Question about 3D image suite plugin

Hi! I have just found this plugin today. I’m still new in the image analysis field but I have the following problem

I have confocal stacks with two channels, and I would like to threshold the images as well as possible. Then I would like to do the following (ideally for user-defined ROIs)

  1. Make measurements of the volume of the signal (mainly there are like dots) for each channel
  2. Quantify the number of colocalizing puncta for each ROI.

I have another way of doing 2), but I didn’t find a good way of performing 1). I have tried with the 3d Object Counter but I’m not quite satisfied with the results given that the thresholding algorithm could be not optimal.

I have read the wiki of the plugin. I chose my ZStack as the spots and used the local maxima function to get the seeds. Then I tried to select the ROIs and when I tested I didn’t get a result. I know that must be a very silly question but I’m not sure if I have the make a roi for each seed or if the rois can be the ones that I would like to use in my further analysis.

Sorry again