Crosstalk or bleedthrough

In Fluorescence Microscopes, crosstalk (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 filters efficiency is not 100%). Therefore, some signal is actually recorded as coming from certain dye when it really comes from a different one.

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 e.g. Fluorescence Resonance Energy Transfer - FRET) require simultaneous acquisition of signal from all the present dyes, with the 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 considered being zero in this simplified case.

Crosstalk within Multi Channel images dramatically affects almost any type of data analysis, such as for example CoLocalization.

In line with our aim to improve microscopic image quality and measurements, we have implemented a Crosstalk Corrector tool in the Huygens Software to correct for this imaging artifact.

The Huygens Crosstalk corrector.
The Huygens Crosstalk corrector.