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首页> 外文期刊>Biophysical Journal >CellSpecks: A Software for Automated Detection and Analysis of Calcium Channels in Live Cells
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CellSpecks: A Software for Automated Detection and Analysis of Calcium Channels in Live Cells

机译：CellSpecks：一种用于自动检测和实时钙通道分析的软件

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摘要

To couple the fidelity of patch-clamp recording with a more high-throughput screening capability, we pioneered a, to our knowledge, novel approach to single-channel recording that we named "optical patch clamp." By using highly sensitive fluorescent Ca2+ indicator dyes in conjunction with total internal fluorescence microscopy techniques, we monitor Ca2+ flux through individual Ca2+-permeable channels. This approach provides information about channel gating analogous to patch-clamp recording at a time resolution of similar to 2 ms with the additional advantage of being massively parallel, providing simultaneous and independent recording from thousands of channels in the native environment. However, manual analysis of the data generated by this technique presents severe challenges because a video recording can include many thousands of frames. To overcome this bottleneck, we developed an image processing and analysis framework called CellSpecks capable of detecting and fully analyzing the kinetics of ion channels within a video sequence. By using randomly generated synthetic data, we tested the ability of CellSpecks to rapidly and efficiently detect and analyze the activity of thousands of ion channels, including openings for a few milliseconds. Here, we report the use of CellSpecks for the analysis of experimental data acquired by imaging muscle nicotinic acetylcholine receptors and the Alzheimer's disease-associated amyloid beta pores with multiconductance levels in the plasma membrane of Xenopus laevis oocytes. We show that CellSpecks can accurately and efficiently generate location maps and create raw and processed fluorescence time traces; histograms of mean open times, mean close times, open probabilities, durations, and maximal amplitudes; and a "channel chip" showing the activity of all channels as a function of time. Although we specifically illustrate the application of CellSpecks for analyzing data from Ca2+ channels, it can be easily customized to analyze other spatially and temporally localized signals.
机译：要通过更高吞吐量的筛选能力耦合Patch-Clamp录制的保真度，我们致力于我们的知识，我们所扮演的单通道录制的新方法，我们命名为“光学贴片夹”。通过使用高敏感的荧光Ca2 +指示剂染料结合全内部荧光显微镜技术，我们通过单独的CA2 +可-折磨通道监测CA2 +通量。该方法提供了有关相似于2ms的时间分辨率的频道门控的信息，其具有额外的优点是批量平行，从本机环境中的数千个通道提供同时和独立地记录。然而，对该技术产生的数据的手动分析提出了严重的挑战，因为视频记录可以包括数千个帧。为了克服这种瓶颈，我们开发了一种称为Cellspecks的图像处理和分析框架，能够检测和充分分析视频序列内离子通道的动力学。通过使用随机生成的合成数据，我们测试了Cellspecks快速有效地检测和分析了数千个离子通道的活动的能力，包括几毫秒的开口。在这里，我们报告使用细胞分析用于分析通过成像肌肉烟碱乙酰胆碱受体和阿尔茨海默病相关的淀粉样蛋白β孔，在Xenopus Laevis卵母细胞的质膜中具有多导水平的阿尔茨海默病相关的淀粉样蛋白β孔。我们表明Cellspecks可以准确且有效地生成位置图，并创建原始和加工的荧光时间迹线;平均开放时间的直方图，意味着关闭时间，开放的概率，持续时间和最大振幅;和“通道芯片”显示所有通道的活动作为时间的函数。尽管我们具体地示出了用于分析来自CA2 +信道的数据的Cellspeck的应用，但是可以容易地定制以分析其他空间和时间局部化信号。

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