Columbus image analysis pipeline

Building block	Specification/details
Input Image	Selected input image is displayed.
Find Nuclei	Nuclei are detected based on Hoechst signal. Output population: I
Calculate Intensity Properties	Intensities of nuclei are calculated.
Calculate Morphology Properties	Area and roundness of nuclei are calculated.
Select Population	Nuclei at the border of the image are removed. Output population: II
Select Population (2)	Additional nuclei are excluded based on individual area, roundness, and intensity parameters. Output population: III
Find Cytoplasm	Cell cytoplasms are defined based on previously selected nuclei (Output population: III).
Calculate Intensity Properties (2)	ΔΨm intensity is calculated based on TMRM channel within the previously defined cell regions.
Define Results	Output results are defined. “Population III” is used to select “Number of Objects” (nuclei) and the mean “ΔΨm intensity” in the cell as results.
Sequence Comment	Comment section for the script.