[{"related_material":{"record":[{"status":"public","id":"12817","relation":"research_data"},{"id":"14770","relation":"shorter_version","status":"public"}],"link":[{"relation":"software","url":"https://github.com/danzllab/LIONESS"}]},"main_file_link":[{"url":"https://doi.org/10.1038/s41592-023-01936-6","open_access":"1"}],"isi":1,"external_id":{"isi":["001025621500001"],"pmid":["37429995"]},"title":"Dense 4D nanoscale reconstruction of living brain tissue","ec_funded":1,"doi":"10.1038/s41592-023-01936-6","year":"2023","acknowledged_ssus":[{"_id":"ScienComp"},{"_id":"Bio"},{"_id":"PreCl"},{"_id":"E-Lib"},{"_id":"LifeSc"},{"_id":"M-Shop"}],"quality_controlled":"1","project":[{"call_identifier":"FWF","_id":"265CB4D0-B435-11E9-9278-68D0E5697425","name":"Optical control of synaptic function via adhesion molecules","grant_number":"I03600"},{"name":"Molecular Drug Targets","_id":"2548AE96-B435-11E9-9278-68D0E5697425","call_identifier":"FWF","grant_number":"W1232-B24"},{"_id":"25C5A090-B435-11E9-9278-68D0E5697425","name":"The Wittgenstein Prize","call_identifier":"FWF","grant_number":"Z00312"},{"name":"High content imaging to decode human immune cell interactions in health and allergic disease","_id":"23889792-32DE-11EA-91FC-C7463DDC885E"},{"call_identifier":"H2020","_id":"2564DBCA-B435-11E9-9278-68D0E5697425","name":"International IST Doctoral Program","grant_number":"665385"},{"grant_number":"715767","_id":"24F9549A-B435-11E9-9278-68D0E5697425","name":"MATERIALIZABLE: Intelligent fabrication-oriented Computational Design and Modeling","call_identifier":"H2020"},{"name":"Probing the Reversibility of Autism Spectrum Disorders by Employing in vivo and in vitro Models","_id":"25444568-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","grant_number":"715508"},{"grant_number":"692692","call_identifier":"H2020","name":"Biophysics and circuit function of a giant cortical glumatergic synapse","_id":"25B7EB9E-B435-11E9-9278-68D0E5697425"},{"name":"Synaptic computations of the hippocampal CA3 circuitry","_id":"fc2be41b-9c52-11eb-aca3-faa90aa144e9","call_identifier":"H2020","grant_number":"101026635"},{"name":"High-speed 3D-nanoscopy to study the role of adhesion during 3D cell migration","_id":"2668BFA0-B435-11E9-9278-68D0E5697425","grant_number":"LT00057"}],"oa_version":"Published Version","acknowledgement":"We thank J. Vorlaufer, N. Agudelo and A. Wartak for microscope maintenance and troubleshooting, C. Kreuzinger and A. Freeman for technical assistance, M. Šuplata for hardware control support and M. Cunha dos Santos for initial exploration of software. We\r\nthank P. Henderson for advice on deep-learning training and M. Sixt, S. Boyd and T. Weiss for discussions and critical reading of the manuscript. L. Lavis (Janelia Research Campus) generously provided the JF585-HaloTag ligand. We acknowledge expert support by IST\r\nAustria’s scientific computing, imaging and optics, preclinical, library and laboratory support facilities and by the Miba machine shop. We gratefully acknowledge funding by the following sources: Austrian Science Fund (F.W.F.) grant no. I3600-B27 (J.G.D.), grant no. DK W1232\r\n(J.G.D. and J.M.M.) and grant no. Z 312-B27, Wittgenstein award (P.J.); the Gesellschaft für Forschungsförderung NÖ grant no. LSC18-022 (J.G.D.); an ISTA Interdisciplinary project grant (J.G.D. and B.B.); the European Union’s Horizon 2020 research and innovation programme,\r\nMarie-Skłodowska Curie grant 665385 (J.M.M. and J.L.); the European Union’s Horizon 2020 research and innovation programme, European Research Council grant no. 715767, MATERIALIZABLE (B.B.); grant no. 715508, REVERSEAUTISM (G.N.); grant no. 695568, SYNNOVATE (S.G.N.G.); and grant no. 692692, GIANTSYN (P.J.); the Simons\r\nFoundation Autism Research Initiative grant no. 529085 (S.G.N.G.); the Wellcome Trust Technology Development