I’m a first-time poster on these forums, and relatively new to the CellProfiler software. I use the developer’s version of CP for Matlab (with the image processing toolbox) on a computer running Vista.
I’ve been analyzing images of epithelial tissues, and I’ve had a few peculiar problems with some of CellProfiler’s analysis.
- Problem with Perimeters
In the MeasureObjectNeighbors module, I’m getting the output for “Percent Touching” (PT), and most of the time it seems to be accurate. Interior cells are generally identified correctly and are assigned a PT value of 100%. Border cells are also generally identified correctly, with a PT value of <100%.
Unfortunately, though, some interior cells are misidentified as having PT < 100%–often as low as 85%. I think that the problem is the way that some borders are developing “bumps” in the interior of the cells. Essentially, these look like extra pixels that are not representative of the cell membrane itself, but rather are spuriously drawn by CP. Because these pixels are present as part of the cell perimeter, and yet they are not shared as pixels with another cell… I believe this is causing the PT value to register as < 100%.
On the image I’ve attached, you can see the yellow cell marked with an ‘x’ as an example. The cell is clearly an interior cell, and yet PT ~ 85% for this cell due to the extra “bumps” of the cell border.
[attachment=0]CP Forum Explanation.PNG[/attachment]
- Problem with Inter-cellular Borders and Neighbor Counts
Again in the MeasureObjectNeighbors module, I’m outputting the values of “number of object neighbors. Each cell is color-coded based on the number of neighboring cells… and this works correctly about 95% of the time. However, some cells are misidentified as having fewer neighbors than they actually do.
As an example, please see the image attached, and look at the green cell #46. This cell clearly has 5 neighbors, but CP is only registering 4 of them. Similarly, the orange cell to its right should have 7 neighbors, but it is only identified as having 6. Something is awry with the border separating the green and orange cells here, and I think I know what the problem is… though I don’t know how to fix it.
It appears that when a border is a perfect “staircase” of pixels (see attached image for an illustration), the border is somehow misidentified by CP. It strikes me as particularly odd that this border is still understood to be a “border”, per se, because these are identified as two separate cells. Moreover, this is all identified as usable perimeter since the “Percent Touching” statistic is reported as 100% for both cells involved here. Yet somehow, this neighbor relationship is not identified.
Now, neither of these problems is an isolated incident. They occur for roughly 1-2% of the cells, and in the statistics I’m trying to compile, that’s significant. Any ideas on what could be the cause of these problems I’m having? Any suggestions on how to fix them?
All help is greatly appreciated. If necessary, I’d be glad to include more images illustrating similar cases.