CellProfiler for RNAscope

Does anyone have previous experience using CellProfiler with RNAscope tissue? I’m currently trying to find an ideal pipeline for this, but I’m running into some issues with it. Any advice or tips would be greatly appreciated. I can supply more information as well if that is necessary, thanks!

Hi @cschratz,

Sharing little more details like your sample image and the problem exactly that you are facing might facilitate to help you better. Nevertheless you can explore the example pipelines here.

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

Read more on our site.
Yokogawa CV8000 - The Ultimate in Confocal HCS
https://www.wakoautomation.com/products/yokogawa-high-content-imaging

Lakshmi,

Thanks for the response. I’ll give some detail to what I’m currently running and what I want to be able to do. I’m using RNAscope to visualize Glutamate, GABA, and cFOS expression within the amygdala of rat brains sliced at 10 microns and stained with DAPI for cell identification. There are 4 channels within our microscope giving us blue, green, red, and white signals. I want to be able to identify cells stained with DAPI and then be able to determine if those cells are expressing any or all of the other tracers. I’ve messed around with the speckle pipeline, but didn’t really understand how to accomplish this task. I’ve been able to identify cell bodies stained with DAPI, but the outlines are not perfect and some cells are being missed. From there I’m unsure of what the next process is since there are multiple secondary objects that need to be identified.

I hope this is enough to give you an idea of what I’m trying to accomplish.

Thanks.

Hi @cschratz,

Thanks for the details. I got the understanding of what you are trying to do. Its a great start to try with the speckle pipeline. Generally, this could be your workflow,

  1. Identify the nuclei using DAPI channel as a primary object (i think you are already doing this)
  2. Identify the cell bodies as a secondary object using your nuclei as primary object.
  3. Identify your fluorescence signals (Glutamate, GABA, cFOS) as seperate primary objects (using 3 primary object module). I assume they have three different fluorophores and they appear as a puncta structure.
  4. Relate these three primary objects to the cell body using Relate object module.

To identify your cellbodies completely you might need play around with the parameters.
In case still you face problem, do you mind sharing your pipeline that you are trying along with sample image.

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

Read more on our site.
Yokogawa CV8000 - The Ultimate in Confocal HCS
https://www.wakoautomation.com/products/yokogawa-high-content-imaging