Hi - I am trying to use CP to measure the fraction of fluorescence from an RFP labelled protein that is in the plasma membrane (PM) of yeast cells. In my experiments the protein relocalizes from the PM to the cytosol and vice-versa depending on various treatments. The cells also expressed a cytosolic GFP. I want to use the GFP signal (which is always cytosolic) to create an outline, then use this outline to measure the fraction of RFP fluorescence in the PM.
Following Mark Bray advice I made the pipeline attached. It uses the following modules:
LoadImages: Load the RFP and GFP images
IdentifyPrimaryObjects: Identify the cells from the GFP image.
MeasureRadialDistribution: This module divides the object into a set of radial bins (sort of like concentric circles from the cell center to the cell perimeter), and then measures the intensity within each bin. One of the measurements made is the fraction of the total stain (RFP) in a given bin, for each bin (FracAtD)
ExportToSpreadsheet: Export the cell measurements to a csv.
I have also added an “ExpandObject” command, but this is not important now.
I attached images of the outlines I get, superimposed on the RFP and GFP images. As you can see in the RFP overlay, the outlines is often well aligned with the fluorescence ring in the PM, but in too many cells it is not. In those same cells it is also not tracking very well the border of the GFP fluorescence.
It would seem that the outline could be much better than this, based on the nice contrast of the GFP images. I thought maybe changing parameters could get what I want. Maybe the choice of thresholding method and its parameters is key. There are so many there and I don’t know which should work best for this. Any help would be appreciated.
I used Otsu Global-Two clasess-Weigthed variance with Threshold CF 1, 0 and 1 boundaries to generate these images.
membrane-pipeline.cp (4.47 KB)