Ferrer-Imbert, G. | Search bioRxiv

bioRxiv2026

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 m), 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 {Delta}F/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. In BriefImaging the same cortical neurons across five axial resolutions and five analysis pipelines, Ji and colleagues show that lower resolution corrupts neuronal tuning and population correlations. No pipeline corrects these artifacts, and pipeline choice yields divergent results even at high resolution. Soma-targeted sensors mitigate but do not eliminate these failures.

neuroscience↗

bioRxiv2026

Tubulin C-terminal tails are pH sensors that regulate microtubule function

Changes in intracellular pH are critical for maintaining homeostasis, mediating signaling pathways, and enabling cellular responses to stress, injury, and disease. There is increasing evidence that clusters of acidic residues, primarily glutamates, are both highly prevalent and conserved in disordered regions of proteins and can play an important role in cellular pH response. Tubulin C-terminal tails (CTTs) are glutamate rich regions which protrude from the microtubule surface. These tails are a primary site of for both post-translational modifications and binding of microtubule-associated proteins. Motivated by these observations, we measured the pH response of tubulin CTTs using NMR spectroscopy, circular dichroism, and computational simulations. We find that glutamate residues in CTTs taken from organisms across eukaryotes exhibit a robust upshift in their pKa values, that the sequential context of glutamate residues creates hot spots for protonation, and that hydrogen bonding between side chains stabilizes interactions that alter the conformation of the CTT. To determine whether the CTT pH response plays a potentially important role in microtubule interactions, we measured the pH dependence of the binding of the yeast kinesin-5, Cin8, to microtubules. We find that Cin8 binding is modulated by pH in a CTT-dependent manner. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish that pH can regulate interactions at the microtubule surface. Significance StatementVariation in cellular pH is important for cell function in changing environmental conditions or developmental states. Here we probe protonation of the glutamate-rich C-terminal tails of tubulin, revealing the existence of and mechanism driving the anomalously high pH response and subsequent regulation of microtubule binding. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish pH-based regulation of interactions at the microtubule surface.

biophysics↗