grant no. 202932 (S.G.N.G.); the Marie Skłodowska-Curie Actions Individual Fellowship no. 101026635 under the EU Horizon 2020 program (J.F.W.);\r\nthe Human Frontier Science Program postdoctoral fellowship LT000557/2018 (W.J.); and the National Science Foundation grant no. IIS-1835231 (H.P.) and NCS-FO-2124179 (H.P.).","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","publication_identifier":{"eissn":["1548-7105"],"issn":["1548-7091"]},"pmid":1,"_id":"13267","article_processing_charge":"Yes","date_updated":"2024-01-10T08:37:48Z","oa":1,"volume":20,"author":[{"full_name":"Velicky, Philipp","last_name":"Velicky","orcid":"0000-0002-2340-7431","first_name":"Philipp","id":"39BDC62C-F248-11E8-B48F-1D18A9856A87"},{"id":"3FB91342-F248-11E8-B48F-1D18A9856A87","first_name":"Eder","orcid":"0000-0001-5665-0430","full_name":"Miguel Villalba, Eder","last_name":"Miguel Villalba"},{"last_name":"Michalska","full_name":"Michalska, Julia M","orcid":"0000-0003-3862-1235","first_name":"Julia M","id":"443DB6DE-F248-11E8-B48F-1D18A9856A87"},{"first_name":"Julia","full_name":"Lyudchik, Julia","last_name":"Lyudchik","id":"46E28B80-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Wei","full_name":"Wei, Donglai","first_name":"Donglai"},{"first_name":"Zudi","last_name":"Lin","full_name":"Lin, Zudi"},{"last_name":"Watson","full_name":"Watson, Jake","orcid":"0000-0002-8698-3823","first_name":"Jake","id":"63836096-4690-11EA-BD4E-32803DDC885E"},{"first_name":"Jakob","full_name":"Troidl, Jakob","last_name":"Troidl"},{"first_name":"Johanna","last_name":"Beyer","full_name":"Beyer, Johanna"},{"id":"43DF3136-F248-11E8-B48F-1D18A9856A87","first_name":"Yoav","full_name":"Ben Simon, Yoav","last_name":"Ben Simon"},{"first_name":"Christoph M","orcid":"0000-0003-1216-9105","last_name":"Sommer","full_name":"Sommer, Christoph M","id":"4DF26D8C-F248-11E8-B48F-1D18A9856A87"},{"first_name":"Wiebke","full_name":"Jahr, Wiebke","last_name":"Jahr","id":"425C1CE8-F248-11E8-B48F-1D18A9856A87"},{"id":"9ac8f577-2357-11eb-997a-e566c5550886","first_name":"Alban","last_name":"Cenameri","full_name":"Cenameri, Alban"},{"full_name":"Broichhagen, Johannes","last_name":"Broichhagen","first_name":"Johannes"},{"last_name":"Grant","full_name":"Grant, Seth G.N.","first_name":"Seth G.N."},{"id":"353C1B58-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-5001-4804","full_name":"Jonas, Peter M","last_name":"Jonas","first_name":"Peter M"},{"id":"3E57A680-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-7673-7178","last_name":"Novarino","full_name":"Novarino, Gaia","first_name":"Gaia"},{"first_name":"Hanspeter","last_name":"Pfister","full_name":"Pfister, Hanspeter"},{"id":"49876194-F248-11E8-B48F-1D18A9856A87","first_name":"Bernd","last_name":"Bickel","full_name":"Bickel, Bernd","orcid":"0000-0001-6511-9385"},{"id":"42EFD3B6-F248-11E8-B48F-1D18A9856A87","last_name":"Danzl","full_name":"Danzl, Johann G","orcid":"0000-0001-8559-3973","first_name":"Johann G"}],"abstract":[{"text":"Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue.","lang":"eng"}],"citation":{"chicago":"Velicky, Philipp, Eder Miguel Villalba, Julia M Michalska, Julia Lyudchik, Donglai Wei, Zudi Lin, Jake Watson, et al. “Dense 4D Nanoscale Reconstruction of Living Brain Tissue.” <i>Nature Methods</i>. Springer Nature, 2023. <a href=\"https://doi.org/10.1038/s41592-023-01936-6\">https://doi.org/10.1038/s41592-023-01936-6</a>.","ieee":"P. Velicky <i>et al.