Can I use diff function to subtract pixel from 2-pixel away from it? So it not neighbour pixel.

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No.
I suggest you use
diff2_col = YourArray(:,3:end) - YourArray(:,1:end-2)
or
diff2_row = YourArray(3:end,:) - YourArray(1:end-2,:)
depending on which dimension you want to proceed across.

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