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How to quantitatively measure, assess and correct the fluorescence crosstalk in the wide-field microscopy

Tie, H. C.; Lu, L.

2020-05-23 biophysics
10.1101/2020.05.20.105627 bioRxiv
Show abstract

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.

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