Extending resolution of structured illumination microscopy with sparse deconvolution

Extending resolution of structured illumination microscopy with sparse deconvolution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Extending resolution of structured illumination microscopy with sparse deconvolution Weisong Zhao, Shiqun Zhao, Liuju Li, Xiaoshuai Huang, Shijia Xing, and 22 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-279271/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The spatial resolutions of live-cell super-resolution microscopes are limited by the maximum collected photon flux. Taking advantage of a priori knowledge of the sparsity and continuity of biological structures, we develop a deconvolution algorithm that further extends the resolution of super-resolution microscopes under the same photon budgets by nearly twofold. As a result, sparse structured illumination microscopy (Sparse-SIM) achieves ~60 nm resolution at a 564 Hz frame rate, allowing it to resolve intricate structural intermediates, including small vesicular fusion pores, ring-shaped nuclear pores formed by different nucleoporins, and relative movements between the inner and outer membranes of mitochondria in live cells. Likewise, sparse deconvolution can be used to increase the three-dimensional resolution and contrast of spinning-disc confocal-based SIM (SD-SIM), and operates under conditions with the insufficient signal-to-noise-ratio, all of which allows routine four-color, three-dimensional, ~90 nm resolution live-cell super-resolution imaging. Overall, sparse deconvolution may be a general tool to push the spatiotemporal resolution limits of live-cell fluorescence microscopy. Optics/Lasers General Cell Biology & Physiology Photon Flux Intricate Structural Intermediates Signal-to-noise Ratio Spatiotemporal Resolution Live-cell Fluorescence Microscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryinformationSparseSIM.pdf Supplementary information 1MovieNup98Longterm.mp4 Supplementary Video 1 2MovieActin.mp4 Supplementary Video 2 3VideoCaveoleTIRF.mp4 Supplementary Video 3 4Movielamplysolipid.mp4 Supplementary Video 4 5MovieFusionPoreinrealtime.mp4 Supplementary Video 5 6MovieFusionPore.mp4 Supplementary Video 6 7MovieMito.mp4 Supplementary Video 7 8MovieMitoER.mp4 Supplementary Video 8 9MovieCCP.mp4 Supplementary Video 9 10Movie4colorlysomitonucltub.mp4 Supplementary Video 10 11MovieTOM203DMito.mp4 Supplementary Video 11 12MovieER.mp4 Supplementary Video 12 13Moviemicrotubulelysosomeperoxisome.mp4 Supplementary Video 13 14Movie3color3D.mp4 Supplementary Video 14 15MovieActinWFSIM.mp4 Supplementary Video 15 16MovieActinCCP.mp4 Supplementary Video 16 17MovielysoER.mp4 Supplementary Video 17 18MovieMTPM.mp4 Supplementary Video 18 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-279271","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":13865182,"identity":"4c8c0ac0-c21d-400d-addd-f935cf994723","order_by":0,"name":"Weisong Zhao","email":"","orcid":"https://orcid.org/0000-0002-5969-1956","institution":"Harbin Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weisong","middleName":"","lastName":"Zhao","suffix":""},{"id":13865183,"identity":"c04f2ffc-a9b0-47ae-9a9e-d9ce1bda6352","order_by":1,"name":"Shiqun Zhao","email":"","orcid":"","institution":"Peking 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University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Liangyi","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2021-02-26 14:25:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-279271/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-279271/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6499642,"identity":"419f619a-5f54-4d11-9e9f-78d3dad86a00","added_by":"auto","created_at":"2021-03-02 00:20:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":606659,"visible":true,"origin":"","legend":"Evaluating Sparse-SIM with DNA origami samples. (a-d) DNA origami samples (Method) with 60 nm (a), 80 nm (b), 100 nm (c), and 