Abstract
Two-photon calcium imaging enables measurements of neural activity from individual neurons and their subcellular processes in awake animals. Axial brain motion can change measured fluorescence independently of neural activity and move small structures out of the imaging plane, confounding two-photon calcium measurements in behaving animals. We developed two complementary post-acquisition methods, selected according to whether the imaged structure remains detectable across the encountered depth range. When it does, z-stack correction estimates imaging depth from a separately acquired reference z-stack and uses an axial fluorescence profile specific to each neural structure to rescale fluorescence to a common reference depth. When a structure can leave a single imaging plane, volume-based z-registration reconstructs a common-depth movie from repeated acquisitions of closely spaced planes. In awake mice, both methods tracked experimentally imposed axial displacements within the sampled range, with depth-estimation root mean squared errors (RMSEs) below 1
µ
m for z-stack correction and 0.69–1.03
µ
m for volume-based z-registration. Correction changed fluorescence substantially for a subset of somata, with larger effects in axonal boutons. Both methods are implemented in
suite2p-in-depth
, a Python package that integrates with Suite2p and provides diagnostics for depth estimation, correction quality and data reliability. Together, these methods enable axial-motion artifacts to be assessed and corrected within established Suite2p workflows.