Normalisation of 3D STACK SBFSEM

Good morning :slight_smile:
I have a tiny question

I try to find a strong protocol for normalisation of my multiple EM 3D stack. Also, I have a staining on these EM 3D stack.
I already tried to do the simple steps

1-normalisation of the contrast trough the stack
2-play with the display with the pic at the middle
And good to go

But this last step, I’m perplex with it because if it’s not consistent for all my stacks my stats at the end would be false. Since I’m doing stat of intensity correlate to the morphology at the end

I also know that each image in electron microscopy could be different for sure.

But I just want to be sure to have strong protocol to normalise my stack in the same way.
If you have any idea.

Thanks

Hi Julie,
during normalization, histogram mean and histogram deviation of each slice are modified to match those of the whole stack or to match a certain predefined value. To make normalization more predictable, you can use the manual mode:
image
as result the histogram of each slice will be matched to the mean and std values that you put in the two edit boxes under the manual dropdown.
To find approximate values to put there, you can run normalization procedure in the automatic mode for few reference slices and the resulting values will be displayed in the command window or you can check them from Path panel->Log:
image
You can round up those numbers and use for all datasets that you have. Remember, that you may have to use the masked mode (Menu->Image->Contrast->Normalize layers based on masked areas) to select areas that have similar histogram to your reference area.

Ilya