Superpositioning of multiple 3-dimensional digital volumes is essentia
l to extending neuroimaging capabilities from single-subject analysis
to group evaluation. In this study, 6 rat heads were cryoplaned and di
gital images of the specimen blockface were organized into data volume
s. The midline plane of each brain guided coronal and horizontal rotat
ions and medial-lateral positioning. Four different methods of determi
ning sagittal rotation, dorsoventral position; and anteroposterior pos
ition were tested. The first technique uses lambda and bregma as fiduc
ial points; the second method employs bony sutures in the base of the
skull; and the third strategy fits both sets of alignments points. The
fourth method differs from the previous landmark-based approaches by
employing cross-correlation of image densities. A composite of midsagi
ttal cutplanes through the volumes aligned by the first method reveale
d significant misalignment. Use of ventral fiducials resulted in impro
ved correlation statistics for orthogonal cutplanes through the volume
s and significantly reduced positional variability of anteroposterior
landmarks. Combining dorsal and ventral fiducials produced a slight im
provement in midsagittal image correlation but had mixed effects on la
ndmark variability. Following the fourth method of registration, corre
lation statistics and the alignment of reference points were similar t
o those obtained with the 4-point approach.