Intravital mesoscale optical imaging: challenges, techniques, and future perspectives

Intravital mesoscale imaging plays a crucial role in bridging the gap between cellular and organ level investigations by enabling high-resolution visualization across large field of view. Continuous advancements in optical microscopy have significantly improved imaging performance, yet fundamental challenges remain. Effective intravital mesoscale imaging requires a balance between spatial resolution, imaging speed, field of view, and while overcoming limitations such as scattering, aberrations, phototoxicity and photobleaching. This review summarizes key challenges in achieving high-performance intravital mesoscale optical imaging and provides an overview of advanced optical imaging techniques, including wide field, laser scanning, as well as computational imaging approaches. Despite these advancements, further improvements are necessary to address existing limitations and unlock new possibilities. Future developments will focus on enhancing imaging depth, further improving space bandwidth product and integrating computational methods for real-time processing and largescale data analysis, further advancing mesoscale imaging for biological research.