Masahiro Okada, Yusuke Sato, Tetsuji Itoh, Seiichi Nishizawa
Quantitative analysis of small extracellular vesicles (sEVs) at single-particle resolution remains challenging due to their nanoscale dimensions and compositional heterogeneity. Existing methods often rely on specialized and costly instrumentation, limiting accessibility for many researchers and necessitating extensive optimization. Here, we present a porous membrane adsorption for capturing small vesicles (PMACS), which employs a porous anodized aluminum oxide (AAO) membrane as nanoscale compartments to spatially isolate individual vesicles. This allows accessible single-particle discrimination using standard wide-field fluorescence microscopy without either advanced equipment or expert skills. Single-particle discrimination is achieved based on the principle that AAO membranes accommodate vesicles at most one particle per pore according to Poisson distribution and that occupied pores are sufficiently separated beyond the optical resolution limit, which ensures reliable attribution of fluorescent spots to individual vesicles. PMACS achieves reliable quantification of culture cell-derived sEVs across a broad dynamic range (103 to 106 particles/μL). This performance exceeds conventional methods without the need for either advanced instrumentation or expert operation. We demonstrate that PMACS allows colocalization analysis of sEV cargos by assessing the tetraspanin (CD9, CD63, and CD81) population of cultured cell-derived sEVs (Hela, A549, and HEK293T cell lines). The heterogeneous protein expressions on sEVs are visualized by means of a simple facile setup. Owing to its simplicity, sensitivity, and accessibility, PMACS provides a practical platform to advance studies of sEV heterogeneity and broaden participation in sEV science.