Sample image and/or code
I am trying to look an nuclear localization of a transcription factor in response to IGF1. These are fixed and labeled with GFP. I also have a dapi stain. Also the cells are embedded in agarose so they are 3D spherical objects. I do have a membrane stain but I forgot to apply it. I will redo this experiment but I wanted to see how far I can get with what I have.
I would like to get the ratio of GFP intensity of nucleus to the cytosol. Activation would be a higher ratio and low the opposite.
What I have tried
So I am new to ICY and I have been working with the HK Means plugin to get my ROIs. I have a good handle on that. What I have so far is a manual process where I extract the channels, do a z projection and do the ROI identification on each separately, so I have a list of nucleus and cells in some random order. When I cut and paste they are sort of matched up but not quite. I have been trying to manually click through and notate which ones are the pairs. This is not a sustainable path forward.
Ideally I am looking for something akin to cell profiler where you could look inside an existing ROI to find the nucleus so there is some linkage. I have never looked in separate channels in cell profiler so I am not sure it has this ability. I would be willing to try and make my own plug in for it. the pseudo code would be
- take image
- Pre process if necessary ( smooth sharpen etc…)
- look in Channel 1 to identify cells
- Lookin Channel 2 to identify nuclei in each of said cells
- calculate the intensity of Channel 3 in just the nuclei
- calculate the intesnity of the cells indentified in step 3 minus the nuclei in step 4
I would need some way of pairing the two ROIs so I would not have to manualy pick and do binary operations to get what I want.
Thanks in advance for the help!