How to deal with dendritic mRNA, when quantifying in situ spots in the nuclei

Hi,
I’m trying to quantify RNAscope data. I have three probes and DAPI(C4). I have identified nuclei and spots.

One of my probes is highly dendritic , why it can be found every where(image C3).
If I put a pseudo nucleus in the image, I will get as many spots as I have for a true nucleus.
If I put a threshold for C3=30 spots, how can I be sure those spots is nuclear and not dendritic.?

C1.tif (4.0 MB)
C2.tif (4.0 MB)
C3.tif (4.0 MB)
C4.tif (4.0 MB)
RNAscopePip.cppipe (117.7 KB)
Best

Hello @Abdukuku,

In general (not seeing your pipeline), you could try masking (MaskImage module, which you could use an image or object to mask an image) and subtracting (ImageMath module).

If these didn’t work, feel free to upload your pipeline so we can take a look.

Thank you for your quick answer.
I have modified the post and added the pipline.
I’ll try to MaskImage.

Best

Could you elaborate on this:

What part of your current pipeline you’re trying to do this?
Could you also explain a little what you would like to count and measure?

Feel free to signup for our office hours this Friday.

Hi @Abdukuku,

I checked your pipeline. My understanding is that you would like to get the spot from C3 in the region of nuclei. To achive this, you should be relating the segmented nuclei (SmallBlockCorrectedNuclei) with the spots (GREEN) identified from the C3 channel. You could try this in the “Relate object module”. Then you could later measure the features of the related objects only.

Hope this helps.

Regards,
Lakshmi
Fujifilm Wako Automation (Consultant)
www.wakoautomation.com
For CellProfiler training or optimised pipeline write to,
lakshmi.balasubramanian.contractor@fujifilm.com

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