{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T03:56:48Z","timestamp":1789099008415,"version":"build-2803163510"},"reference-count":42,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2024,8,29]],"date-time":"2024-08-29T00:00:00Z","timestamp":1724889600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2024,8,29]],"date-time":"2024-08-29T00:00:00Z","timestamp":1724889600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Nat Commun"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Activation of caged fluorophores in microscopy has mostly relied on the absorption of a single ultraviolet (UV) photon of \u2272400\u2009nm wavelength or on the simultaneous absorption of two near-infrared (NIR) photons &gt;700\u2009nm. Here, we show that two green photons (515\u2009nm) can substitute for a single photon (~260\u2009nm) to activate popular silicon-rhodamine (Si-R) dyes. Activation in the green range eliminates the chromatic aberrations that plague activation by UV or NIR light. Thus, in confocal fluorescence microscopy, the activation focal volume can be matched with that of confocal detection. Besides, detrimental losses of UV and NIR light in the optical system are avoided. We apply two-photon activation (2PA) of three Si-R dyes in different superresolution approaches. STED microscopy of thick samples is improved through optical sectioning and photobleaching reduced by confining active fluorophores to a thin layer. 2PA of individualized fluorophores enables MINSTED nanoscopy with nanometer-resolution.<\/jats:p>","DOI":"10.1038\/s41467-024-51160-9","type":"journal-article","created":{"date-parts":[[2024,8,29]],"date-time":"2024-08-29T09:02:34Z","timestamp":1724922154000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Bleaching protection and axial sectioning in fluorescence nanoscopy through two-photon activation at 515\u2009nm"],"prefix":"10.1038","volume":"15","author":[{"given":"Jan-Erik","family":"Bredfeldt","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7189-0491","authenticated-orcid":false,"given":"Joanna","family":"Oracz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-2207-8007","authenticated-orcid":false,"given":"Kamila A.","family":"Kiszka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-7040-0612","authenticated-orcid":false,"given":"Thea","family":"Moosmayer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9680-7543","authenticated-orcid":false,"given":"Michael","family":"Weber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2917-0778","authenticated-orcid":false,"given":"Steffen J.","family":"Sahl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9638-5077","authenticated-orcid":false,"given":"Stefan W.","family":"Hell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,8,29]]},"reference":[{"key":"51160_CR1","doi-asserted-by":"crossref","unstructured":"Pawley, J. B. Handbook of Biological Confocal Microscopy. Springer (2006).","DOI":"10.1007\/978-0-387-45524-2"},{"key":"51160_CR2","unstructured":"Minsky, M. Microscopy apparatus. Patent US3013467A (1961)."},{"key":"51160_CR3","doi-asserted-by":"crossref","unstructured":"Sheppard, C. J. R. Scanning optical microscopy. in Advances in Imaging and Electron Physics, 213 (eds. H\u00fftch, M. & Hawkes, P. W.) 227\u2013325 (Elsevier, 2020).","DOI":"10.1016\/bs.aiep.2019.11.001"},{"key":"51160_CR4","doi-asserted-by":"publisher","first-page":"685","DOI":"10.1038\/nrm.2017.71","volume":"18","author":"SJ Sahl","year":"2017","unstructured":"Sahl, S. J., Hell, S. W. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat. Rev. Mol. Cell Biol. 18, 685\u2013701 (2017).","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"51160_CR5","doi-asserted-by":"publisher","first-page":"1723","DOI":"10.1002\/anie.201509649","volume":"55","author":"JB Grimm","year":"2016","unstructured":"Grimm, J. B. et al. Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy. Angew. Chem. Int. Ed. 55, 1723\u20131727 (2016).","journal-title":"Angew. Chem. Int. Ed."},{"key":"51160_CR6","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-12480-3","volume":"10","author":"MS Frei","year":"2019","unstructured":"Frei, M. S. et al. Photoactivation of silicon rhodamines via a light-induced protonation. Nat. Commun. 