Large-Field Mesoscopic Spinning-Disk Confocal Microscopy Enhances Quantitative Fidelity for In Vivo Cerebrovascular and Calcium Sensing.

Reliable spatiotemporal quantification of fluorescent reporter signals in thick biological specimens remains a central challenge for analytical imaging, because out-of-focus background fluorescence in conventional single-photon wide-field microscopy degrades contrast, obscures signal identification, and compromises measurement fidelity. Here, we present a mesoscopic spinning-disk confocal (MSDC) imaging platform that physically suppresses background fluorescence while preserving the throughput required for large-scale functional imaging. By systematically optimizing the mesoscopic spatial filtering configuration and hardware synchronization scheme, the MSDC system achieves a field of view of ∼6.8 × 6.8 mm2 with uniform ∼8 μm lateral and ∼50 μm axial sectioning thicknesses at full-frame rates up to 100 fps. Ex vivo evaluations demonstrate that the platform successfully suppresses out-of-focus fluorescence by ∼90%, thereby preserving structural contrast and improving imaging fidelity in the presence of strong background signals. In vivo cerebrovascular imaging shows that the MSDC platform substantially reduces background-induced dark-vessel artifacts and enables positive-contrast three-dimensional reconstruction of cerebrovascular networks across cortical depths. When integrated with extended constrained nonnegative matrix factorization analysis, the platform further supports extraction of high-signal-to-noise calcium dynamics from hundreds of active Layer 2/3 neurons distributed across multiple functional brain regions. Together, these results establish MSDC as a robust and cost-effective analytical imaging platform for high-fidelity structural and functional interrogation of fluorescent signals in complex biological specimens.