Huygens Crosstalk Corrector

Estimate and correct crosstalk for reliable analysis

Crosstalk (a.k.a. optical crosstalk, bleed-through, spectral unmixing or crossover) is a phenomenon in multi-channel microscopy where signal from one dye is collected as signal from another dye, i.e., signal seems to come from one dye when it really comes from another. This poses a problem when performing multi-channel analyses like colocalization or object-based analysis. Huygens CrossTalk Corrector can quickly estimate and correct crosstalk between up to 32 channels simultaneously, and is therefore also ideally suited for images acquired for 'Spatial Biology' studies. Since nearly any multichannel datasets also suffers from chromatic aberration, you may want to have a look at the Chromatic Aberration Corrector as well.

Image description
Mitochondrial (TMRM) red signal is also detected as signal in the green Lipid Droplet (BODIPY) channel due to crosstalk. This crosstalk will have a serious impact on the analysis of lipid droplet structure. Both channels are shown in the upper part; only the green channel is shown in the lower part. The spinning disk image was corrected with Huygens Crosstalk Corrector (right). Courtesy of Kevin Knoops (Microscopy CORE Lab), Sabine Daemen and Matthijs Hesselink, Maastricht University, The Netherlands.

Crosstalk corrected

Automatic and Easy

Quickly assess if your image suffers from crosstalk and correct with just two button clicks.

Accurate correction

2D histograms show the estimated and corrected crosstalk with a MIP of the corrected image.

Improved analysis

Multi-channel analysis, like colocalization or object-based analysis, is more reliable after crosstalk correction.

In the main window of the Crosstalk Corrector, the signal from two channels is shown in a 2D histogram (see figure). In this histogram, points close to the axes represent pixels or voxels containing signal from only one of the channels. In the case of crosstalk, signal from one channel (one dye) is always paired with signal from the other, wrongfully recorded dye. As a result, all points are placed some distance from the first channel's axis. The Crosstalk Corrector uses this distance to automatically estimate the crosstalk factor. This is done for all possible channel pairs in the image. If desired, the estimates can be improved manually; a preview of the corrected image is shown to help with this. The Corrector then uses the crosstalk factors to correct the image.

Multi-channel images often suffer from some form of chromatic aberration, which should be corrected before performing analyses. Huygens offers a wide range of analysis options, for example the assessment of the colocalization between different molecules.

Chromatic Aberration Corrector Colocalization Analyzer

All show Crosstalk
All Crosstalk corrected
This two channel multiphoton image shows a live mouse brain (anesthetized animal), expressing GFP in microglia cells and CFP in astrocytes. Because the emission spectra overlap and NDD detectors were used, there is a very serious problem with crosstalk/bleedthrough falsely suggesting there are double stained cells (see arrowheads). Huygens Crosstalk Corrector succesfully corrects the bleedthrough. Image kindly provided by Dr. Dimitra Thomaidou group, Neurobiology department and Light Microscopy Unit, Hellenic Pasteur Institute, Greece.

See more: Images in the field of NeuroBiology