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. Schematic representation of the cell swelling and upright mounting (CSUM)–based imaging approach for high-resolution visualization of intracellular trafficking across the blood–brain barrier (BBB) using conventional confocal microscopy. Created with BioRender.com.受体介导的转胞吞作用（Receptor-mediated transcytosis, RMT）是一种极具潜力的跨血脑屏障（blood–brain barrier, BBB）递送大分子与胶体治疗剂的策略。然而，由于亚细胞运输通路的可视化难度较高，RMT的机制阐释仍受限于此。传统成像手段要么空间分辨率不足，要么需要昂贵且技术复杂度极高的仪器设备。本文报道一种基于细胞肿胀与直立固定的（cell swelling and upright mounting, CSUM）成像方法，该方法可通过标准共聚焦显微镜（confocal microscopy）将Z轴重定向至高分辨率XY平面，无需计算重构或专用硬件即可直接可视化RMT过程。 我们借助该CSUM基成像方法，结合细胞区室特异性标志物（compartment-specific markers）与时间分辨共定位分析，追踪了转铁蛋白（transferrin, Tf）与抗转铁蛋白受体抗体（anti-transferrin receptor antibody, anti-TfR Ab）这两种模型递送货物的细胞内运输过程。该方法可解析携带递送货物的囊泡从顶膜向基底膜转运的全过程。转铁蛋白可在15分钟内完成转胞吞作用，而抗转铁蛋白受体抗体在受体饱和条件下，最初会进入内体溶酶体通路，随后重新定向至转胞吞途径。 CSUM方法为膜运输与囊泡动力学的高分辨率可视化提供了一种简便高效的研究平台，可广泛应用于药物递送研究与脑靶向治疗剂的开发。 基于常规共聚焦显微镜、用于可视化跨血脑屏障细胞内运输的细胞肿胀与直立固定（CSUM）成像方法示意图。由BioRender.com制作。