I have a taken series of photos, on a stereoscope, from plates with fluorescent E. coli. The colonies formed by E. coli vary markedly in size and often I can visually identify up to 3 different sizes. I have been trying to use cellprofiler to automatise this process and to get size distributions. I have attached the pipeline I have been using (Ramiro_09102013_Ecoli.cp), which is derived from the ExampleYeastColonies_BT.cp and have also attached the input files (test_input) and the OutlinedColonies image.
The main problem that I am encountering is that the program seems to be oversplitting the colonies. I can reduce oversplitting by changing two settings on the module IdentifyPrimaryObjects to: 1) “Method to distinguish clumped objects” to Shape and 2) “Method to draw dividing lines between clumped objects” to Intensity. However, while this reduces oversplitting, I still get it for some of the colonies. Would you know how I can reduce/eleminate overspliting?
Moreover, I have a few other questions:
- some of my colonies are at the edges of the image - is there a way to eliminate these from the analysis?
- is it possible to measure the intensity of white? (as this is related to how fluorescent my bacteria are)
thanks for any help,
cellprofiler.zip (306 KB)