From EM to AI: Multiscale imaging of the secretory pathway with insights from plant cells.

The secretory pathway is a central and evolutionarily conserved feature of eukaryotic cells, responsible for protein and lipid trafficking, membrane biogenesis, signalling, and cellular homeostasis. Its complexity, dynamic behaviour, and nanoscale organisation have made it a longstanding target of microscopy-driven investigation. In this review, we trace the parallel evolution of our understanding of the secretory pathway and imaging technologies, with a particular emphasis on plant cells, where unique architectural and functional features have challenged and enriched mechanistic models. We highlight the foundational role of electron microscopy (EM) in defining the ultrastructural organisation of secretory organelles and establishing directional models of intracellular transport, followed by the fluorescence microscopy revolution that enabled direct visualisation of cargo flux and organelle dynamics in living cells. The advent of super-resolution fluorescence techniques bridged the long-standing resolution gap between light microscopy and EM, revealing nanoscale compartmentalisation, membrane contact sites, and trafficking intermediates previously inaccessible in living cells. More recently, the integration of functional assays, optogenetics, and artificial intelligence-driven segmentation, denoising, and adaptive imaging now enables quantitative and high-throughput analysis of secretory architecture. Together, these advances have transformed the secretory pathway from a static morphological concept into a dynamic, increasingly mechanistically defined system. We conclude by discussing emerging integrative strategies, particularly correlative and AI-enhanced approaches that promise to unify ultrastructural precision with molecular specificity and temporal resolution in future studies of endomembrane organisation.