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%). As a result, some signal is actually recorded as coming from certain dye when it really comes from a different one.


The animation on the left shows red signal entering the green detector in a two channel image. The sample is composed of two non-overlapping objects dyed red and green. The higher the crosstalk factor, the more yellow 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 to be zero in this simplified case.

To avoid crosstalk, microscopes usually excite each dye alternatively, making sure that all the detected radiation comes from a single dye type. However, some experiments (e.g. Fluorescence Resonance Energy Transfer - FRET) require simultaneous acquisition of signal from all the present dyes, with the possible risk of crosstalk. Crosstalk in Multi Channel images dramatically affects nearly all types of data analysis, for example colocalization analysis. It should therefore be corrected through image restoration.

To improve microscopy image quality and thereby allow for reliable image analysis, Huygens offers a Crosstalk Corrector tool to correct for this imaging artifact.

The main window of the Huygens Crosstalk Corrector before (left) and after (right) estimating the crosstalk.