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How to quantitatively measure, assess and correct the fluorescence crosstalk in the wide-field microscopy
bioRxiv - Biophysics Pub Date : 2020-05-23 , DOI: 10.1101/2020.05.20.105627
Hieng Chiong Tie , Lei Lu

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。
更新日期:2020-05-23
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