In cases like this it's best to post a representative image. It makes it easier to reply with concrete answers, and allows readers to immediately relate to the problem without having to go through long posts (like this one) just to understand the issue at hand. For clarity, I'll split your post into a list of questions:
How to display sampling shells as circular ROIs?
Last saturday I released v3.6.5 that adds a new Overlay 2D shells checkbox in the Output section of the main prompt to allow this. Please run Help>Update... to fetch the update. A couple of things:
- ROIs are added to the image overlay as either circular ROIs, or composite ROIs when using hemicircles, and are named according to their radius.
- Thickness is defined by the value specified in # Samples. Their color by whatever selection color you have specified in IJ, i.e., the one reported in the (Multi)Point Tool, in the main IJ window.
- Right now it is limited to 2D images (which I'm guessing you are using). It is not immediate to me how to extend the rendering of these ROIs to 3D images.
How to perform the analysis in pixels in spatially calibrated images?
If your images are spatially calibrated, analysis is performed in physical units, otherwise in pixels. If you want to impose distances in pixels: Run Analyze>Set Scale.., click on Click to Remove Scale button, than activate the Global option. The Global option is explained in the user guide. Note that this is an ImageJ feature, and is not specific to the Sholl plugin.
NB1: I would strongly advise against disabling spatial calibration on 3D images. If you do so, sampling distances will be calculated assuming an isotropic voxel size, even if, your images are likely anisotropic due to the lower axial resolution of conventional microscopes.
NB2: You shouldn't need to discard an image's metada to achieve consistency in a processing workflow. Perhaps some of the procedure(s) you're using should be revised?
How to analyze RGB images?
You convert them to multichannel images, and then run Sholl on a selected channel. I was convinced I had made this point clear when you prompted me to release v3.6.4 last month. I really regret you are still having problems with it. In short:
- Open your RGB image
- Activate the Channels Tool by pressing Shift+Z (Image>Color>Channels Tool...)
- Choose Make Composite Image from the More>> dropdown menu
- Set the Display mode to "Grayscale"
- Activate the channel to be analyzed using the "C" slider underneath the image canvas.
NB: Step 4. is required because "Composite mode images cannot be tresholded" (This is the actual error message you'll read in the status bar when trying to segment a multichannel image displayed as "Composite").
However there should be no need to use RGB images. You can either 1) use your DAPI image to define the central point ROI and transfer it to the analysis image using Shift+E (Edit>Selection>Restore Selection), or 2) Merge the single channels directly into a Multi-channel image using the Create composite option in Image>Color>Merge Channels...
Was this helpful? It would be great if you could update the documentation page with this info (Perhaps in the FAQs), so that others can profit from it.
I have to admit I dot not understand the question. Could you elaborate? What is not working for you?