{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T08:04:39Z","timestamp":1789632279306,"version":"build-2803163510"},"reference-count":37,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Small Methods"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>The functional accessibility of membrane proteins is a fundamental determinant of biological activity but cannot be accurately inferred from protein abundance alone. Current extracellular vesicle (EV) analyses primarily quantify expression levels, providing limited information on whether surface ligands are physically accessible for molecular interactions. Here, we establish high\u2010speed atomic force microscopy (HS\u2010AFM) as a label\u2010free, real\u2010time approach for directly visualizing and quantifying ligand accessibility on individual small extracellular vesicles (sEVs) under near\u2010physiological conditions. Using programmed death\u2010ligand 1 (PD\u2010L1) as a model immune checkpoint, we characterize antibody binding dynamics on sEVs derived from luminal breast cancer cells, triple\u2010negative breast cancer (TNBC) cells, and patient\u2010derived TNBC cells. PD\u2010L1\u2010high TNBC sEVs exhibit frequent, stable antibody docking with prolonged dwell times, whereas PD\u2010L1\u2010low luminal sEVs display predominantly transient interactions despite detectable PD\u2010L1 expression. Patient\u2010derived sEVs show intermediate binding dynamics, revealing functional heterogeneity that is not captured by bulk protein measurements. By integrating binding frequency and interaction dwell time, we establish a quantitative framework for nanoscale functional phenotyping of individual vesicles. These findings identify ligand accessibility as a distinct biophysical property complementary to protein abundance and establish HS\u2010AFM as a platform for investigating membrane protein function at single\u2010vesicle resolution.<\/jats:p>","DOI":"10.1002\/smtd.71041","type":"journal-article","created":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T07:25:01Z","timestamp":1789629901000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Single\u2010Vesicle Functional Nanophenotyping Reveals Heterogeneous PD\u2010L1 Accessibility in Triple\u2010Negative Breast Cancer"],"prefix":"10.1002","author":[{"given":"Reon","family":"Imakawa","sequence":"first","affiliation":[{"name":"Division of Nano Life Science, Graduate School of Frontier Science Initiative Kanazawa University  Kanazawa 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