I will try to be more specific. These are the pipelines that I am using:
IlluminationCorrection_Alexa.cpproj (701.7 KB)
IlluminationCorrection_DAPI.cpproj (701.7 KB)
CellCycleAnalysis_corretilu_3.cpproj (1.1 MB)
First and second are illumination corrections that I a applied in the third pipeline. I have cells stained with DAPI and Alexa 488. Here there is one example of an image that I am trying to analyze (I can not doanload in here the file because it says that the format is not authorized (.czi), but if you want I can send you by email) :
My objective is to measure just the Integrated Intensity of cells DAPI-positive, Alexa 488-negative. First of all I need to select these cells, and exclude the remaining ones. For this purpose I used the module Mask Image. The image to be masked is the one with the cells I want to keep, so I selected the image with the channel DAPI (Dapi_corr). Then I want to remove the cells stained with Alexa. My mask has to contain the cells I want to remove, so my objects for mask are the nucleis stained with Alexa (Nuclei_alexa), previously identified with the module IdentifyPrimaryObjects.
After this I thought I would obtain an image with cells DAPI-positive, Alexa 488-negative, so I measured the integrated intensity of the cells present in this image. After running the software, I obtained in a folder the image with the intensities, and I realized that the selected cells with the values of intensities are mainly DAPI-positive, Alexa 488-positive and not DAPI-positive, Alexa 488-negative:
DAPIpos_ALEXneg0001.zip (715.7 KB)
Following is an example identified with a white arrow.
I know that the software selected these cells to measure integrated intensity because i used the module DisplayDataOnImage, and these cells have the value of the Integrated Intensity:
If there is something you can’t understand, please tell me. Thank you for your help!