Using the Volume Fraction function and the Analyse Particle function to find cortical porosity


I’m an undergraduate student trying to find the cortical porosity of the tibiofibular junction. I have a 25-stack image. I’ve used the Analyse Particle function in the bonej plugin to find the total pore volume and I believe these values to be correct as they correspond to the literature (567520 μm^3). However I am struggling to understand how to get a correct total cortical volume value. I am using the Volume Fraction function on bonej to obtain this.

I have been making my image binary and then inverting it so the background and the pores have a pixel intensity of 0 and the bone has a pixel intensity of 255 then using the Volume Fraction function. I’ve attached an image of the input used

The numbers I’m producing do not seem right, though I could be wrong as my subject knowledge isn’t very advanced. The reason I think they are incorrect, is that to me it makes sense that if I were to take total pore volume and subtract this from total cortical volume, I should get a value similar to the total bone volume, however it is quite different:

From BoneJ > Fraction > Area/Volume Fraction:
BV = 1.48E+07μm^3, TV=5.45E+07μm^3, BV/TV = 0.27

However, if I subtract the total pore volume from the Analyse Particle function:
Bone Volume = Total Volume - Total Pore Volume = 5.45E+07μm^3 - 567520μm^3 = 5.39E+07μm^3

There is quite a large discrepancy between the two values for Bone Volume, but they are in the same order of magnitude: 1.48E+07μm^3 vs 5.39E+07μm^3

I am wondering if this discrepancy is expected. Is the image I input into the Volume Fraction function incorrect or is my calculation incorrect?

Thank you.

Just to note, I have also already tried increasing the level of preciseness for measuring the pores to 9 decimal places and it didn’t change the outcome.