</i>, “Dense 4D nanoscale reconstruction of living brain tissue,” <i>Nature Methods</i>, vol. 20. Springer Nature, pp. 1256–1265, 2023.","apa":"Velicky, P., Miguel Villalba, E., Michalska, J. M., Lyudchik, J., Wei, D., Lin, Z., … Danzl, J. G. (2023). Dense 4D nanoscale reconstruction of living brain tissue. <i>Nature Methods</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41592-023-01936-6\">https://doi.org/10.1038/s41592-023-01936-6</a>","short":"P. Velicky, E. Miguel Villalba, J.M. Michalska, J. Lyudchik, D. Wei, Z. Lin, J. Watson, J. Troidl, J. Beyer, Y. Ben Simon, C.M. Sommer, W. Jahr, A. Cenameri, J. Broichhagen, S.G.N. Grant, P.M. Jonas, G. Novarino, H. Pfister, B. Bickel, J.G. Danzl, Nature Methods 20 (2023) 1256–1265.","ista":"Velicky P, Miguel Villalba E, Michalska JM, Lyudchik J, Wei D, Lin Z, Watson J, Troidl J, Beyer J, Ben Simon Y, Sommer CM, Jahr W, Cenameri A, Broichhagen J, Grant SGN, Jonas PM, Novarino G, Pfister H, Bickel B, Danzl JG. 2023. Dense 4D nanoscale reconstruction of living brain tissue. Nature Methods. 20, 1256–1265.","ama":"Velicky P, Miguel Villalba E, Michalska JM, et al. Dense 4D nanoscale reconstruction of living brain tissue. <i>Nature Methods</i>. 2023;20:1256-1265. doi:<a href=\"https://doi.org/10.1038/s41592-023-01936-6\">10.1038/s41592-023-01936-6</a>","mla":"Velicky, Philipp, et al. “Dense 4D Nanoscale Reconstruction of Living Brain Tissue.” <i>Nature Methods</i>, vol. 20, Springer Nature, 2023, pp. 1256–65, doi:<a href=\"https://doi.org/10.1038/s41592-023-01936-6\">10.1038/s41592-023-01936-6</a>."},"publication_status":"published","date_created":"2023-07-23T22:01:13Z","department":[{"_id":"PeJo"},{"_id":"GaNo"},{"_id":"BeBi"},{"_id":"JoDa"},{"_id":"Bio"}],"language":[{"iso":"eng"}],"scopus_import":"1","publisher":"Springer Nature","date_published":"2023-08-01T00:00:00Z","article_type":"original","month":"08","page":"1256-1265","publication":"Nature Methods","status":"public","intvolume":"        20","type":"journal_article","day":"01"},{"date_created":"2022-08-23T11:07:59Z","related_material":{"record":[{"id":"12470","relation":"dissertation_contains","status":"public"}]},"department":[{"_id":"PeJo"},{"_id":"GaNo"},{"_id":"BeBi"},{"_id":"JoDa"}],"main_file_link":[{"open_access":"1","url":"https://doi.org/10.1101/2022.03.16.484431"}],"language":[{"iso":"eng"}],"publisher":"Cold Spring Harbor Laboratory","title":"Saturated reconstruction of living brain tissue","date_published":"2022-05-09T00:00:00Z","doi":"10.1101/2022.03.16.484431","year":"2022","month":"05","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","oa_version":"Preprint","_id":"11943","date_updated":"2024-03-25T23:30:11Z","oa":1,"article_processing_charge":"No","publication":"bioRxiv","status":"public","author":[{"id":"39BDC62C-F248-11E8-B48F-1D18A9856A87","first_name":"Philipp","last_name":"Velicky","full_name":"Velicky, Philipp","orcid":"0000-0002-2340-7431"},{"id":"3FB91342-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-5665-0430","full_name":"Miguel Villalba, Eder","last_name":"Miguel Villalba","first_name":"Eder"},{"id":"443DB6DE-F248-11E8-B48F-1D18A9856A87","first_name":"Julia M","orcid":"0000-0003-3862-1235","full_name":"Michalska, Julia M","last_name":"Michalska"},{"first_name":"Donglai","full_name":"Wei, Donglai","last_name":"Wei"},{"first_name":"Zudi","last_name":"Lin","full_name":"Lin, Zudi"},{"full_name":"Watson, Jake","last_name":"Watson","orcid":"0000-0002-8698-3823","first_name":"Jake","id":"63836096-4690-11EA-BD4E-32803DDC885E"},{"last_name":"Troidl","full_name":"Troidl, Jakob","first_name":"Jakob"},{"first_name":"Johanna","last_name":"Beyer","full_name":"Beyer, Johanna"},{"id":"43DF3136-F248-11E8-B48F-1D18A9856A87","full_name":"Ben Simon, Yoav","last_name":"Ben Simon","first_name":"Yoav"},{"id":"4DF26D8C-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-1216-9105","full_name":"Sommer, Christoph M","last_name":"Sommer","first_name":"Christoph M"},{"id":"425C1CE8-F248-11E8-B48F-1D18A9856A87","last_name":"Jahr","full_name":"Jahr, Wiebke","first_name":"Wiebke"},{"first_name":"Alban","full_name":"Cenameri, Alban","last_name":"Cenameri","id":"9ac8f577-2357-11eb-997a-e566c5550886"},{"first_name":"Johannes","last_name":"Broichhagen","full_name":"Broichhagen, Johannes"},{"last_name":"Grant","full_name":"Grant, Seth G. N.","first_name":"Seth G. N."