High Axial Resolution Is Necessary for Quantitative Two-Photon Calcium Imaging of Neuronal Populations

Two-photon calcium imaging is a standard tool for measuring neuronal population activity in vivo, yet how axial resolution, sensor expression strategy, and analysis pipeline jointly affect data accuracy remains poorly characterized. Here, we imaged the same L2/3 neurons in mouse primary visual cortex at five axial resolutions (3.6 - 21.0 μm), spanning current two-photon systems from benchtop microscopes to large-field-of-view and miniaturized designs. We expressed cytosolic, transgenic, and soma-targeted GCaMP variants and applied five analysis pipelines. Reducing axial resolution systematically attenuated ΔF/F0, corrupted visual responsiveness and orientation tuning classifications, and biased pairwise correlations. No pipeline corrected these resolution-dependent artifacts, and pipeline choice alone produced quantitatively divergent results even at the highest resolution. Soma-targeted sensors mitigated but did not eliminate these artifacts. Our findings demonstrate that high axial resolution is necessary for accurate quantitative population imaging, and that robust separation of somatic from neuropil signals remains an unresolved challenge.