{"doi":"10.1101/2024.03.04.583337","title":"Quantitative Superresolution Imaging of F-Actin in the Cell Body and Cytoskeletal Protrusions Using Phalloidin-Based Single-Molecule Labeling and Localization Microscopy","abstract":"ABSTRACT We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy ( d STORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":494958,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9532,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":886228,"name":"Thilini Perera","orcid":"0000-0002-0452-945X","position":1,"is_corresponding":false},{"id":1341485,"name":"Chih-Jia Chao","orcid":"0009-0006-1892-7754","position":2,"is_corresponding":false},{"id":1091751,"name":"J Bruno","orcid":"0000-0003-2261-9421","position":3,"is_corresponding":false},{"id":886227,"name":"Badeia Saed","orcid":"0000-0002-5522-6694","position":4,"is_corresponding":false},{"id":1112406,"name":"Jesse Anderson","orcid":"0000-0001-5731-5511","position":5,"is_corresponding":false},{"id":107100,"name":"Zongmin Zhao","orcid":"0000-0001-8979-844X","position":6,"is_corresponding":false},{"id":490385,"name":"Ying Hu","orcid":"0000-0002-3912-7063","position":7,"is_corresponding":false},{"id":1112405,"name":"Hirushi Gunasekara","orcid":"0000-0002-3750-2309","position":0,"is_corresponding":true}],"reference_count":63,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:09:15.528853Z","pmid":"38496456","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":[]}