{"doi":"10.1101/2025.04.07.647376","title":"SpeedyTrack: Direct microsecond wide-field single-molecule tracking and super-resolution mapping via CCD vertical shift","abstract":"Abstract We introduce spatially-encoded dynamics tracking (SpeedyTrack), a strategy to enable direct microsecond wide-field single-molecule tracking/imaging on common microscopy setups. Capitalizing on the native sub-microsecond vertical charge shifting capability of popular electron-multiplying charge-coupled devices (EM-CCDs), SpeedyTrack staggers wide-field single-molecule images along the CCD chip at ∼10-row spacings between consecutive timepoints, effectively projecting the time domain to the spatial domain. Wide-field tracking is thus achieved for freely diffusing molecules at down to 50 µs temporal resolutions for >30 timepoints, permitting trajectory analysis to quantify diffusion coefficients up to 1,000 µm 2 /s. Concurrent acquisition of single-molecule diffusion trajectories and Förster resonance energy transfer (FRET) time traces further elucidates conformational dynamics and binding states for diffusing molecules. Moreover, with a temporally patterned vertical shifting scheme, we deconvolve the spatial and temporal information to map long, fast single-molecule trajectories at the super-resolution level, thus resolving the diffusion mode of a fluorescent protein in live cells with nanoscale resolution. While these demonstrated capabilities substantially outperform existing approaches, SpeedyTrack further stands out for its simplicity by directly working off the built-in functionalities of EM-CCDs without the need to modify existing optics or electronics. We thus provide a facile solution to the microsecond tracking/imaging of single molecules and their super-resolution mapping in the wide field.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":556136,"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":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9573,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":218681,"name":"Ke Xu","orcid":"0000-0002-2788-194X","position":1,"is_corresponding":false},{"id":1014551,"name":"Megan A. Steves","orcid":"0000-0002-1410-5650","position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":null,"created_at":"2026-07-19T02:55:03.976486Z","pmid":"40291653","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":[]}