Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy
Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy
复制标题
DOI：
10.1021/acsnano.6b06945
复制
发表时间：
2017-01-01
期刊：
影响因子：
17.1
通讯作者：
Popescu, Gabriel
中科院分区：
文献类型：
--
作者：
Kandel, Mikhail E.;Teng, Kai Wen;Popescu, Gabriel
由于单个微管的直径只有24 nm，因此在不使用外部造影剂的情况下成像是非常困难的。因此，荧光标记法是显示其运动能力的常用方法。然而，这类研究受到光漂白和光毒性的限制。我们在实验中展示了将无标记空间光干涉显微镜(SLIM)与数值处理相结合的能力，用于在滑动实验中对单个微管进行成像。SLIM将四个不同强度的图像组合在一起，以获得与样本相关的光路长度图。由于使用了宽带光场，在没有时间平均的情况下，对光程长度的灵敏度优于1 nm，而在平均的情况下，对光程长度的灵敏度优于0.1 nm。我们的结果表明，SLIM可以在200×200微米(2)的全视场中连续数小时成像微管的动力学。用扩散-平流方程模拟微管输运时，我们发现色散关系得到了速度分布的标准差，而不需要跟踪单个管子。有趣的是，在2小时的窗口中，微管开始减速，在20分钟内100 PM/S(2)。因此，SLIM很可能成为了解分子马达活性的有用工具，特别是在大时间尺度上，荧光方法的用处有限。
Due to their diameter, of only 24 nm, single microtubules are extremely challenging to image without the use of extrinsic contrast agents. As a result, fluorescence tagging is the common method to visualize their motility. However, such investigation is limited by photobleaching and phototoxicity. We experimentally demonstrate the capability of combining label-free spatial light interference microscopy (SLIM) with numerical processing for imaging single microtubules in a gliding assay. SLIM combines four different intensity images to obtain the optical path length map associated with the sample. Because of the use of broadband fields, the sensitivity to path length is better than 1 nm without (temporal) averaging and better than 0.1 nm upon averaging. Our results indicate that SLIM can image the dynamics of microtubules in a full field of view, of 200 X 200 mu m(2), over many hours. Modeling the microtubule transport via the diffusion-advection equation, we found that the dispersion relation yields the standard deviation of the velocity distribution, without the need for tracking individual tubes. Interestingly, during a 2 h window, the microtubules begin to decelerate, at 100 pm/s(2) over a 20 min period. Thus, SLIM is likely to serve as a useful tool for understanding molecular motor activity, especially over large time scales, where fluorescence methods are of limited utility.