Basal Layer Issues

Hello,
Thank you for all your work on CellProfiler, it really is a wonderful tool to use.
I seem to have reached an impasse in regards to identifying the specific section I want.

I am only interested in the basal layer of the skin tissue, which is the bottommost line of nuclei (not including any free-floating keratinocytes). Using this basal layer, I was attempting to use the program to count the number of DAPI stained nuclei and the related number of Ki67 stained nuclei as well.

What I was attempting to do was to crop out all of the unnecessary layers and preserve the basal layer. The problem arose when I tried to use the crop function to do this. The image is not perfectly straight up and down so using the ellipse and rectangle crop functions did not work.

The next step I tried was to use the module IdentifyObjectsManually. Unfortunately, I got an error when I tried to save the resulting crop: “object cpi is not defined”. Being unable to save the resulting cropped image made IdentifyObjectsManually not useful to me.

Attached are example images I have tried working around with. Any comments/suggestions would be much appreciated!

Thanks,
Brian
Involucrin_Ki67_June28.cp (8.54 KB)





You may want to consider using the FlipOrRotate module to get the image into the position you want prior to cropping.

See this post for the explanation and workaround for the problem.

Regards,
-Mark

Hi Mark,

Sorry about the late response. I tried both of your suggestions, the FlipOrRotate module giving me the most help. Unfortunately, the basal layer that I am interested in is not a straight line so it is more difficult to identify using the standard rectangle or ellipse cropping feature.

If I uncheck the “Retain outlines…” setting under IdentifyObjectsManually, I am unable to refer to the image that I identified later in the pipeline. Is there any way where I can draw a circle around the basal layer and have the pipeline identify just that circle as the area of interest?

Thanks,
Brian

Even if you uncheck the outlines settings, you still have the manually identified object that you can work with.

Assuming that you have used this module to draw a circular object and called it ‘RegionOfInterest’ (for the sake of discussion), you can use the MaskImage module to select the input image of choice, select masking with Object (rather than Image) and then select RegionOfInterest as the object.

The result will be an image in which the region within the RegionOfinterest object will be retaining, and everything else will be masked out (that is, removed from future consideration). Even though the masked pixels are still there, there will be ignored in all subsequent operations, such as object thresholding and identification.

Regards,
-Mark