My aim is to quantify the intensities of the protein stained in the red channel. The images are of cells stained with Hoescht (blue channel-D) to label nuclei and for a bone marker (red channel-R). The images are part of a high-throughout system whereby cells are grown on ‘microspots’ of various chemicals, and unfortunately some of these spots are auto-fluorescent, causing uneven background illumination that changes from one spot to the next.
1- I understand that I need CorrectIlluminationCalculate and CorrectIlluminationApply modules to get accurate quantification of the staining. Are there certain things to avoid when setting up these modules, i.e.: settings that would affect fluorescence quantification? E.g.: Does the re-scaling that automatically happens with these modules affect subsequent quantification?
2- I have been told that illumination correction modules should be done in a separate pipeline and then the corrected images loaded into the new pipeline for segmentation and measurement? Is this true? How would that be advantageous?
3- I plan to use “integrated” intensity for quantification of protein expression per cell (i.e fluorescence), then divide that by cell area to avoid any introduction of bias based on cell size. Is there a better/easier way of doing this, e.g.: median intensity?
OCN-Finalpipeline-MAmer.cpproj (1.3 MB)