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The composition of tight junctions (TJs) in epithelia is well understood, whereas that one in endothelial cells at the blood-brain barrier (BBB) is controversial. Although freeze fracture EM remains the gold standard for assessing TJ nano-structures, advances in fluorescence microscopy, such as stimulated emission depletion (STED) together with image analysis tools, allow immunostaining-based quantitative analysis of the protein composition and the nano-organization of TJ strands. Here, we present two approaches for quantitative analysis of the BBB TJ meshwork utilizing confocal and STED imaging together with open access image analysis tools CellProfiler, iLastik and ImageJ. For the first use case, a BBB model based on brain capillary endothelial-like cells (BCELCs) differentiated from human induced pluripotent stem cells (hiPSC) was employed. For the second one, monolayers of human primary brain microvascular endothelial cells (pBMVECs) were used. STED analysis of BCELC monolayers revealed that here claudin-5 co-polymerizes with claudin-4 and − 6 into continuous TJ strand meshworks. Treatments with established TJ openers (1.4 M mannitol or C. perfringens enterotoxin-based claudin binders) strongly decreased transendothelial resistance (TEER) accompanied by a reduction in the nanoscopic co-localization of claudin-5/-4, -5/-6 and amount of junctional claudin-5. These findings suggested that in hiPSC-based BBB models multiple claudins together constitute TJs, leading to a tight paracellular barrier against solutes. Using STED imaging of pBMVECs immunostained for Cldn5, extensive meshworks of continuous TJ strands were detected. In contrast to the hiPSC-based model, other claudins were not detected. TEER and morphometric analyses of Cldn5-positive strands revealed that the barrier defect caused by claudin-5 binders is due to impaired TJ structural integrity. The findings suggest that Cldn5 is sufficient to form the tight paracellular barrier at the BBB. The presence of claudin-4, -5 and − 6 in hiPSC-derived cells reflects a mixed phenotype depending on their differentiation process. In sum, we demonstrated that, unlike confocal imaging, STED enables monitoring the composition and structural integrity of BBB TJ strands.