Adding a couple of hopefully helpful comments here. 1. There is a lot of terminological abuse in this area, but fusion is properly a display (or production of

Adding a couple of hopefully helpful comments here. 1. There is a lot of terminological abuse in this area, but fusion is properly a display (or production of new images) technique, (co-) registration is the mapping of one image to another. 2. You almost certainly do not want deformative or even affine transforms in the registration of same patient bony anatomy in CT (because it is stereo-tactically accurate). You want a rigid transform here. Also because of CT you can rely on the units are meaningful so you don't need to normalize. 3. Because you are doing rigid transform only , you should not resample the moving image here (could be both images move, more often as you describe). Most package will get this wrong. Save the resampling for the end and do it once. 4. You will have to resample one or both images, as their underlying lattices will not be aligned. There is a bit of subtlety here, but in the OPs case probably best to sample both to a similar isotropic grid (e.g. 1mmx1mmx1mm) and subtract. 5. You can do the resampling explicitly or implicitly (i.e. as part of computing the subtraction) but either way use a proper smoothing filter not tri-linear interpolation. 6. Oh, a note - your original images are probably 12 bit encoded in 16 bit ints, make sure your loader isn't truncating or rescaling to 8 bit.