Shading Compensation Filters

Undesirable shading across the field-of-view, which can be a prominent phenomenon in microscopy and may occur due to non-uniform illumination, inhomogeneous detector sensitivity, or non-specific sample staining, can be largely eliminated with the shading compensation filters. Eliminating shading is frequently necessary for subsequent processing, especially when quantification is the final goal.

A good indication that a dataset requires shading correction can be provided by the intensity profile of rulers drawn across the image. Eliminating shading may be required if linear regression, as shown below, is apparent (see Profiling Intensity).

Intensity profile before (in red) and after shading correction (in blue)

Typically used to correct problems created by non-uniform illumination, this filter uses a radial basis function to correct image shading. You should note that the Manual RBF filter requires at least three(3) seed points to be placed in representative regions on an image slice and that corrections are applied in the X and Y axes only. If corrections are required in another direction, you will need to transform the data.

The Manual RBF filter is only available in Dragonfly Pro. Contact Object Research Systems for information about the availability of Dragonfly Pro.

Do the following to add seed points:

  • Scroll to a representative image slice in the window with the original dataset and then adjust the zoom factor, pan position, and window level, if required.
  • Click the Points tool, if it is not already selected.
  • Click on the image to add seed points. Add as many points as required to representative regions on the selected image slice.

  • Click the Compute Selected Preview button to view the effect of filtering on the selected image slice.
  • Reposition, add, or remove seed points, as required, and evaluate the computed preview.

    Click the Go to Ref button to return to the reference slice if you navigated from the slice to which you added your seed points.

Do the following to edit seed points:

  • Select a seed point and then drag to reposition it.
  • Select a seed point and then click Delete Selected to remove it.
  • Click the Delete All button to remove all seed points.

Default configured actions for the RBF filter
Action State Key Mouse

Create points for RBF filter

OrsHandlePointForRBFFilter

 

Left mouse

Edit points for RBF filter

OrsHandlePointForFilterEdit

 

Left mouse

This filter uses a third degree polynomial to correct uneven shading.