120 nm (d) designed distances imaged by TIRF (top), TIRF-SIM (middle), and Sparse-SIM ×2 (bottom) configurations. (e, f) Enlarged regions enclosed by the white boxes in (a-d) under TIRF-SIM (e) and Sparse-SIM ×2 (f) configurations. (g) Corresponding intensity profiles and multiple Gaussian fitting of the DNA origami structures indicated by the white arrows in (e), respectively. The numbers represent the FWHM and distance between peaks. (h) Average fitted distances of different DNA origami samples. Each measurement was repeated 12 times. Scale bars: (d) 500 nm; (e) 50 nm. ","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-279271/v1/0cbef8c29e5d95358ef9fa49.png"},{"id":6499965,"identity":"64cfe362-832f-471c-b818-8da20afbdeea","added_by":"auto","created_at":"2021-03-02 00:23:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1232712,"visible":true,"origin":"","legend":"Sparse-SIM resolves known structures of ~60 nm in size. (a-b) Separation of two fluorescent lines with interline spacing down to 60 nm by the Sparse-SIM. Raw images were either reconstructed with Wiener algorithm (2D-SIM), or Fourier interpolated before reconstructed with Wiener algorithm followed by Fourier interpolated before reconstructed with the RL deconvolution for 20 (20 RL × 2), 50 (50 RL × 2) iterations, or the Sparse deconvolution pipeline (Sparse ×2) (a). The region enclosed by the yellow box was magnified and shown in (b). (c) A representative example of dynamic ring-shaped nuclear pores labeled with Nup98-GFP in a live COS-7 cell was observed with Sparse-SIM for more than ten mins. Images under the 2D-SIM and the Sparse-SIM ×2 configurations were shown in the upper and bottom panels, respectively. (d) The snapshot of the nuclear pore structure enclosed by the cyan box in c was compared with a 100-nm fluorescent bead under different reconstruction methods (2D-SIM, 20 RL × 2, 50 RL × 2, Sparse ×2). (e) Because the sizes of nuclear pores were comparable to the resolution of Sparse-SIM and the size of the pixel, we followed the protocol in Supplementary Note 9.1 to derive the actual diameters of nuclear pore structures labeled by Nup35-GFP (red), Nup98-GFP (yellow), Nup93-GFP (green), and Nup107-GFP (cyan), respectively. (f) Average diameters of rings formed by Nup35 (66 ± 3 nm, n = 30 from 3 cells), Nup98 (75 ± 6 nm, n = 40 from 3 cells), Nup93 (79 ± 4 nm, n = 40 from 3 cells), or Nup107 (97 ± 5 nm, n = 40 from 3 cells). Left and right montages showed the results after Wiener reconstruction or Sparse deconvolution. (g) The magenta box in (c) is enlarged and shown at six time points. Scale bars: (c) 500 nm; (d, g) 100 nm. ","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-279271/v1/ac42effa10b17ce8e3e30297.png"},{"id":6499650,"identity":"476937f8-148e-454a-9fd9-fad6cdfd362c","added_by":"auto","created_at":"2021-03-02 00:21:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":955458,"visible":true,"origin":"","legend":"Sparse deconvolution assisted expansion microscopy (Sparse-ExM). (a) The COS-7 cell was immunostained with a primary antibody against α-tubulin, and a second antibody conjugated with AF488. We showed the 4.5 times expanded cell (ExM-4.5×) in the background and the Sparse ExM-4.5× image in the center. (b) Magnified views of the regions in (a) under ExM-4.5×, Sparse ExM-4.5×, and ExM-4.5× under the 2D-SIM (2D-SIM ExM-4.5×). (c) Intensity profiles and multiple Gaussian fitting of the filaments are indicated by the white arrows in (c), respectively. The numbers represent the distances between peaks. (d) ExM images of Sec61β-GFP in a COS-7 cell. We showed the 4.5 times expanded cell (ExM-4.5×) in the background and the Sparse ExM-4.5× image in the center. (e) Enlarged regions enclosed by the white box in (a) seen under ExM-4.5×, Sparse ExM-4.5×, and 2D-SIM ExM-4.5×. (f) Highlighted regions from white boxes in (e). Scale bars: (a, d) 1 μm; (b) 300 nm; (e) 500 nm; (f) 200 nm.