},{"full_name":"Jonas, Peter M","last_name":"Jonas","orcid":"0000-0001-5001-4804","first_name":"Peter M","id":"353C1B58-F248-11E8-B48F-1D18A9856A87"},{"id":"3E57A680-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-7673-7178","last_name":"Novarino","full_name":"Novarino, Gaia","first_name":"Gaia"},{"last_name":"Pfister","full_name":"Pfister, Hanspeter","first_name":"Hanspeter"},{"first_name":"Bernd","last_name":"Bickel","full_name":"Bickel, Bernd","orcid":"0000-0001-6511-9385","id":"49876194-F248-11E8-B48F-1D18A9856A87"},{"first_name":"Johann G","last_name":"Danzl","full_name":"Danzl, Johann G","orcid":"0000-0001-8559-3973","id":"42EFD3B6-F248-11E8-B48F-1D18A9856A87"}],"abstract":[{"text":"Complex wiring between neurons underlies the information-processing network enabling all brain functions, including cognition and memory. For understanding how the network is structured, processes information, and changes over time, comprehensive visualization of the architecture of living brain tissue with its cellular and molecular components would open up major opportunities. However, electron microscopy (EM) provides nanometre-scale resolution required for full <jats:italic>in-silico</jats:italic> reconstruction<jats:sup>1–5</jats:sup>, yet is limited to fixed specimens and static representations. Light microscopy allows live observation, with super-resolution approaches<jats:sup>6–12</jats:sup> facilitating nanoscale visualization, but comprehensive 3D-reconstruction of living brain tissue has been hindered by tissue photo-burden, photobleaching, insufficient 3D-resolution, and inadequate signal-to-noise ratio (SNR). Here we demonstrate saturated reconstruction of living brain tissue. We developed an integrated imaging and analysis technology, adapting stimulated emission depletion (STED) microscopy<jats:sup>6,13</jats:sup> in extracellularly labelled tissue<jats:sup>14</jats:sup> for high SNR and near-isotropic resolution. Centrally, a two-stage deep-learning approach leveraged previously obtained information on sample structure to drastically reduce photo-burden and enable automated volumetric reconstruction down to single synapse level. Live reconstruction provides unbiased analysis of tissue architecture across time in relation to functional activity and targeted activation, and contextual understanding of molecular labelling. This adoptable technology will facilitate novel insights into the dynamic functional architecture of living brain tissue.","lang":"eng"}],"type":"preprint","publication_status":"submitted","day":"09","citation":{"chicago":"Velicky, Philipp, Eder Miguel Villalba, Julia M Michalska, Donglai Wei, Zudi Lin, Jake Watson, Jakob Troidl, et al. “Saturated Reconstruction of Living Brain Tissue.” <i>BioRxiv</i>. Cold Spring Harbor Laboratory, n.d. <a href=\"https://doi.org/10.1101/2022.03.16.484431\">https://doi.org/10.1101/2022.03.16.484431</a>.","ieee":"P. Velicky <i>et al.</i>, “Saturated reconstruction of living brain tissue,” <i>bioRxiv</i>. Cold Spring Harbor Laboratory.","apa":"Velicky, P., Miguel Villalba, E., Michalska, J. M., Wei, D., Lin, Z., Watson, J., … Danzl, J. G. (n.d.). Saturated reconstruction of living brain tissue. <i>bioRxiv</i>. Cold Spring Harbor Laboratory. <a href=\"https://doi.org/10.1101/2022.03.16.484431\">https://doi.org/10.1101/2022.03.16.484431</a>","short":"P. Velicky, E. Miguel Villalba, J.M. Michalska, D. Wei, Z. Lin, J. Watson, J. Troidl, J. Beyer, Y. Ben Simon, C.M. Sommer, W. Jahr, A. Cenameri, J. Broichhagen, S.G.N. Grant, P.M. Jonas, G. Novarino, H. Pfister, B. Bickel, J.G. Danzl, BioRxiv (n.d.).","ista":"Velicky P, Miguel Villalba E, Michalska JM, Wei D, Lin Z, Watson J, Troidl J, Beyer J, Ben Simon Y, Sommer CM, Jahr W, Cenameri A, Broichhagen J, Grant SGN, Jonas