AbstractBenefitting from the advantages of high imaging throughput and low cost, wide-field microscopy has become indispensable in biomedical studies. However, it remains challenging to record biodynamics with a large field of view and high spatiotemporal resolution due to the limited space–bandwidth product. Here we propose random-access wide-field (RA-WiFi) mesoscopy for the imaging of in vivo biodynamics over a 163.84 mm² area with a spatial resolution of ~2.18 μm. We extend the field of view beyond the nominal value of the objective by enlarging the object distance, which leads to a lower field angle, followed by the correction of optical aberrations. We also implement random-access scanning with structured illumination, which enables optical-sectioning capability and high imaging contrast. The multi-plane imaging capability also makes the technique suitable for curved-surface samples. We demonstrate RA-WiFi mesoscopy in multi-modal imaging, including bright-field, dark-field and multi-colour fluorescence imaging. Specifically, we apply RA-WiFi mesoscopy to calcium imaging of cortex-wide neural network activities in awake mice in vivo, under both physiological and pathological conditions. We also show its unique capability in the three-dimensional random access of irregular regions of interest via the biodynamic imaging of mouse spinal cords in vivo. As a compact, low-cost mesoscope with optical-sectioning capability, RA-WiFi mesoscopy will enable broad applications in the biodynamic study of biological systems.