文章基本信息

标题：PiQSARS: A pipeline for quantitative and statistical analyses of ratiometric fluorescent biosensors
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作者：Elise Lévy ; Florence Jaffrézic ; Denis Laloë 等
期刊名称：MethodsX
印刷版ISSN：2215-0161
电子版ISSN：2215-0161
出版年度：2020
卷号：7
页码：1-23
DOI：10.1016/j.mex.2020.101034
语种：English
出版社：Elsevier
摘要：AbstractGenetically encoded ratiometric fluorescent probes are cutting-edge tools in biology. They allow precise and dynamic measurement of various physiological parameters within cell compartments. Because data extraction and analysis are time consuming and may lead to inconsistencies between results, we describe here a standardized pipeline for•Semi-automated treatment of time-lapse fluorescence microscopy images.•Quantification of individual cell signal.•Statistical analysis of the data.First, a dedicated macro was developed using the FIJI software to reproducibly quantify the fluorescence ratio as a function of time. Raw data are then exported and analyzed using R and MATLAB softwares. Calculation and statistical analysis of selected graphic parameters are performed. In addition, a functional principal component analysis allows summarizing the dataset. Finally, a principal component analysis is performed to check consistency and final analysis is presented as a visual diagram. The method is adapted to any ratiometric fluorescent probe. As an example, the analysis of the cytoplasmic HyPer probe in response to an acute cell treatment with increasing amounts of hydrogen peroxide is shown. In conclusion, the pipeline allows to save time and analyze a larger amount of samples while reducing manual interventions and consequently increasing the robustness of the analysis.Graphical abstractDisplay Omitted
关键词：Ratiometric fluorescent probes;Automated image analysis;Biostatistics;Data analysis pipeline;Oxidative stress