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This plugin performs attenuation correction.

Philippe Andrey, Eric Biot, Souad Blila Modeling and Digital Imaging Group Institut Jean-Pierre Bourgin, INRA Versailles, France.

Contact: philippe dot andrey at versailles dot inra dot fr.

It is frequently observed that fluorescence intensity decreases with depth in confocal microscopy image stacks. This plugin applies an attenuation correction to rectify the fluorescence levels throughout the stack. The implemented algorithm relies on the assumption that the background is stationnary in the absence of attenuation. The correction thus consists in applying a linear transformation of grey levels to each slice image, in order to make the average and standard-deviation of the background constant (equal to that of a reference slice) throughout the image stack.

The background is estimated by applying a grey level morphological opening to each slice. It is thus assumed that the fluorescent objects are small compared to image size. The radius of the opening should be chosen according to the object size (diameter, thickness).

Further details on the method can be found in Biot et al. (2008).

The first image displays a montage of an image stack with a strong attenuation of fluorescence intensity. The second image displays the same stack after correction with this plugin (the first slice was selected as reference).

Input	Output

The plugin accepts 8, 16 and 32 bits grey level image stacks.

The plugin lets the user select the following two parameters:

• Opening radius: radius of the morphological opening used to estimate background images.
  Hint: The radius should be as large as the radius (or thickness) of the smallest objects.
• Reference slice: index of the slice selected as reference (this slice will not be modified).
  Hint: The reference slice will generally be the first (upper-most) slice containing objects.

Preprocessing: to prevent noise (when present) from being enhanced by the correction, it is recommended to apply a noise reduction filter before running the plugin.

Download the attenuation_correction.class.zip file and extract the Attenuation_Correction.class file it contains into your ImageJ plugin directory.
The plugin will appear as Attenuation Correction in the Plugins menu.

When reporting or publishing results obtained using this plugin, please refer to:

Biot E, Crowell E, Höfte H, Maurin Y, Vernhettes S & Andrey P (2008). A new filter for spot extraction in N-dimensional biological imaging. In Fifth IEEE International Symposium on Biomedical Imaging (ISBI'08): From Nano to Macro, pp. 975-978.

2012.10.10: first release.

2012.12.12: fixed a bug that caused saturation at 255 in 16 and 32 bits images.

2014.07.17: the spatial calibration of the original image is preserved in the corrected image.