Cell swelling and upright mounting-based imaging for high-resolution visualization of intracellular trafficking across the BBB using conventional confocal microscopy.

Receptor-mediated transcytosis (RMT) represents a promising strategy for delivering macromolecular and colloidal therapeutics across the blood-brain barrier (BBB). However, mechanistic elucidation of RMT remains limited by the difficulty of visualizing subcellular trafficking pathways. Conventional imaging approaches either lack sufficient spatial resolution or require costly, technically complex instrumentation. Here, we report a cell swelling and upright mounting-based (CSUM-based) imaging approach that reorients the Z-axis into the high-resolution XY-plane using standard confocal microscopy, enabling direct RMT visualization without computational reconstruction or specialized hardware. We tracked intracellular trafficking of transferrin (Tf) and anti-transferrin receptor antibody (anti-TfR Ab) as model cargos using our CSUM-based imaging approach via compartment-specific markers and time-resolved co-localization analysis. This approach resolved cargo-containing vesicles traversing from the apical to basolateral membranes. Tf completed transcytosis within 15 min, whereas anti-TfR Ab initially entered the endolysosomal pathway before rerouting to transcytosis under receptor saturation conditions. The CSUM approach provides a simple yet effective platform for high-resolution visualization of membrane transport and vesicle dynamics, offering broad applicability to drug delivery research and the design of brain-targeted therapeutics.