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-279271/v1/ee81f7c364f554913f3efbc7.png"},{"id":6499645,"identity":"f675009e-2680-4e1c-b645-cd3d140f9d76","added_by":"auto","created_at":"2021-03-02 00:21:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1159296,"visible":true,"origin":"","legend":"Sparse-SIM achieves ~60 nm and millisecond spatiotemporal resolution in live cells. (a) A representative COS-7 cell labeled with LifeAct-EGFP. (b-c) Enlarged regions enclosed by the white box in a (b), and the corresponding profiles along lines (c). Each box in (c) denotes the intensity of one pixel. (d) Average contrasts of two peaks with different distances. (e) FRC maps of actin filaments. (f) Resolutions measured as the FWHMs and by the FRC. (g) Time-dependent minimal FRC resolutions. Black triangles represent the theoretical resolution limit of 2D-SIM. (h) The magnified view of actin filaments in the yellow box from (a) (left) and the segmented version (right, Method). (i) Cumulative distributions of pore sizes within the actin meshes in (h). (j) A representative COS-7 cell labeled with caveolin-EGFP. (k-l) From top to bottom are magnified views of the white box in (j) reconstructed by TIRF-SIM, Sparse-SIM, and Sparse-SIM ×2 with upsampling (k), and their fluorescence profiles are shown in (l). (m) Lysosomes were labeled with LAMP1-EGFP (left, yellow) or LysoView (middle, cyan), while lipid droplets were labeled with LipidSpot (right, magenta). (n) Average diameters of different vesicles. (o) Representative montages of a vesicle fusion event. (p) Kymographs from lines in TIRF-SIM (upper) and Sparse-SIM ×2 (lower) images are shown in (o). (q) Average opening time (left), diameters (middle), and duration time of early (yellow) and stationary (green) fusion pores (right). Centerline, medians; limits, 75% and 25%; whiskers, maximum and minimum; error bars, s.e.m.; Cumul. Freq., cumulative frequency; scale bars: (a, e, and m top) 5 μm; (b and j) 500 nm; (h and m bottom) 1 μm; (k, o) 100 nm; (p) 500 ms.","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-279271/v1/cb1102e7f9136f1e5c8fc2a6.png"},{"id":6499648,"identity":"8830a7aa-bb9f-483e-9bdc-65ca855512ac","added_by":"auto","created_at":"2021-03-02 00:21:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1160558,"visible":true,"origin":"","legend":"Sparse SD-SIM enables three-dimensional, multi-color, and sub-90-nm resolution for live-cell SR imaging. (a) A snapshot of CCPs in a COS-7 cell. (b) The profile corresponding to the resolved central ring of the CCP. (c) Average minimal resolutions by the FRC method. (d) Histogram of the diameters of CCPs. (e-f) Magnified view of the white boxed region (e) and yellow boxed region (f) in (a). (g) Temporal projections of CCPs within 16 mins. (h) A representative example of four-color (LAMP1-mCherry, yellow; MitoTracker, green; Hoechst, blue; Tubulin-EGFP, brown), live-cell SR imaging. (i) Magnified view of the white boxed region in (h). (j) Average resolutions by the FRC method. (k) Three-dimensional distributions of all mitochondria (labeled with TOM20-mCherry) of a live COS-7 cell. (l) Color-coded horizontal (left) and vertical sections (right) from the white boxed region in (k). Centerline, medians; limits, 75% and 25%; whiskers, maximum and minimum; error bars, s.e.m; scale bars: (a, i) 3 μm; (e, f) 300 nm; (g, l) 1 μm; (h, k) 5 μm. ","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-279271/v1/8958802b18be290cc818bbb7.png"},{"id":6499662,"identity":"981b10cb-af08-4392-8ff8-46882497d6ae","added_by":"auto","created_at":"2021-03-02 00:21:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2465468,"visible":true,"origin":"","legend":"Upsampling enables Sparse SD-SIM to overcome the Nyquist sampling limit to achieve multi-color 3D SR imaging in live cells. (a) ER tubules in a COS-7 cell seen under different configurations. (b) Average resolutions by the FRC method. (c) Magnified views of ER tubules from (a). (d) Fourier transforms of images. Black arrows indicate the OTFs of corresponding images. (e) A snapshot of a HeLa cell labeled