PM, Novarino G, Pfister H, Bickel B, Danzl JG. Saturated reconstruction of living brain tissue. bioRxiv, <a href=\"https://doi.org/10.1101/2022.03.16.484431\">10.1101/2022.03.16.484431</a>.","mla":"Velicky, Philipp, et al. “Saturated Reconstruction of Living Brain Tissue.” <i>BioRxiv</i>, Cold Spring Harbor Laboratory, doi:<a href=\"https://doi.org/10.1101/2022.03.16.484431\">10.1101/2022.03.16.484431</a>.","ama":"Velicky P, Miguel Villalba E, Michalska JM, et al. Saturated reconstruction of living brain tissue. <i>bioRxiv</i>. doi:<a href=\"https://doi.org/10.1101/2022.03.16.484431\">10.1101/2022.03.16.484431</a>"}},{"publisher":"Springer Nature","scopus_import":"1","language":[{"iso":"eng"}],"month":"03","date_published":"2021-03-01T00:00:00Z","article_type":"letter_note","date_created":"2021-03-21T23:01:20Z","department":[{"_id":"JoDa"}],"intvolume":"        18","status":"public","day":"01","type":"journal_article","issue":"3","publication":"Nature Methods","page":"226-228","title":"Pycro-Manager: Open-source software for customized and reproducible microscope control","external_id":{"pmid":["33674797"],"isi":["000625600600007"]},"doi":"10.1038/s41592-021-01087-6","year":"2021","main_file_link":[{"url":"https://doi.org/10.1038/s41592-021-01087-6","open_access":"1"}],"isi":1,"author":[{"first_name":"Henry","full_name":"Pinkard, Henry","last_name":"Pinkard"},{"first_name":"Nico","full_name":"Stuurman, Nico","last_name":"Stuurman"},{"first_name":"Ivan E.","last_name":"Ivanov","full_name":"Ivanov, Ivan E."},{"first_name":"Nicholas M.","last_name":"Anthony","full_name":"Anthony, Nicholas M."},{"first_name":"Wei","full_name":"Ouyang, Wei","last_name":"Ouyang"},{"full_name":"Li, Bin","last_name":"Li","first_name":"Bin"},{"last_name":"Yang","full_name":"Yang, Bin","first_name":"Bin"},{"full_name":"Tsuchida, Mark A.","last_name":"Tsuchida","first_name":"Mark A."},{"first_name":"Bryant","full_name":"Chhun, Bryant","last_name":"Chhun"},{"first_name":"Grace","full_name":"Zhang, Grace","last_name":"Zhang"},{"first_name":"Ryan","last_name":"Mei","full_name":"Mei, Ryan"},{"first_name":"Michael","full_name":"Anderson, Michael","last_name":"Anderson"},{"first_name":"Douglas P.","last_name":"Shepherd","full_name":"Shepherd, Douglas P."},{"full_name":"Hunt-Isaak, Ian","last_name":"Hunt-Isaak","first_name":"Ian"},{"first_name":"Raymond L.","last_name":"Dunn","full_name":"Dunn, Raymond L."},{"last_name":"Jahr","full_name":"Jahr, Wiebke","first_name":"Wiebke","id":"425C1CE8-F248-11E8-B48F-1D18A9856A87"},{"first_name":"Saul","full_name":"Kato, Saul","last_name":"Kato"},{"last_name":"Royer","full_name":"Royer, Loïc A.","first_name":"Loïc A."},{"full_name":"Thiagarajah, Jay R.","last_name":"Thiagarajah","first_name":"Jay R."},{"first_name":"Kevin W.","full_name":"Eliceiri, Kevin W.","last_name":"Eliceiri"},{"first_name":"Emma","full_name":"Lundberg, Emma","last_name":"Lundberg"},{"last_name":"Mehta","full_name":"Mehta, Shalin B.","first_name":"Shalin B."},{"first_name":"Laura","last_name":"Waller","full_name":"Waller, Laura"}],"publication_status":"published","citation":{"ista":"Pinkard H, Stuurman N, Ivanov IE, Anthony NM, Ouyang W, Li B, Yang B, Tsuchida MA, Chhun B, Zhang G, Mei R, Anderson M, Shepherd DP, Hunt-Isaak I, Dunn RL, Jahr W, Kato S, Royer LA, Thiagarajah JR, Eliceiri KW, Lundberg E, Mehta SB, Waller L. 2021. Pycro-Manager: Open-source software for customized and reproducible microscope control. Nature Methods. 