Hello,

I am having a problem to measure the diameter of capillaries in 3D on ImageJ, and I need some help please.

I have a stack of images (8-bits) of capillaries that I threshold, on ImageJ, to got a binary stack :

These images are taken with a light sheet microscope so pixel dimensions are different in x,y and z (0.5, 0.5, 2) µm.

What I want, it is to measure the diameter of these capillaries in 3D on ImageJ. So after some research on internet, I found that there is a pluggin called 3D Suites created by @ThomasBoudier that allows to make 3D Distance Mapping on binary stack and that take in consideration the calibration of voxels.

So, after using the pluggin, I get a EDT stack in 32-bits, that I convert in 8-bits :

Now, as I want quantitative informations, I analyze the grey-level histogramm of the stack. (Because, on ImageJ, it is said that the euclidean distances after distance mapping are coded into grey values).

The problem is that I don’t understand what I get. The minimum grey value that I get is 7 which should correspond to a 7 µm radius (= 14 µm diameter). However, it is biologically impossible as capillaries have a 3-10µm diameter.

This is what I get :

So my questions are : using this pluggin, can I get quantitative informations ? if yes, how can I get them ? And using my technique, are the informations obtained with the histogramm really correspond to euclidean distances ? and if yes, are they in voxels or converted in µm ?

Thank you in advance for your help.