Fluorescence lifetime imaging microscopy (FLIM) is used in diverse disciplines, including biology, chemistry and biophysics, but its use has been limited by the complexity of the data analysis. The phasor approach to FLIM has the potential to markedly reduce this complexity and at the same time provide a powerful visualization of the data content. Phasor plots for fluorescence lifetime analysis were originally developed as a graphical representation of excited-state fluorescence lifetimes for in vitro systems. The method's simple mathematics and specific rules avoid errors and confusion common in the study of complex and heterogeneous fluorescence. In the case of FLIM, the phasor approach has become a powerful method for simple and fit-free analyses of the information contained in the many thousands of pixels constituting an image. At present, the phasor plot is used not only for FLIM, but also for hyperspectral imaging, wherein phasors provide an unprecedented understanding of heterogeneous fluorescence. Undoubtedly, phasor plots will be increasingly important in the future analysis and understanding of FLIM and hyperspectral confocal imaging. This protocol presents the principle of the method and guides users through one of the popular interfaces for FLIM phasor analysis, namely, the SimFCS software. Implementation of the analysis takes only minutes to complete for a dataset containing hundreds of files.

中文翻译：

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使用相量方法对荧光寿命成像数据进行无拟合分析。

荧光寿命成像显微镜 (FLIM) 用于多种学科，包括生物学、化学和生物物理学，但其使用受到数据分析复杂性的限制。FLIM 的相量方法有可能显着降低这种复杂性，同时提供强大的数据内容可视化。用于荧光寿命分析的相量图最初是作为体外系统激发态荧光寿命的图形表示而开发的。该方法简单的数学原理和具体的规则避免了复杂和异质荧光研究中常见的错误和混乱。在 FLIM 的情况下，相量方法已成为一种强大的方法，可以对构成图像的数千个像素中包含的信息进行简单且无拟合的分析。现在，相量图不仅用于 FLIM，还用于高光谱成像，其中相量提供了对异质荧光的前所未有的理解。毫无疑问，相量图在未来对 FLIM 和高光谱共聚焦成像的分析和理解中将变得越来越重要。该协议介绍了该方法的原理，并通过一种流行的 FLIM 相量分析界面（即 SimFCS 软件）引导用户。对于包含数百个文件的数据集，分析的实施只需几分钟即可完成。相量图在未来对 FLIM 和高光谱共聚焦成像的分析和理解中将变得越来越重要。该协议介绍了该方法的原理，并通过一种流行的 FLIM 相量分析界面（即 SimFCS 软件）引导用户。对于包含数百个文件的数据集，分析的实施只需几分钟即可完成。相量图在未来对 FLIM 和高光谱共聚焦成像的分析和理解中将变得越来越重要。该协议介绍了该方法的原理，并通过一种流行的 FLIM 相量分析界面（即 SimFCS 软件）引导用户。对于包含数百个文件的数据集，分析的实施只需几分钟即可完成。