A simple method for automatic tracking of actin filaments in the motility assay
A simple method for automatic tracking of actin filaments in the motility assay
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DOI：
10.1007/bf00123365
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发表时间：
1996-08-01
影响因子：
2.7
通讯作者：
Roper, G
中科院分区：
文献类型：
--
作者：
Marston, SB;Fraser, IDC;Roper, G
从基本的Kron和Spudich体外运动测定中可以获得大量关于肌动球蛋白相互作用的定量信息，只要注意获得实验之间的一致性，并且对数据进行全面而非选择性的检查。根据对各种条件下的细丝运动的观察，我们提出了这样一个假设，即在短时间内运动的大量细丝与在较长时间内运动的较少细丝是无法区分的。用这个设计了一个简单的自动化灯丝检测程序。通过帧捕获器将一系列图像数字化。如果对连续的帧进行比较，程序将搜索并检测每个灯丝的新位置，并在屏幕上显示其矢量。计算速度并显示为频率直方图。该程序定期检测每对帧中移动的100多个细丝；通常一个多达15对帧的序列被研究，总共产生500-1000个向量。该算法不能处理相遇、交叉或分裂的细丝，但当细丝在帧间移动小于2 μ m时，这只是整体的一小部分。程序输出细丝的运动分数、平均速度随标准差和细丝密度（细丝mu m(-2)）。累积频率直方图可以直观直观地显示菌丝种群的表现。直接比较表明，除了非移动细丝的视速度较小外，自动跟踪产生的数据与人工跟踪没有什么区别。检测过程大约需要5分钟，几乎不需要技巧或判断力。这可以大大提高运动工作中数据分析的速度。
A great deal of quantitative information about the actomyosin interaction can be obtained from the basic Kron and Spudich in vitro motility assay provided that care is taken to obtain consistency between experiments and that the data is examined comprehensively and not selectively. From observations of filament movement under a wide variety of conditions we have formulated the hypothesis that a large number of filaments moving over a short time period is indistinguishable from fewer filaments moving over a longer sequence of frames. This has been used to devise a simple automation of filament detection procedures. A sequence of images is digitized through a frame-grabber. If successive pairs of frames are compared the program will search for and detect the new position of every filament and show its vector on screen. Velocity is calculated and shown as a frequency histogram. The program regularly detects over 100 filaments moving in each pair of frames; usually a sequence of up to 15 pairs of frames are studied yielding 500-1000 vectors in total. The algorithm cannot deal with filaments that meet, cross or divide, however, when filaments are moving less than 2 mu m between frames this is only a small proportion of the whole.The program outputs fraction of filaments motile, mean velocity with standard deviation and density of filaments (filaments mu m(-2)). A cumulative frequency histogram gives an immediate visual indication of the performance of the population of filaments. Direct comparisons show that the data produced by automatic tracking is indistinguishable from manual tracking apart from the small apparent velocity of non-mobile filaments. The detection process takes about 5 min and requires little skill or judgement. This can lead to great increases in the rate of data analysis in motility work.