18(3), 226–228.","short":"H. Pinkard, N. Stuurman, I.E. Ivanov, N.M. Anthony, W. Ouyang, B. Li, B. Yang, M.A. Tsuchida, B. Chhun, G. Zhang, R. Mei, M. Anderson, D.P. Shepherd, I. Hunt-Isaak, R.L. Dunn, W. Jahr, S. Kato, L.A. Royer, J.R. Thiagarajah, K.W. Eliceiri, E. Lundberg, S.B. Mehta, L. Waller, Nature Methods 18 (2021) 226–228.","mla":"Pinkard, Henry, et al. “Pycro-Manager: Open-Source Software for Customized and Reproducible Microscope Control.” <i>Nature Methods</i>, vol. 18, no. 3, Springer Nature, 2021, pp. 226–28, doi:<a href=\"https://doi.org/10.1038/s41592-021-01087-6\">10.1038/s41592-021-01087-6</a>.","ama":"Pinkard H, Stuurman N, Ivanov IE, et al. Pycro-Manager: Open-source software for customized and reproducible microscope control. <i>Nature Methods</i>. 2021;18(3):226-228. doi:<a href=\"https://doi.org/10.1038/s41592-021-01087-6\">10.1038/s41592-021-01087-6</a>","chicago":"Pinkard, Henry, Nico Stuurman, Ivan E. Ivanov, Nicholas M. Anthony, Wei Ouyang, Bin Li, Bin Yang, et al. “Pycro-Manager: Open-Source Software for Customized and Reproducible Microscope Control.” <i>Nature Methods</i>. Springer Nature, 2021. <a href=\"https://doi.org/10.1038/s41592-021-01087-6\">https://doi.org/10.1038/s41592-021-01087-6</a>.","apa":"Pinkard, H., Stuurman, N., Ivanov, I. E., Anthony, N. M., Ouyang, W., Li, B., … Waller, L. (2021). Pycro-Manager: Open-source software for customized and reproducible microscope control. <i>Nature Methods</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41592-021-01087-6\">https://doi.org/10.1038/s41592-021-01087-6</a>","ieee":"H. Pinkard <i>et al.</i>, “Pycro-Manager: Open-source software for customized and reproducible microscope control,” <i>Nature Methods</i>, vol. 18, no. 3. Springer Nature, pp. 226–228, 2021."},"_id":"9258","pmid":1,"publication_identifier":{"eissn":["1548-7105"],"issn":["1548-7091"]},"acknowledgement":"We thank S. van der Walt and K. Marchuk for discussion during development. This project was funded by Packard Fellowship and Chan Zuckerberg Biohub Investigator Awards to L.W.; STROBE: A NSF Science and Technology Center; an NSF Graduate Research Fellowship awarded to H.P.; a Berkeley Institute for Data Science/UCSF Bakar Computational Health Sciences Institute Fellowship awarded to H.P. with support from the Koret Foundation, the Gordon and Betty Moore Foundation, and the Alfred P. Sloan Foundation to the University of California, Berkeley. K.W.E., B.L. and M.T. were funded by the Chan Zuckerberg Initiative and NIH grant P41GM135019.","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","quality_controlled":"1","oa_version":"Published Version","date_updated":"2023-08-07T14:19:08Z","volume":18,"oa":1,"article_processing_charge":"No"},{"abstract":[{"lang":"eng","text":"Super-resolution fluorescence microscopy has become an important catalyst for discovery in the life sciences. In STimulated Emission Depletion (STED) microscopy, a pattern of light drives fluorophores from a signal-emitting on-state to a non-signalling off-state. Only emitters residing in a sub-diffraction volume around an intensity minimum are allowed to fluoresce, rendering them distinguishable from the nearby, but dark fluorophores. STED routinely achieves resolution in the few tens of nanometers range in biological samples and is suitable for live imaging. Here, we review the working principle of STED and provide general guidelines for successful STED imaging. The strive for ever higher resolution comes at the cost of increased light burden. We discuss techniques to reduce light exposure and mitigate its detrimental effects on the specimen. These include specialized illumination strategies as well as protecting fluorophores from photobleaching mediated by high-intensity STED light. This opens up the prospect of volumetric imaging in living cells and tissues with diffraction-unlimited resolution in all three spatial dimensions."}],"author":[{"id":"425C1CE8-F248-11E8-B48F-1D18A9856A87","first_name":"Wiebke","full_name":"Jahr, Wiebke","last_name":"Jahr"},{"first_name":"Philipp","orcid":"0000-0002-2340-7431","full_name":"Velicky, Philipp","last_name":"Velicky","id":"39BDC62C-F248-11E8-B48F-1D18A9856A87"},{"id":"42EFD3B6-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-8559-3973","full_name":"Danzl, Johann G","last_name":"Danzl","first_name":"Johann G"}],"publication_status":"published","citation":{"ista":"Jahr W, Velicky P, Danzl JG. 2020. Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens. Methods. 174(3), 27–41.","short":"W. Jahr, P. Velicky, J.G. Danzl, Methods 174 (2020) 27–41.","mla":"Jahr, Wiebke, et al. “Strategies to Maximize Performance in STimulated Emission Depletion (STED) Nanoscopy of Biological Specimens.” <i>Methods</i>, vol. 174, no. 3, Elsevier, 2020, pp. 27–41, doi:<a href=\"https://doi.org/10.1016/j.ymeth.2019.07.019\">10.1016/j.ymeth.2019.07.019</a>.","ama":"Jahr W, Velicky P, Danzl JG. Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens. <i>Methods</i>. 2020;174(3):27-41. doi:<a href=\"https://doi.org/10.1016/j.ymeth.2019.07.019\">10.1016/j.ymeth.2019.07.019</a>","chicago":"Jahr, Wiebke, Philipp Velicky, and Johann G Danzl. “Strategies to Maximize Performance in STimulated Emission Depletion (STED) Nanoscopy of Biological Specimens.” <i>Methods</i>. Elsevier, 2020. <a href=\"https://doi.org/10.1016/j.ymeth.2019.07.019\">https://doi.org/10.1016/j.ymeth.2019.07.019</a>.","ieee":"W. Jahr, P. Velicky, and J. G. Danzl, “Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens,” <i>Methods</i>, vol. 174, no. 3. Elsevier, pp. 27–41, 2020.","apa":"Jahr, W., Velicky, P., &#38; Danzl, J. G. (2020). Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens. <i>Methods</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.ymeth.2019.07.019\">https://doi.org/10.1016/j.ymeth.2019.07.019</a>"},"pmid":1,"_id":"6808","publication_identifier":{"issn":["1046-2023"]},"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","project":[{"call_identifier":"FWF","name":"Optical control of synaptic function via adhesion molecules","_id":"265CB4D0-B435-11E9-9278-68D0E5697425","grant_number":"I03600"},{"grant_number":"LT00057","name":"High-speed 3D-nanoscopy to study the role of adhesion during 3D cell migration","_id":"2668BFA0-B435-11E9-9278-68D0E5697425"}],"quality_controlled":"1","oa_version":"Submitted Version","volume":174,"date_updated":"2023-08-17T13:59:57Z","oa":1,"article_processing_charge":"No","external_id":{"isi":["000525860400005"],"pmid":["31344404"]},"title":"Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens","year":"2020","doi":"10.1016/j.ymeth.2019.07.019","main_file_link":[{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7100895/","open_access":"1"}],"isi":1,"intvolume":"       174","status":"public","day":"01","type":"journal_article","issue":"3","publication":"Methods","page":"27-41","publisher":"Elsevier","scopus_import":"1","language":[{"iso":"eng"}],"month":"03","article_type":"original","date_published":"2020-03-01T00:00:00Z","date_created":"2019-08-12T16:36:32Z","department":[{"_id":"JoDa"}]},{"status":"public","intvolume":"         6","type":"journal_article","day":"24","file_date_updated":"2020-07-14T12:47:17Z","publication":"Frontiers in Chemistry","language":[{"iso":"eng"}],"scopus_import":"1","publisher":"Frontiers Media S.A.","date_published":"2019-01-24T00:00:00Z","month":"01","date_created":"2019-02-17T22:59:24Z","file":[{"access_level":"open_access","date_updated":"2020-07-14T12:47:17Z","file_size":1766820,"file_name":"2019_frontiers_Lindner.pdf","checksum":"7841301d7c53b56ef873791b4b6f7b24","date_created":"2019-02-18T15:10:34Z","relation":"main_file","content_type":"application/pdf","creator":"dernst","file_id":"6039"}],"department":[{"_id":"JoDa"}],"has_accepted_license":"1","author":[{"first_name":"Marco","last_name":"Lindner","full_name":"Lindner, Marco"},{"first_name":"Aliz","full_name":"Tresztenyak, Aliz","last_name":"Tresztenyak"},{"first_name":"Gergö","full_name":"Fülöp, Gergö","last_name":"Fülöp"},{"id":"425C1CE8-F248-11E8-B48F-1D18A9856A87","full_name":"Jahr, Wiebke","last_name":"Jahr","first_name":"Wiebke"},{"first_name":"Adrian","last_name":"Prinz","full_name":"Prinz, Adrian"},{"full_name":"Prinz, Iris","last_name":"Prinz","first_name":"Iris"},{"orcid":"0000-0001-8559-3973","last_name":"Danzl","full_name":"Danzl, Johann G","first_name":"Johann G","id":"42EFD3B6-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Schütz, Gerhard J.","last_name":"Schütz","first_name":"Gerhard J."