10, 4580 (2019).","journal-title":"Nat. Commun."},{"key":"51160_CR7","doi-asserted-by":"publisher","first-page":"451","DOI":"10.1002\/chem.202004645","volume":"27","author":"M Weber","year":"2021","unstructured":"Weber, M. et al. Photoactivatable fluorophore for stimulated emission depletion (STED) microscopy and bioconjugation technique for hydrophobic labels. Chem. A Eur. J. 27, 451\u2013458 (2021).","journal-title":"Chem. A Eur. J."},{"key":"51160_CR8","doi-asserted-by":"publisher","first-page":"18388","DOI":"10.1021\/jacs.1c09999","volume":"143","author":"AN Butkevich","year":"2021","unstructured":"Butkevich, A. N. et al. Photoactivatable fluorescent dyes with hydrophilic caging groups and their use in multicolor nanoscopy. J. Am. Chem. Soc. 143, 18388\u201318393 (2021).","journal-title":"J. Am. Chem. Soc."},{"key":"51160_CR9","doi-asserted-by":"publisher","first-page":"4653","DOI":"10.1021\/ja036958m","volume":"126","author":"Y Zhao","year":"2004","unstructured":"Zhao, Y. et al. New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications. J. Am. Chem. Soc. 126, 4653\u20134663 (2004).","journal-title":"J. Am. Chem. Soc."},{"key":"51160_CR10","doi-asserted-by":"publisher","first-page":"1346","DOI":"10.1529\/biophysj.104.054502","volume":"89","author":"M Schneider","year":"2005","unstructured":"Schneider, M., Barozzi, S., Testa, I., Faretta, M. & Diaspro, A. Two-photon activation and excitation properties of PA-GFP in the 720\u2013920-nm region. Biophys. J. 89, 1346\u20131352 (2005).","journal-title":"Biophys. J."},{"key":"51160_CR11","doi-asserted-by":"publisher","DOI":"10.1038\/srep23866","volume":"6","author":"SJJ Kwok","year":"2016","unstructured":"Kwok, S. J. J. et al. Two-photon excited photoconversion of cyanine-based dyes. Sci. Rep. 6, 23866 (2016).","journal-title":"Sci. Rep."},{"key":"51160_CR12","doi-asserted-by":"publisher","first-page":"022001","DOI":"10.1088\/2050-6120\/ab7365","volume":"8","author":"AP Demchenko","year":"2020","unstructured":"Demchenko, A. P. Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection. Methods Appl. Fluorescence 8, 022001 (2020).","journal-title":"Methods Appl. Fluorescence"},{"key":"51160_CR13","doi-asserted-by":"publisher","first-page":"780","DOI":"10.1364\/OL.19.000780","volume":"19","author":"SW Hell","year":"1994","unstructured":"Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780\u2013782 (1994).","journal-title":"Opt. Lett."},{"key":"51160_CR14","doi-asserted-by":"publisher","first-page":"8206","DOI":"10.1073\/pnas.97.15.8206","volume":"97","author":"TA Klar","year":"2000","unstructured":"Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206\u20138210 (2000).","journal-title":"Proc. Natl Acad. Sci. USA"},{"key":"51160_CR15","doi-asserted-by":"publisher","first-page":"935","DOI":"10.1038\/nature04592","volume":"440","author":"KI Willig","year":"2006","unstructured":"Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935\u2013939 (2006).","journal-title":"Nature"},{"key":"51160_CR16","doi-asserted-by":"publisher","first-page":"1347","DOI":"10.1038\/nbt895","volume":"21","author":"SW Hell","year":"2003","unstructured":"Hell, S. W. Toward fluorescence nanoscopy. Nat. Biotech. 21, 1347\u20131355 (2003).","journal-title":"Nat. Biotech."},{"key":"51160_CR17","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1038\/nmeth.1291","volume":"6","author":"SW Hell","year":"2009","unstructured":"Hell, S. W. Microscopy and its focal switch. Nat. Methods 6, 24\u201332 (2009).","journal-title":"Nat. Methods"},{"key":"51160_CR18","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-09902-x","volume":"7","author":"J Oracz","year":"2017","unstructured":"Oracz, J., Westphal, V., Radzewicz, C., Sahl, S. J. & Hell, S. W. Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore. Sci. Rep. 7, 11354 (2017).","journal-title":"Sci. Rep."