During the multi-color fluorescence imaging, a fluorophore can be detected in the non-corresponding channel due to the broad spectra of the fluorophore and filters, resulting in the artefact called the crosstalk (crossover or bleed-through). Unfortunately, the fluorescence crosstalk is ubiquitous for the conventional fluorescence microscopy. Significant crosstalk can distort the image and lead to incorrect interpretation. Here, we introduce a simple quantitative metric, the crosstalk factor, to measure the crosstalk effect of a fluorophore on a channel in the wide-field fluorescence microscopy. We describe a cell biologist friendly protocol which should be easily implemented by cell biologists using commonly available image processing software such as ImageJ.

中文翻译：

---

如何在宽视场显微镜中定量测量，评估和校正荧光串扰

在多色荧光成像过程中，由于荧光团和滤光片的光谱较广，可以在非对应的通道中检测到荧光团，从而导致了所谓的串扰（交叉或渗漏）。不幸的是，荧光串扰在常规荧光显微镜检查中无处不在。严重的串扰会使图像失真并导致错误的解释。在这里，我们介绍一种简单的量化指标，即串扰因子，以在宽视野荧光显微镜下测量荧光团在通道上的串扰效应。我们描述了一种细胞生物学家友好的协议，应该由细胞生物学家使用常见的图像处理软件（例如ImageJ）轻松实现。