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Scientific Volume Imaging B.V.

Scientific Volume Imaging

Deconvolution - Visualization - Analysis


™Huygens Crosstalk & Autofluorescence Corrector

Easy to use Wizard with automated estimation and correction

Crosstalk (also known as bleed-through) 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. Autofluorescence, the natural emission of light by biological structures, can similarly interfere with signals collected from specific dyes. Crosstalk presents a significant obstacle in single-channel analyses and multi-channel techniques such as colocalization or object-based analysis, where channel distinction is crucial. The new Huygens CrossTalk & Autofluorescence Corrector Wizard effectively estimates and corrects crosstalk between up to 32 channels at once, making it particulary useful for 'Spatial Biology' imaging studies. Since many multichannel datasets also suffer from chromatic aberration, you may want to have a look at the Chromatic Aberration Corrector to ensure optimal channel alignment.

Image description

Mitochondrial (TMRM) red signal is also detected in the green Lipid Droplet (BODIPY) channel due to crosstalk. Upper part: both channels; Lower part: only the green channel. The spinning disk image is shown before and after correction with Huygens Crosstalk Corrector. Courtesy: Kevin Knoops (Microscopy CORE Lab), Sabine Daemen and Matthijs Hesselink, Maastricht University, The Netherlands.

Before
Crosstalk corrected

Automatic and Easy

Quickly assesses if your image suffers from crosstalk and autofluorescence via a step by step process.

Accurate correction

Compare 2D histograms and MIPs of the original and corrected images.

More accurate analysis

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

Crosstalk Corrector

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.

Autofluorescence Corrector

When choosing autofluorescence correction in the Crosstalk & Autofluorescence Corrector Wizard, you'll be asked to provide a reference image of the autofluorescence of the sample. The wizard will then estimate the autofluorescence within every channel and subtract it from the other channel. Any additional crosstalk between the used dyes, will also be corrected by this same wizard.

Autofluorescence channelAutofluorescence Channel Crop2
Comparison
Autofluorescence Corrected
Before Corrected
Left: Only the autofluorescence channel; Right: Overlay of 6 different channels before and after correction with Huygens. This 7 channel widefield image of FFPE tissue was first stitched, then the high level of autofluorescence and crosstalk in six channels was corrected with the Crosstalk & Autofluorescence Corrector, and finally the image was deconvolved.

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

More information

Crosstalk Support page

Crosstalk Corrector webinar
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