Error for module "ConvertImagetoObject"

Vaidehi_Gupta · February 23, 2021, 8:04pm

Hello!

I am trying to use the pipeline below to measure the dendrite and axon length of the neurons that I have imaged. I am experiencing some issues with measuring the area of the skeletonized image. I have two specific questions:

Is there a way to convert the branchpoint image from the module “Measure object skeleton” into a binary image? This would most ideal, since I would like to measure the area occupied by the skeletonized image.
Alternatively, I am trying to convert my image to objects and then measure the area. However, the pipeline gives the error : “Error while processing ConvertImageToObject: too many values to unpack (expected2)”. I don’t understand what I am supposed to change to fix the issue.

Any help would be greatly appreciated. I am attaching the pipeline below, along with the sample set of images. I am using version 4.0.4.

Thank you,
Vaidehi
[KirwanHCS_Jan2021_Staining 1.cpproj|attachment]
(upload://xOChcrBvqXff5QBkMf68YX8geI3.cpproj) (1.0 MB) Replicate2_P1_1DPR_Shank46_HuCDRbR_MAP2chG_SMI31msFR_DAPIB_B07_s1_w4.TIF (2.9 MB) Replicate2_P1_1DPR_Shank46_HuCDRbR_MAP2chG_SMI31msFR_DAPIB_B07_s1_w1.TIF (1.7 MB) Replicate2_P1_1DPR_Shank46_HuCDRbR_MAP2chG_SMI31msFR_DAPIB_B07_s1_w2.TIF (2.8 MB) Replicate2_P1_1DPR_Shank46_HuCDRbR_MAP2chG_SMI31msFR_DAPIB_B07_s1_w3.TIF (2.7 MB)

pearl-ryder · February 23, 2021, 8:54pm

Hi @Vaidehi_Gupta,

It looks like something happened to your pipeline upload – I’m not able to download that file. Could you try again and verify that you see a link to that file in your message?

Thanks!
Pearl

Vaidehi_Gupta · February 23, 2021, 9:08pm

Hi Pearl,

Sorry about that. Here you go. Thank you!
KirwanHCS_Jan2021_Staining 1.cpproj (1.0 MB)

pearl-ryder · February 23, 2021, 10:18pm

The issues you raise here are in fact, related @Vaidehi_Gupta. The output of the MeasureObjectSkeleton module is an RGB image. If you’d like to threshold the image, I’d recommend:

Convert the image to individual channels using ColorToGray

Screen Shot 2021-02-23 at 5.10.38 PM740×394 31.4 KB

Screen Shot 2021-02-23 at 5.07.01 PM1049×848 39.9 KB
Add the individual channels together w/ ImageMath
Then you can threshold the output of this ImageMath module and complete any measurements, convert to objects, etc. I found that an Otsu three-class method with the middle intensity class assigned to the background worked well. The goal is to get rid of your nuclei from the final threshold.

Screen Shot 2021-02-23 at 5.06.26 PM803×785 40.9 KB

I hope this helps!

Vaidehi_Gupta · February 23, 2021, 10:56pm

Thank you so much @pearl-ryder!