Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells

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dc.citation.firstpage3050en_US
dc.citation.issueNumber5en_US
dc.citation.journalTitleBiomedical Optics Expressen_US
dc.citation.lastpage3063en_US
dc.citation.volumeNumber15en_US
dc.contributor.authorNelson, Tyleren_US
dc.contributor.authorVargas-Hernández, Sofíaen_US
dc.contributor.authorFreire, Margarethen_US
dc.contributor.authorCheng, Siyangen_US
dc.contributor.authorGustavsson, Anna-Karinen_US
dc.contributor.orgSmalley-Curl Institute;Institute of Biosciences & Bioengineering;Center for Nanoscale Imaging Sciencesen_US
dc.date.accessioned2024-08-22T15:28:49Zen_US
dc.date.available2024-08-22T15:28:49Zen_US
dc.date.issued2024en_US
dc.description.abstractSingle-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin – a protein located in the focal adhesion complex – and actin in human osteosarcoma cells.en_US
dc.identifier.citationNelson, T., Vargas-Hernández, S., Freire, M., Cheng, S., & Gustavsson, A.-K. (2024). Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells. Biomedical Optics Express, 15(5), 3050–3063. https://doi.org/10.1364/BOE.521362en_US
dc.identifier.digitalboe-15-5-3050en_US
dc.identifier.doihttps://doi.org/10.1364/BOE.521362en_US
dc.identifier.urihttps://hdl.handle.net/1911/117695en_US
dc.language.isoengen_US
dc.publisherOptica Publishing Groupen_US
dc.rightsPublished under the terms of the Optica Open Access Publishing Agreementen_US
dc.rights.urihttps://opg.optica.org/library/license_v2.cfm#VOR-OAen_US
dc.titleMultimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cellsen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
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