{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T01:50:45Z","timestamp":1788313845195,"version":"build-2803163510"},"posted":{"date-parts":[[2024,3,6]]},"group-title":"Cell Biology","reference-count":62,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,3,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  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 (\n                  <jats:italic>d<\/jats:italic>\n                  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.\n                <\/jats:p>","DOI":"10.1101\/2024.03.04.583337","type":"posted-content","created":{"date-parts":[[2024,3,6]],"date-time":"2024-03-06T18:46:12Z","timestamp":1709750772000},"source":"Crossref","is-referenced-by-count":1,"title":["Quantitative Superresolution Imaging of F-Actin in the Cell Body and Cytoskeletal Protrusions Using Phalloidin-Based Single-Molecule Labeling and Localization Microscopy"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3750-2309","authenticated-orcid":false,"given":"Hirushi","family":"Gunasekara","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0452-945X","authenticated-orcid":false,"given":"Thilini","family":"Perera","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-1892-7754","authenticated-orcid":false,"given":"Chih-Jia","family":"Chao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2261-9421","authenticated-orcid":false,"given":"Joshua","family":"Bruno","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5522-6694","authenticated-orcid":false,"given":"Badeia","family":"Saed","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5731-5511","authenticated-orcid":false,"given":"Jesse","family":"Anderson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8979-844X","authenticated-orcid":false,"given":"Zongmin","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3912-7063","authenticated-orcid":false,"given":"Ying S.","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2024030902550600000_2024.03.04.583337v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1841"},{"key":"2024030902550600000_2024.03.04.583337v1.2","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E12-12-0856"},{"key":"2024030902550600000_2024.03.04.583337v1.3","doi-asserted-by":"crossref","first-page":"4363","DOI":"10.1038\/s41467-022-32038-0","article-title":"Actin Nano-Architecture of Phagocytic Podosomes","volume":"13","year":"2022","journal-title":"Nat. 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