Correct for bleaching and illumination instability in the Z and Time dimension
The Bleaching Corrector for fluorescence microscopy is available in Huygens Essential and Huygens Professional since version 17.04 and is included there free of charge.
The Bleaching Corrector allows you to correct for the fading of the emission intensity of the sample under study. Depending on the image dimensions and microscope type, the emission intensity can fade with respect to the Z and/or T dimension of the image.
The Bleaching Corrector can correct for bleaching and illumination instability in the Z and T direction. A constant emission intensity throughout the image is important for image restoration because of the following reasons:
- Large differences in intensity between the first and last plane/frame of the image can result in wrap-around effects. Because the discrete Fourier transform in the deconvolution algorithm requires the signal of the image to be periodic, the intensity values at the boundaries of the image should be the same. If this is not the case, the deconvolved image will acquire incorrect intensity values at the edges called wrap-around effects.
- Illumination instability, i.e. lamp jitter, can give rise to artifacts in the restoration result. The Bleaching Corrector can be used to correct this.
However, detection of bleaching effects is not always possible. Bleaching detection is possible in fluorescence microscopy images that are either Widefield 3D or contain at least two time frames. Here is a summary of the types of images that are supported by Huygens and in which bleaching effects can be corrected:
- Widefield 3D
- Widefield time series
- Confocal time series
- STED time series
- Spinning Disk time series
WindowThe Bleaching Corrector window can be opened by selecting an image and going to Deconvolution > Bleaching Corrector.
How to use itAn image can be corrected for bleaching through the following procedure:
- Choose a dimension (Z, T or Z&T) in which you want to correct for bleaching
- Check the box 'Include lamp jitters' if you want to correct for illumination instability
- Choose the plane and frame range in which you want to correct for bleaching
- Calculate the corrected intensity and corresponding bleaching factors by pressing 'Estimate'
- Adjust the bleaching factors
- Correct the image according to the bleaching factors by pressing 'Correct'
Important functionalitiesImage Slicer
A slice of the image is shown in the upper left corner of the window. More information regarding the scene and view options can be (un)selected under the View menu in the taskbar.
The bottom of the “Bleaching Corrector” window contains widgets coupled to the rendered image. They are divided into four tabs:
- Time. Change the displayed time frame.
- Channels & Colors. Changes the active channels and color scheme of the image and data plot.
- Slicer. Change the displayed XY-plane.
- Contrast. Adjust the brightness, contrast and compression of the image.
Both the original (straight) and corrected (dashed) intensities are plotted in the upper right corner of the window. The plots are divided into three tabs which makes it possible to toggle between intensity curves in the Z, T and Z&T direction, depending on the estimation that is made.
In the middle of the window it is possible to adjust the bleaching factors after the estimation is made. A bleaching factor can be selected by clicking on a cell and can be altered either with keyboard or the slider above the table. By double clicking on one of the channel row titles a whole row of bleaching factors can be selected and subsequently altered with the slider. The bullet button left of the slider resets the selected cells to their initial estimated bleaching factors.
Close and Load
It is possible to open the corrected images with either the Twin Slicer or the Deconvolution Wizard.