Two-step workflow integrating automatic registration and manual refinement for the accurate alignment of serial...

The accurate alignment of serial histological sections is essential for preserving anatomical continuity in 3D reconstruction. Although automated registration tools can efficiently correct global alignment errors, they often fail to resolve local misalignments caused by sectioning artifacts, tissue deformation, staining variability, or missing slices. Thus, we propose a practical two-step registration workflow that uses MultiStackReg (an ImageJ/Fiji plugin) for automatic alignment and AlignRef (a standalone application for interactive adjustment) for manual refinement. In the first step, MultiStackReg performs global registration by using rigid body transformations. In the second step, AlignRef corrects residual misalignments through semi-transparent overlay visualization, keyboard-based translation and rotation, and batch propagation of recorded transformations across selected slice ranges. We applied our workflow to 135 serial sections of a Carnegie Stage 15 embryo from the Virtual Human Embryo dataset that were stained with hematoxylin and eosin. MultiStackReg resolved most global inconsistencies, whereas AlignRef enabled the precise adjustment of subtle local deviations, particularly in curved structures such as neural tubes and limb buds. After automatic registration with MultiStackReg, the subsequent manual refinement step using AlignRef was completed in approximately 30 min and produced a suitable stack for 3D reconstruction. This two-step workflow balances automation with expert-guided correction and provides an accessible, reproducible, and anatomically precise method for the serial section alignment of morphological and developmental anatomy.