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Crosstalk or bleedthrough

In Fluorescence Microscopes, crosstalk (also known as crossover or bleedthrough) can occur when acquiring a Multi Channel image. In that case, the emission radiation of a given Emission Wavelength is detected by the wrong detector because part of the photons go through the wrong optical path inside the microscope (e.g. because the spectral profiles of the fluorophores overlap and filters cannot separate the channels a 100% efficient). Therefore some signal is actually recorded as coming from certain dye when it really comes from a different one. Autofluorescence can similarly interfere with signals collected from specific dyes, although it is caused by the natural emission of light by biological structures.

To avoid this situation microscopes usually excite each dye alternatively, making sure that all the detected radiation comes from a single dye type. But some experiments (like FluorescenceResonanceEnergyTransfer) require simultaneous acquisition of signal from all the present dyes, with possible risk of crosstalk. This can in principle be corrected during the Image Restoration if properly calibrated.

The following animation shows a varying crosstalk factor of the red signal entering the green detector in a Two Channel image. The sample is composed of two non-overlapping objects dyed in red and green. The higher the crosstalk factor, the more yellowish the red object looks, because its signal is recorded not only in the red channel but also in the green one. The crosstalk from the green to the red channel is always zero in this simplified case.

Image

Crosstalk will dramatically affect almost any data analysis in Multi Channel images, like CoLocalization.

Can crosstalk be removed computationally? Find some notes about the general case in Spectral Image Deconvolution.