Computational analysis of F-actin turnover in cortical actin meshworks using fluorescent speckle microscopy
Computational analysis of F-actin turnover in cortical actin meshworks using fluorescent speckle microscopy
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DOI：
10.1016/s0006-3495(03)70058-7
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发表时间：
2003-05-01
影响因子：
3.4
通讯作者：
Danuser, G
中科院分区：
文献类型：
--
作者：
Ponti, A;Vallotton, P;Danuser, G
荧光散斑显微镜（FSM）是一种新的成像技术，具有同时可视化聚合物结构的移位和动态周转的潜力。然而，由于缺乏专门的软件来分析FSM延时序列中数十万个斑点的位置和光度波动，以及将这些数据转化为生物学相关信息，FSM的使用受到了限制。在本文中，我们提出了一个软件的自动分析FSM电影的第一个版本。我们专注于绘制聚合物网络的组装和拆卸动力学。作为一个模型系统，我们在活蝾螈肺上皮细胞中使用了皮质f -肌动蛋白网。详细介绍了该算法，并给出了分析结果。我们地图的高空间和时间分辨率揭示了f -肌动蛋白的动力学循环，其中聚合阶段以空间协调的方式交替与解聚。当使用低剂量的药物latrunculin a处理细胞时，周期率会发生变化，这表明该技术在未来定量筛选影响肌动蛋白细胞骨架的药物方面具有潜力。各种对照实验表明，该算法对于由噪声和光漂白引起的强度变化具有鲁棒性，并且可以通过在图像采集过程中手动重新聚焦来消除焦平面漂移的影响。
Fluorescent speckle microscopy (FSM) is a new imaging technique with the potential for simultaneous visualization of translocation and dynamic turnover of polymer structures. However, the use of FSM has been limited by the lack of specialized software for analysis of the positional and photometric fluctuations of hundreds of thousand speckles in an FSM time-lapse series, and for translating this data into biologically relevant information. In this paper we present a first version of a software for automated analysis of FSM movies. We focus on mapping the assembly and disassembly kinetics of a polymer meshwork. As a model system we have employed cortical F-actin meshworks in live newt lung epithelial cells. We lay out the algorithm in detail and present results of our analysis. The high spatial and temporal resolution of our maps reveals a kinetic cycling of F-actin, where phases of polymerization alternate with depolymerization in a spatially coordinated fashion. The cycle rates change when treating cells with a low dose of the drug latrunculin A. This shows the potential of this technique for future quantitative screening of drugs affecting the actin cytoskeleton Various control experiments demonstrate that the algorithm is robust with respect to intensity variations due to noise and photobleaching and that effects of focus plane drifts can be eliminated by manual refocusing during image acquisition.