},{"key":"51160_CR19","doi-asserted-by":"publisher","first-page":"122","DOI":"10.1038\/nphoton.2015.266","volume":"10","author":"JG Danzl","year":"2016","unstructured":"Danzl, J. G. et al. Coordinate-targeted fluorescence nanoscopy with multiple off states. Nat. Photonics 10, 122\u2013128 (2016).","journal-title":"Nat. Photonics"},{"key":"51160_CR20","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1038\/nmeth.1413","volume":"7","author":"S Kantevari","year":"2010","unstructured":"Kantevari, S., Matsuzaki, M., Kanemoto, Y., Kasai, H. & Ellis-Davies, G. C. R. Two-color, two-photon uncaging of glutamate and GABA. Nat. Methods 7, 123\u2013125 (2010).","journal-title":"Nat. Methods"},{"key":"51160_CR21","doi-asserted-by":"crossref","unstructured":"Ellis-Davies, G. C. R. Two-Photon Uncaging of Glutamate. Front. Synap. Neurosci. 10(2019).","DOI":"10.3389\/fnsyn.2018.00048"},{"key":"51160_CR22","doi-asserted-by":"crossref","unstructured":"Oron, D., Papagiakoumou, E., Anselmi, F. & Emiliani, V. Two-photon optogenetics. in Progress in Brain Research, 196 (eds.Kn\u00f6pfel, T. & Boyden, E. S.) 119-143 (Elsevier, 2012).","DOI":"10.1016\/B978-0-444-59426-6.00007-0"},{"key":"51160_CR23","doi-asserted-by":"publisher","first-page":"1202","DOI":"10.1038\/nmeth.2249","volume":"9","author":"AM Packer","year":"2012","unstructured":"Packer, A. M. et al. Two-photon optogenetics of dendritic spines and neural circuits. Nat. Methods 9, 1202\u20131205 (2012).","journal-title":"Nat. Methods"},{"key":"51160_CR24","doi-asserted-by":"publisher","first-page":"2666","DOI":"10.1021\/acsphotonics.1c00685","volume":"8","author":"T Kubo","year":"2021","unstructured":"Kubo, T. et al. Visible-wavelength multiphoton activation confocal microscopy. ACS Photonics 8, 2666\u20132673 (2021).","journal-title":"ACS Photonics"},{"key":"51160_CR25","doi-asserted-by":"publisher","first-page":"8862","DOI":"10.1364\/OE.24.008862","volume":"24","author":"BR Patton","year":"2016","unstructured":"Patton, B. R. et al. Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics. Opt. Express 24, 8862\u20138876 (2016).","journal-title":"Opt. Express"},{"key":"51160_CR26","doi-asserted-by":"publisher","first-page":"3631","DOI":"10.1364\/OL.41.003631","volume":"41","author":"J Antonello","year":"2016","unstructured":"Antonello, J., Kromann, E. B., Burke, D., Bewersdorf, J. & Booth, M. J. Coma aberrations in combined two- and three-dimensional STED nanoscopy. Opt. Lett. 41, 3631\u20133634 (2016).","journal-title":"Opt. Lett."},{"key":"51160_CR27","doi-asserted-by":"publisher","first-page":"361","DOI":"10.1038\/s41566-021-00774-2","volume":"15","author":"M Weber","year":"2021","unstructured":"Weber, M. et al. MINSTED fluorescence localization and nanoscopy. Nat. Photonics 15, 361\u2013366 (2021).","journal-title":"Nat. Photonics"},{"key":"51160_CR28","doi-asserted-by":"publisher","first-page":"4242","DOI":"10.1364\/OE.19.004242","volume":"19","author":"JR Moffitt","year":"2011","unstructured":"Moffitt, J. R., Osseforth, C. & Michaelis, J. Time-gating improves the spatial resolution of STED microscopy. Opt. Express 19, 4242\u20134254 (2011).","journal-title":"Opt. Express"},{"key":"51160_CR29","doi-asserted-by":"publisher","first-page":"571","DOI":"10.1038\/nmeth.1624","volume":"8","author":"G Vicidomini","year":"2011","unstructured":"Vicidomini, G. et al. Sharper low-power STED nanoscopy by time gating. Nat. Methods 8, 571\u2013573 (2011).","journal-title":"Nat. Methods"},{"key":"51160_CR30","doi-asserted-by":"publisher","first-page":"10226","DOI":"10.1039\/D0NR00227E","volume":"12","author":"S Sograte-Idrissi","year":"2020","unstructured":"Sograte-Idrissi, S. et al. Circumvention of common labelling artefacts using secondary nanobodies. Nanoscale 12, 10226\u201310239 (2020).","journal-title":"Nanoscale"},{"key":"51160_CR31","doi-asserted-by":"publisher","first-page":"605","DOI":"10.1038\/nmeth.1220","volume":"5","author":"J Riedl","year":"2008","unstructured":"Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605\u2013607 (2008).","journal-title":"Nat. Methods"},{"key":"51160_CR32","first-page":"12.18.1","volume":"50","author":"KN Fish","year":"2009","unstructured":"Fish, K. N. Total internal reflection fluorescence (TIRF) microscopy. Curr. Protoc. Cytom. 50, 12.18.1\u201312.18.13 (2009).","journal-title":"Curr. Protoc. Cytom."