I have been trying to implement the ‘CorrectIllumination_Calculate’ module in my pipeline. So far I have been unable to produce a satisfactory illumination correction function, and am trying to figure out why. I have a few questions about this module which I would greatly appreciate answers to (I have read the manual and the scientific articles also).
The following questions concern the various options for CorrectIllumination_Calculate:
For REGULAR INTENSITY: If the incoming images are binary …
Am I correct to assume that this setting is used only as a specialized method for binary images? That is, for my grayscale images, I would not use this setting?
For MEDIAN FILTER or GAUSSIAN FILTER …
If I am using the ‘Regular’ option, then I imagine that I need to smooth out the actual cell structures in the image. Therefore should I enter the approximate cell width in pixels here?
If you want override the above width of artifacts …
I am curious how this setting differs from the one above.
Some general questions:
In the 2008 article by Vokes and Carpenter (Current Protocols in Molecular Biology), there is a description of the CorrectIllumination_Calculate module on page 12. Reference is made here to the ‘smoothing size’. Would this correspond to the median/Gaussian filter setting? Also it is stated that the ‘smoothing size’ is set automatically and displayed in the figure window. I would like to know which window (and where) the smoothing size can be found.
I have experimented on a single image with about 100 cells. Is it feasible to create a workable smoothing function using the ‘Background’ method for a single image? (Segmentation using ‘IdentifySecondary’ indicates that 34.1% of the image consists of object). When I try to use this method, the illumination correction function resembles a blurred version of the original image of cells for some reason. Would it be feasible to create a workable smoothing function on this image using the ‘Regular’ option and appropriate smoothing (so far I have been unable to produce a sufficiently smooth correction function)? … any pointers would be welcome.
In the CorrectIllumination_Calculate Display window, a ‘Min Value’ and ‘Max Value’ is listed. I would like to know if possible what these values correspond to.
Thanks very much,