Multi-colour localization microscopy has enabled sub-diffraction studies of colocalization between multiple biological species and quantification of their correlation at length scales previously inaccessible with conventional fluorescence microscopy. However, bleed-through, or misidentification of probe species, creates false colocalization and artificially increases certain types of correlation between two imaged species, affecting the reliability of information provided by colocalization and quantified correlation. Despite the potential risk of these artefacts of bleed-through, neither the effect of bleed-through on correlation nor methods of its correction in correlation analyses has been systematically studied at typical rates of bleed-through reported to affect multi-colour imaging. Here, we present a reliable method of bleed-through correction applicable to image rendering and correlation analysis of multi-colour localization microscopy. Application of our bleed-through correction shows our method accurately corrects the artificial increase in both types of correlations studied (Pearson coefficient and pair correlation), at all rates of bleed-through tested, in all types of correlations examined. In particular, anti-correlation could not be quantified without our bleed-through correction, even at rates of bleed-through as low as 2%. Demonstrated with dichroic-based multi-colour FPALM here, our presented method of bleed-through correction can be applied to all types of localization microscopy (PALM, STORM, dSTORM, GSDIM, etc.), including both simultaneous and sequential multi-colour modalities, provided the rate of bleed-through can be reliably determined.

中文翻译：

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多色定位显微镜中渲染和相关分析的渗色校正

多色定位显微镜已经实现了多个生物物种之间共定位的亚衍射研究，并在以前无法用传统荧光显微镜进行的长度尺度上量化它们的相关性。然而，探针物种的渗漏或错误识别会产生错误的共定位并人为地增加两个成像物种之间的某些类型的相关性，影响共定位和量化相关性提供的信息的可靠性。尽管这些渗出的伪影存在潜在风险，但在相关分析中渗出对相关性的影响及其校正方法均未以据报道影响多色成像的典型渗出率进行系统研究。这里，我们提出了一种可靠的渗色校正方法，适用于多色定位显微镜的图像渲染和相关分析。我们的渗透校正的应用表明，我们的方法准确地校正了所研究的两种类型的相关性（Pearson 系数和对相关）的人为增加，在所有测试的渗透率下，在所有类型的相关性中。特别是，如果没有我们的渗漏校正，即使渗漏率低至 2%，也无法量化反相关性。此处展示了基于二色性的多色 FPALM，我们提出的渗色校正方法可应用于所有类型的定位显微镜（PALM、STORM、dSTORM、GSDIM 等），包括同时和顺序多色模式,