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What is fluorescence crosstalk?

Posted November 1, 2019


Sometimes referred to as ‘crossover’, this common microscopy problem refers to overlapping excitation and emission wavelengths of two or more fluorescent dyes, which muddy the signal and interfere with accurate measurement of experimental results. Overlapping spectra can give false negatives or positives, or otherwise obscure data.

To prevent this issue for multiparameter visualizations, dyes with good separation between their excitation and emission spectra should be chosen. Some dyes have wider spectra bands than others, so the researcher must take this into account. If one or more dyes must be used that will potentially overlap, choosing multiple controls (negative controls, single-dye, and others) will help compensate for the issue during data analysis. Another method of compensation is to use more narrow bandpass filters, which will help sanitize the signals, but at the cost of lower overall signal levels.

Careful dye selection and instrumentation, along with the use of experimental controls, will minimize the presence of fluorescence crosstalk.

AAT Bioquest's Interactive Spectrum Viewer allows easy visual comparison of the presence and degree of spectral overlap between hundreds of commonly selected fluorophores.

Additional resources

Assaywise Article: Optimized Fluorescence Labeling Solutions

iFluor™ Dye Selection Guide

Multiplexing Cell Proliferation and Cytotoxicity Assays Using Calcein Red and CytoCalceins

Fluorescence Imaging Assay Development Services