Z-stack slice number changes my measurements

Hi all,

I’m hoping someone can help me with this issue.

I have a 96 z-stack image of some micro-particles and I want to measure the volume, surface area, diameter, etc of these particles. The protocol is as follow:

  1. open .tiff image with bio-formats (96 stacks image)
  2. go to slice number 48 (which should be the most focused slice)
  3. adjust threshold using Otsu thresholding & create a binary image
  4. image segmentation with MorphoLibJ
  5. open the segmented image with 3D ROI manager and get my measurements

What I have noticed however, is that based on the slice of my z-stack I select in step 2 (so the slice on which the thresholding is based upon), my measurements will change at the end, so for instance the value for the volume, surface area, etc.

Unfortunately the microscope I am using is not automatized so most of the time the most focused slice ends up being number 48 (as it should be); however sometimes it is number 50 or 52 for instance and so my measurements at the end would not be correct.

My question is, is there a way to automatically pre-select the most focused slice (e.g. the slice with the brightest point) so that I do not have to manually check which slice I should select prior to running my image segmentation protocol?

Hope it makes sense & Thanks in advance!

Hi @tal,

The only thing that could change your measurements is the thresholding, two different values of the threshold values will yield to two different binary images and hence to different measurements. Did you check that you are using 3D histogram in the threshold window and also be sure to uncheck compute threshold for all slices, you want the same threshold throughout your stack.

You can try to use 3D objects counter to select your threshold.