},{"first_name":"Eva","last_name":"Sevcsik","full_name":"Sevcsik, Eva"}],"abstract":[{"lang":"eng","text":"Protein micropatterning has become an important tool for many biomedical applications as well as in academic research. Current techniques that allow to reduce the feature size of patterns below 1 μm are, however, often costly and require sophisticated equipment. We present here a straightforward and convenient method to generate highly condensed nanopatterns of proteins without the need for clean room facilities or expensive equipment. Our approach is based on nanocontact printing and allows for the fabrication of protein patterns with feature sizes of 80 nm and periodicities down to 140 nm. This was made possible by the use of the material X-poly(dimethylsiloxane) (X-PDMS) in a two-layer stamp layout for protein printing. In a proof of principle, different proteins at various scales were printed and the pattern quality was evaluated by atomic force microscopy (AFM) and super-resolution fluorescence microscopy."}],"citation":{"chicago":"Lindner, Marco, Aliz Tresztenyak, Gergö Fülöp, Wiebke Jahr, Adrian Prinz, Iris Prinz, Johann G Danzl, Gerhard J. Schütz, and Eva Sevcsik. “A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns.” <i>Frontiers in Chemistry</i>. Frontiers Media S.A., 2019. <a href=\"https://doi.org/10.3389/fchem.2018.00655\">https://doi.org/10.3389/fchem.2018.00655</a>.","ieee":"M. Lindner <i>et al.</i>, “A fast and simple contact printing approach to generate 2D protein nanopatterns,” <i>Frontiers in Chemistry</i>, vol. 6. Frontiers Media S.A., 2019.","apa":"Lindner, M., Tresztenyak, A., Fülöp, G., Jahr, W., Prinz, A., Prinz, I., … Sevcsik, E. (2019). A fast and simple contact printing approach to generate 2D protein nanopatterns. <i>Frontiers in Chemistry</i>. Frontiers Media S.A. <a href=\"https://doi.org/10.3389/fchem.2018.00655\">https://doi.org/10.3389/fchem.2018.00655</a>","short":"M. Lindner, A. Tresztenyak, G. Fülöp, W. Jahr, A. Prinz, I. Prinz, J.G. Danzl, G.J. Schütz, E. Sevcsik, Frontiers in Chemistry 6 (2019).","ista":"Lindner M, Tresztenyak A, Fülöp G, Jahr W, Prinz A, Prinz I, Danzl JG, Schütz GJ, Sevcsik E. 2019. A fast and simple contact printing approach to generate 2D protein nanopatterns. Frontiers in Chemistry. 6, 655.","ama":"Lindner M, Tresztenyak A, Fülöp G, et al. A fast and simple contact printing approach to generate 2D protein nanopatterns. <i>Frontiers in Chemistry</i>. 2019;6. doi:<a href=\"https://doi.org/10.3389/fchem.2018.00655\">10.3389/fchem.2018.00655</a>","mla":"Lindner, Marco, et al. “A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns.” <i>Frontiers in Chemistry</i>, vol. 6, 655, Frontiers Media S.A., 2019, doi:<a href=\"https://doi.org/10.3389/fchem.2018.00655\">10.3389/fchem.2018.00655</a>."},"publication_status":"published","oa_version":"Published Version","quality_controlled":"1","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","publication_identifier":{"eissn":["22962646"]},"_id":"6029","article_processing_charge":"No","oa":1,"date_updated":"2023-08-24T14:45:38Z","volume":6,"title":"A fast and simple contact printing approach to generate 2D protein nanopatterns","external_id":{"isi":["000456718000001"]},"doi":"10.3389/fchem.2018.00655","year":"2019","ddc":["540"],"isi":1,"article_number":"655","tmp":{"image":"/images/cc_by.png","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)"}}]