Quantitative Light Imaging Laboratory, Department of Electrical and Computer Engineering, Beckman Institute of Advanced Science and Technology, ‡Center for the Physics of Living Cells, §Center for Biophysics and Quantitative Biology, ∥Department of Physics, and ⊥Department of Bioengineering, University of Illinois , Urbana, Illinois 61801, United States.
ACS Nano. 2017 Jan 24;11(1):647-655. doi: 10.1021/acsnano.6b06945. Epub 2016 Dec 27.
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 × 200 μm, 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 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.
由于直径仅为 24nm，单根微管在没有外在对比剂的情况下极难成像。因此，荧光标记是可视化其运动的常用方法。然而，这种研究受到荧光漂白和光毒性的限制。我们通过实验证明了将无标记空间光干涉显微镜 (SLIM) 与数值处理相结合，在滑行实验中对单根微管进行成像的能力。SLIM 结合了四个不同的强度图像，以获得与样品相关的光程图。由于使用了宽带场，因此对光程的灵敏度在没有（时间）平均的情况下优于 1nm，在平均情况下优于 0.1nm。我们的结果表明，SLIM 可以在 200×200μm 的全视场中对微管的动力学进行成像，时间长达数小时。通过扩散-输运方程对微管的输运进行建模，我们发现，色散关系得出了速度分布的标准偏差，而无需跟踪单个微管。有趣的是，在 2 小时的窗口内，微管开始减速，在 20 分钟内以 100pm/s 的速度减速。因此，SLIM 可能成为理解分子马达活性的有用工具，特别是在荧光方法用途有限的较大时间尺度上。
