell membrane in single molecule tracking
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DOI：
10.1529/biophysj.104.054106
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发表时间：
2005-03-01
影响因子：
3.4
通讯作者：
Kusumi, A
中科院分区：
文献类型：
--
作者：
Ritchie, K;Shan, XY;Kusumi, A
分子在质膜中经历非布朗扩散，但这种异常扩散背后的机制是有争议的。为了表征质膜复杂系统中的异常扩散并了解其潜在的机制，单分子/粒子方法已经被证明是非常有效的，这种方法使研究人员能够避免系综平均。然而，时间平均(分辨率)和观测频率的内在问题尚未得到探讨。这些对于简单布朗粒子的观测并不重要，但它们确实强烈地影响了对经历异常扩散的分子的观测。我们使用蒙特卡罗模拟方法研究了这些对简单、完全受限或跳跃扩散的分子的表观运动的影响，其中包括相机单帧期间时间平均(曝光时间)和观测频率(帧速率)的影响。这些与时间相关的实验参数与本征扩散参数之间的复杂关系被阐明，这表明通过系统地改变帧时间和帧速率，可以清楚地检测和表征异常扩散。在此基础上，对活体PtK2细胞质膜转铁蛋白受体进行了单粒子示踪，在0.025-33ms(0.03-40 kHz)之间变化，借助于单荧光分子视频成像，揭示了受体在47 nm(平均停留时间为1.7ms)间隔之间的跳跃扩散。
Molecules undergo non-Brownian diffusion in the plasma membrane, but the mechanism behind this anomalous diffusion is controversial. To characterize the anomalous diffusion in the complex system of the plasma membrane and to understand its underlying mechanism, single-molecule/particle methods that allow researchers to avoid ensemble averaging have turned out to be highly effective. However, the intrinsic problems of time-averaging ( resolution) and the frequency of the observations have not been explored. These would not matter for the observations of simple Brownian particles, but they do strongly affect the observation of molecules undergoing anomalous diffusion. We examined these effects on the apparent motion of molecules undergoing simple, totally confined, or hop diffusion, using Monte Carlo simulations of particles undergoing short-term confined diffusion within a compartment and long-term hop diffusion between these compartments, explicitly including the effects of time-averaging during a single frame of the camera ( exposure time) and the frequency of observations ( frame rate). The intricate relationships of these time-related experimental parameters with the intrinsic diffusion parameters have been clarified, which indicated that by systematically varying the frame time and rate, the anomalous diffusion can be clearly detected and characterized. Based on these results, single-particle tracking of transferrin receptor in the plasma membrane of live PtK2 cells were carried out, varying the frame time between 0.025 and 33 ms (0.03 - 40 kHz), which revealed the hop diffusion of the receptor between 47- nm ( average) compartments with an average residency time of 1.7 ms, with the aid of single fluorescent-molecule video imaging.