with tubulin-EGFP (magenta), Pex11a-BFP (cyan), and Lamp1-mCherry (yellow). (f) Magnified views in (e). As highlighted by white arrows in the bottom panel, only Sparse SD-SIM ×2 can dissect the lysosome's deformation by a neighboring peroxisome. (g) Time-lapse images of another example of the co-movement of both a lysosome and a peroxisome along a microtubule. (h) Live-cell three-color (Tubulin-EGFP, green; Hoechst, cyan; MitoTracker Deep Red, magenta) 3D imaging by Sparse SD-SIM ×2. (i) The z-axial view from (h). (j) Three horizontal sections of the cellular nucleus (top) and mitochondria merged with microtubules (bottom). (k) Color-coded volumes of nuclei, mitochondria, and microtubules. Scale bars: (a, d, e, h, i, j, and k) 5 μm; (b and f top, and g) 3 μm; (c) 1 μm; (f middle and bottom) 500 nm.","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-279271/v1/c750ff7f860f79e56102c15f.png"},{"id":13598725,"identity":"384337c6-a62c-424a-8fb2-52d4c23d1c11","added_by":"auto","created_at":"2021-09-17 05:36:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7617287,"visible":true,"origin":"","legend":"Article File","description":"","filename":"MaintextSparseSIM.pdf","url":"https://assets-eu.researchsquare.com/files/rs-279271/v1_covered.pdf"},{"id":6500193,"identity":"82b1a4be-c920-44c3-bb8f-318199535826","added_by":"auto","created_at":"2021-03-02 00:30:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7352093,"visible":true,"origin":"","legend":"Article 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However, the latest manuscript can be downloaded and \u003ca href='/article/rs-279271/latest.pdf' target='_blank'\u003e accessed as a PDF.\u003c/a\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Photon Flux, Intricate Structural Intermediates, Signal-to-noise Ratio, Spatiotemporal Resolution, Live-cell Fluorescence Microscopy","lastPublishedDoi":"10.21203/rs.3.rs-279271/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-279271/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The spatial resolutions of live-cell super-resolution microscopes are limited by the maximum collected photon flux. Taking advantage of a priori knowledge of the sparsity and continuity of biological structures, we develop a deconvolution algorithm that further extends the resolution of super-resolution microscopes under the same photon budgets by nearly twofold. As a result, sparse structured illumination microscopy (Sparse-SIM) achieves ~60 nm resolution at a 564 Hz frame rate, allowing it to resolve intricate structural intermediates, including small vesicular fusion pores, ring-shaped nuclear pores formed by different nucleoporins, and relative movements between the inner and outer membranes of mitochondria in live cells. Likewise, sparse deconvolution can be used to increase the three-dimensional resolution and contrast of spinning-disc confocal-based SIM (SD-SIM), and operates under conditions with the insufficient signal-to-noise-ratio, all of which allows routine four-color, three-dimensional, ~90 nm resolution live-cell super-resolution imaging. Overall, sparse deconvolution may be a general tool to push the spatiotemporal resolution limits of live-cell fluorescence microscopy.","manuscriptTitle":"Extending resolution of structured illumination microscopy with sparse deconvolution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-03-02 00:20:55","doi":"10.21203/rs.3.rs-279271/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"31021006-cd79-4772-b68b-197a384baa22","owner":[],"postedDate":"March 2nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2675496,"name":"Optics/Lasers"},{"id":2675497,"name":"General Cell Biology \u0026 Physiology"}],"tags":[],"updatedAt":"2021-06-09T10:31:19+00:00","versionOfRecord":[],"versionCreatedAt":"2021-03-02 00:20:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-279271","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-279271","identity":"rs-279271","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}