},{"key":"51160_CR33","doi-asserted-by":"publisher","first-page":"606","DOI":"10.1126\/science.aak9913","volume":"355","author":"F Balzarotti","year":"2017","unstructured":"Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606\u2013612 (2017).","journal-title":"Science"},{"key":"51160_CR34","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1038\/s41592-019-0688-0","volume":"17","author":"KC Gwosch","year":"2020","unstructured":"Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217\u2013224 (2020).","journal-title":"Nat. Methods"},{"key":"51160_CR35","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21652-z","volume":"12","author":"R Schmidt","year":"2021","unstructured":"Schmidt, R. et al. MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nat. Commun. 12, 1478 (2021).","journal-title":"Nat. Commun."},{"key":"51160_CR36","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1126\/science.2321027","volume":"248","author":"W Denk","year":"1990","unstructured":"Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73\u201376 (1990).","journal-title":"Science"},{"key":"51160_CR37","doi-asserted-by":"publisher","first-page":"932","DOI":"10.1038\/nmeth818","volume":"2","author":"F Helmchen","year":"2005","unstructured":"Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932\u2013940 (2005).","journal-title":"Nat. Methods"},{"key":"51160_CR38","doi-asserted-by":"publisher","first-page":"E8047","DOI":"10.1073\/pnas.1807104115","volume":"115","author":"J-M Masch","year":"2018","unstructured":"Masch, J.-M. et al. Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling. Proc. Natl Acad. Sci. USA 115, E8047\u2013E8056 (2018).","journal-title":"Proc. Natl Acad. Sci. USA"},{"key":"51160_CR39","doi-asserted-by":"publisher","first-page":"311","DOI":"10.1002\/9780470942390.mo150101","volume":"5","author":"PC Molyneux","year":"2015","unstructured":"Molyneux, P. C., Pyle, L. A., Dillon, M. & Harrington, M. E. A mouse primary hepatocyte culture model for studies of circadian oscillation. Curr. Protoc. Mouse Biol. 5, 311\u2013329 (2015).","journal-title":"Curr. Protoc. Mouse Biol."},{"key":"51160_CR40","doi-asserted-by":"publisher","DOI":"10.1186\/1471-213X-8-23","volume":"8","author":"TC Tuoc","year":"2008","unstructured":"Tuoc, T. C. & Stoykova, A. Er81is a downstream target of Pax6 in cortical progenitors. BMC Dev. Biol. 8, 23 (2008).","journal-title":"BMC Dev. Biol."},{"key":"51160_CR41","first-page":"4851","volume":"27","author":"J Guy","year":"2016","unstructured":"Guy, J. et al. Intracortical network effects preserve thalamocortical input efficacy in a cortex without layers. Cereb. Cortex 27, 4851\u20134866 (2016).","journal-title":"Cereb. Cortex"},{"key":"51160_CR42","doi-asserted-by":"publisher","first-page":"L01","DOI":"10.1016\/j.bpj.2013.11.1119","volume":"106","author":"Willig","year":"2014","unstructured":"Willig et al. Nanoscopy of filamentous actin in cortical dendrites of a living mouse. Biophys. J. 106, L01\u2013L03 (2014).","journal-title":"Biophys. J."}],"container-title":["Nature Communications"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41467-024-51160-9.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41467-024-51160-9","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41467-024-51160-9.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,8,29]],"date-time":"2024-08-29T09:03:04Z","timestamp":1724922184000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41467-024-51160-9"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,8,29]]},"references-count":42,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["51160"],"URL":"https:\/\/doi.org\/10.1038\/s41467-024-51160-9","relation":{},"ISSN":["2041-1723"],"issn-type":[{"value":"2041-1723","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,29]]},"assertion":[{"value":"18 October 2022","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"31 July 2024","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"29 August 2024","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"S.W.H. owns shares of the companies Abberior and Abberior Instruments GmbH, producing superresolution STED dyes and microscopes, respectively. The remaining authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"7472"}}