A software solution for recording circadian oscillator features in time-lapse live cell microscopy.
A software solution for recording circadian oscillator features in time-lapse live cell microscopy.
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DOI：
10.1186/1747-1028-5-17
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发表时间：
2010-07-06
期刊：
影响因子：
2.3
通讯作者：
Dibner C
中科院分区：
文献类型：
--
作者：
Sage D;Unser M;Salmon P;Dibner C
荧光和生物发光延时显微镜方法已成功用于在单细胞水平研究哺乳动物昼夜节律振荡器的分子机制。然而，大多数基于强度阈值分割和帧到帧跟踪的可用软件和常用方法不适用于这些实验。这是由于昼夜节律周期中细胞运动和荧光/生物发光报告蛋白的巨大变化，最低表达水平非常接近背景强度。目前，分析从延时显微镜获得的数据集的标准方法是手动跟踪或应用通用图像处理软件/专用跟踪软件。据我们所知，这些现有的手动和自动跟踪软件解决方案在跟踪单个细胞（如果其平面发生移动）时具有很大的局限性。为了改进现有的大量移动细胞延时跟踪方法，我们开发了一种半自动软件包。它通过跟踪细胞的位移来提取细胞的轨迹，勾画出细胞核或整个细胞，并最终测量各种特征，如报告蛋白表达水平或细胞位移。作为一个例子，我们在这里展示表达荧光昼夜节律报告蛋白的 NIH3T3 小鼠成纤维细胞的单细胞昼夜节律模式和运动分析。使用 Circadian Gene Express 插件，我们对大型荧光延时显微镜数据集进行了快速且无偏见的分析。我们的软件解决方案 Circadian Gene Express (CGE) 易于使用，可以在较长时间内精确、半自动地跟踪运动细胞。尽管蛋白质表达存在显着的昼夜节律变化，谷期表达水平极低，但 CGE 可以准确有效地记录大量细胞参数，包括报告蛋白表达水平、速度、运动方向等。事实证明，CGE 对于分析宽场荧光显微镜数据集以及生物发光成像非常有用。此外，通过手动选择时间序列的各个时间点的每个 z 堆栈的焦平面之一，它可以轻松地适用于共焦图像分析。 CGE是ImageJ的Java插件；可在以下网址免费获取：http://bigwww.epfl.ch/sage/soft/circadian/。
Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series. CGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/.
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