Quantifying fluorescence using 3D ROI manager- RAM issue?

Hi there, I’m trying to quantify fluorescence using the 3D ROI manager and I’m run into several issues. I first use segmentation editor to select my ROIs across slices and interpolate between. For each brain, I have 2 ROIs (left and right) isolated across slices. After saving an ROI, I open 3D ROI Manager so I can quantify fluorescence within each of my ROIs. The biggest issue is that 3D ROI Manager only works some of the time. Most of the time, it freezes as soon as I try to add an image. Sometimes I can add one ROI, but I can’t add the other. I have over 100GB of memory available and 8GB of RAM. Should I upgrade my RAM to get the 3D Manager to work, or am I doing something wrong?

Thank you SO much in advance!!

Hi @KScaplen,

I am not sure to understand what is your input image for 3DManager, when using the add Image command, 3D Manager expects a image with different labels, not really ROIs. You should fill the Rois with different colors. About memory 3D Manager may require like twice the memory of the input image.

Hope this helps



Hi Thomas,

Thank you so much for your response!

Hmm. I think this is what I have. I’ve used segmentation editor to outline (using the selection brush tool) a structure across multiple slices, interpolated across these slices, and then added that selection to a designated label. I do this twice for each side of the brain and so I have two separate labels to import into 3D ROI Manager. Then I open the ROI Manager and select “add image”. If all goes well, two labels appear and the log says “adding image:min-max 1 2”. Unfortunately, it seems to be hit or miss. When it doesn’t work the log says “adding image: min-max 31-65535” and no labels appear in the ROI Manager. I should be okay with memory since the files are around 50MB.

I just noticed you were an author of the plug-in. Am I using it correctly? Will “quantif 3D” actually quantify fluorescence within each labeled selection or should I be doing something else? I tried using the 3D object counter but I couldn’t find a threshold level that was limited to the brain region I would like to quantify, which is why I decided to trace it in segmentation editor.

Thank you in advance for your time and advice! I really appreciate your insight.


Hi @KScaplen,

Maybe the problem is just that you try to import the wrong image. I assume you have two images, one with your labeled Roi and one image with the intensity you want to quantify. Before Add Image you need to select the labeled image, so it gives you the message adding image:min-max 1 2. In case you select the intensity image and try to add it it will display adding image: min-max 31-65535 because it will try to extract object from this intensity image.
So to summarize :

  1. select the labeled image
  2. add image from 3D Manager
  3. select your objects in 3D Manager
  4. select the intensity image
  5. perform quantification in 3D Manager

hope this helps



This worked perfectly! Thank you!!