I apologize for the length of this post as I try to understand just how the MeasureRadialDistribution module works and I appreciate any help that can be provided! I am a research technician trying to determine whether a stain for EGFR spreads out farther throughout the interior of cells before and after treatment. I believe it aggregates more without treatment (thus less dispersion) and spreads out more after treatment and would like to quantitatively demonstrate that.
FracAtD value is the sum of the intensity values for ONE circular bin divided by the sum of the intensity values for ALL the bins. In addition, the MeanPixelFraction is the average intensity per pixel within a given circular bin calculated by dividing the FracAtD value by the fraction of total object pixels within the bin. Is my understanding correct?
For the MeanPixelFrac, if the above is true, are the “object pixels” used for the calculation simply the pixels within the image/cells with a non-zero intensity? Or are these object pixels (aka EGFR puncta) supposed to have been identified using the IdentifyPrimaryObjects module and then analyzed with MeasureRadDist? I believe its the former but I am unsure.
Is it possible to get the fraction of total number pixels (not total intensity) for each bin by dividing FracAtD by MeanPixelFrac? What would I have to do to get the actual pixel number? I am not sure if you could answer this, but would there really be an advantage or disadvantage measuring the fraction of total stain pixels per bin versus the fraction of the total intensity per bin?
Lastly, how is the distance of the largest bin determined? Is it simply the point on the edge farthest from the indicated center?
If it helps, this is a brief explanation of what I’m doing. I load three images; a mask for the cells, a mask for the centers of mass of the signal within each cell, and the original image itself with my stain of interest. I run the IdentifyPrimaryObjects for the cell mask and the center of mass. I then apply a threshold to the image with the stain to remove any background. For MeasureRadDist, I set the mask for the center of mass as my center (NewCenters.tif) and the object to be analyzed as the cell mask (Cells.tif). The image loaded is the thresholded image with my stain (B.ome.tif). I assume that the module analyzes the intensity values for the thresholded image within the perimeter of the cell mask.
I have attached the pipeline, and the relevant images. I ran this pipeline already and would upload the data but the site will not let me
I hope I was able to explain it well enough and I appreciate any help you could offer!
Image with stain
Centers of Mass
Image with Stain
Centers of Mass
Pipeline: Dispersion Analysis Pipeline.cp (16.6 KB)