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Scientific Volume Imaging B.V.

Scientific Volume Imaging

Deconvolution - Visualization - Analysis


Huygens™ Bleaching Corrector

Correct for bleaching and illumination instability in the Z and Time dimension

Bleaching Optimized
Image description:

Three-channel SoRa spinning disk image corrected for bleaching over 12,840 time frames with Huygens' Bleaching Corrector. Both the corrected and uncorrected views are shown using the exact same LUT and contrast. Courtesy of Dr. Kanie Tomoharu and Beibei Liu, Department of Cell Biology, College of Medicine, University of Oklahoma.

The Bleaching Corrector for fluorescence microscopy has been available in Huygens Essential and Professional since version 17.04, free of charge.

It allows you to correct for fading emission intensities of your samples. Depending on the microscope type and parameters, emission intensity can diminish over Z and/or time. Correct for bleaching and illumination instability in both the Z and T direction. A constant emission intensity throughout the image is crucial for image restoration to:

  • Gain accurate deconvolution results. The best algorithms require the signal of the image to be periodic, so the intensity values at the boundaries of the image should be the same. This includes 3D stacks and time series! If this is not the case, the deconvolved image will contain incorrect intensity values at the edges, which are called wrap-around effects.
  • Reduce artifacts and increase analysis reliability. Illumination instability, i.e. lamp jitter, can give rise to artifacts in intensity based analyses, for instance in Förster Resonance Energy Transfer (FRET).

Correcting Z-based bleaching is only possible in widefield images, as the pinhole in confocal systems blocks out background light and thus necessary information. Fortunately, bleaching over timeframes is easily corrected by the Huygens bleaching corrector for all systems!

Window

Open the window by selecting an image and going to Deconvolution & Restoration > Bleaching. Here you can select which dimensions to correct in, see the amount of bleaching and suggested correction. For fine-grained control and reproducible processing, there is an included table with correction amounts.

Tool window. The slicer views show an XY MIP projection, with standard slicer controls to inspect the image. The average intensities can be seen in the graph for each channel, along with the corrected channels.
Tool window. The slicer views show an XY MIP projection, with standard slicer controls to inspect the image. The average intensities can be seen in the graph for each channel, along with the corrected channels.



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