{"doi":"10.1021/acs.jpcb.2c09053","title":"Error-Correction Method for High-Throughput Sizing of Nanoscale Vesicles with Single-Molecule Localization Microscopy","abstract":"Single-molecule localization microscopy (SMLM) allows super-resolution imaging, mapping, counting, and sizing of biological nanostructures such as cell organelles and extracellular vesicles (EVs), but sizing structures smaller than ∼100 nm can be inaccurate due to single-molecule localization error caused by distortion of the point spread function and limited photon number. Here we demonstrate a method to correct localization error when sizing vesicles and other spherical nanoparticles with SMLM and compare sizing results using two vesicle labeling schemes. We use mean approximation theory to derive a simple equation using full width at half-maximum (FWHM) for correcting particle sizes measured by two-dimensional SMLM, validate the method by sizing streptavidin-coated polystyrene nanobeads with the SMLM technique d STORM with and without error correction, using transmission electron microscopy (TEM) for comparison, and then apply the method to sizing small seminal EVs. Nanobead sizes measured by d STORM became increasingly less accurate (larger than TEM values) for beads smaller than 50 nm. The error-correction method reduced the size difference versus TEM from 15% without error correction to 7% with error correction for 40 nm beads, from 44% to 9% for 30 nm beads, and from 66% to 15% for 20 nm beads. Seminal EVs were labeled with a lipophilic membrane dye (MemBright 700) and with an Alexa Fluor 488-anti-CD63 antibody conjugate, and were sized separately using both dyes by d STORM. Error-corrected exosome diameters were smaller than uncorrected values: 72 nm vs 79 nm mean diameter with membrane dyes; 84 nm vs 97 nm with the antibody-conjugated dyes. The mean error-corrected diameter was 12 nm smaller when using the membrane dye than when using the antibody-conjugated dye likely due to the large size of the antibody. Thus, both the error-correction method and the compact membrane labeling scheme reduce overestimation of vesicle size by SMLM. This error-correction method has a low computational cost as it does not require correction of individual blinking events, and it is compatible with all SMLM techniques (e.g., PALM, STORM, and DNA-PAINT).","journal":"The Journal of Physical Chemistry B","year":2023,"id":369941,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9524,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":97948,"name":"James Kim","orcid":"0000-0002-2992-9222","position":1,"is_corresponding":false},{"id":407079,"name":"Lucia Vojtech","orcid":"0000-0003-2343-0719","position":2,"is_corresponding":false},{"id":279206,"name":"Joshua C. Vaughan","orcid":"0000-0002-6550-8935","position":3,"is_corresponding":false},{"id":483362,"name":"Daniel T. Chiu","orcid":"0000-0003-2964-9578","position":4,"is_corresponding":false},{"id":501223,"name":"Seung‐Ryoung Jung","orcid":"0000-0002-1870-1433","position":0,"is_corresponding":true}],"reference_count":26,"raw_metadata":null,"created_at":"2026-07-19T01:15:32.670954Z","pmid":"36944080","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}