I am looking to accurately measure the intensity of cell borders, on a per-cell basis, of human endothelial cells that will either exist in pseudo monolayers or in isolation depending on the treatment. My strategy thus far has been to:
- Identify primary objects (nuclei) by DAPI staining (IdentifyPrimaryObjects module)
- Identify secondary objects (cells) by HCS CellMask staining (IdentifySecondaryObjects module)
- Expand the “cell” objects by a specified number of pixels (ExpandOrShrinkObjects module)
- Identify tertiary objects (putative cell borders) by selecting the larger identified objects as “expanded cells” and the smaller identified objects as “cells” (IdentifyTertiaryObjects module)
- Measure the intensity of the “cell borders” (MeasureObjectIntensity module)
Although this strategy is somewhat effective, I have realized that it is limited by several factors including:
The cell-cell variability in how well the morphology of the identified cell objects matches the morphology of the actual cell borders.
The cell-cell variability in how close the identified cell objects are to the actual cell borders.
The cell-cell variability in how thick the actual cell borders are.
To circumvent these issues, I was thinking that line scans of a user-defined length could be created perpendicular to the identified cell object’s edge in a radial manner in order to measure the integrated intensity of the cell borders on a per-cell basis (see cartoon below).
Does anyone know if these sorts of analyses or anything similar would be possible to do in CellProfiler? Any thoughts or suggestions would be greatly appreciated!