I am using CellProfiler to characterize bacteria containing vacuoles in infected epithelial cells. I have GFP-tagged bacteria and I am using a Cy5-tagged antibody (Lamp1) to identify the bacteria containing vacuoles. In addition to that I have Hoechst to label host cell nuclei and I am using a Cy3-tagged celltracker dye to label my epithelial cells.
I have set up a pipeline to identify (plenty of) objects in my experiment and I can clearly identify my Cy5-labeled bacteria-containing vacuoles as well as GFP-labeled bacteria.
I’m exporting all data in an SQL lite database so that I can then use the Classifier in Analyst to rank “normal” versus “small” or “large” vacuoles etc…so far so good.
Now, what I would like to do is to calculate the GFP intensity WITHIN my Cy5-labeled identified objects as a way to determine “how many” bacteria I have in each vacuole. I’d like to have this measure included in the characteristics of my bacteria-containing vacuoles so that it would be a parameter in my “vacuoles database” and Analyst can use it to further classify the vacuoles.
Any idea on how to do so? Is there a way to tweak the MeasureObjectIntensity module so that I can calculate the GFP intensity of previously identified Cy5 objects?
Or alternatively (it may be easier…) to use two databases (the one containing the infos on my vacuoles and the one containing the infos on my bacteria) in Analyst Classifier?
If it can help I am attaching my pipeline
Thanks in advance
siRNA Screen Higher Threshold Colonies.cpproj (1.5 MB)