[{"title":"Super-resolution expansion microscopy in plant roots","month":"04","_id":"19003","citation":{"apa":"Gallei, M. C., Truckenbrodt, S. M., Kreuzinger, C., Inumella, S., Vistunou, V., Sommer, C. M., … Danzl, J. G. (2025). Super-resolution expansion microscopy in plant roots. <i>The Plant Cell</i>. Oxford University Press. <a href=\"https://doi.org/10.1093/plcell/koaf006\">https://doi.org/10.1093/plcell/koaf006</a>","ama":"Gallei MC, Truckenbrodt SM, Kreuzinger C, et al. Super-resolution expansion microscopy in plant roots. <i>The Plant Cell</i>. 2025;37(4). doi:<a href=\"https://doi.org/10.1093/plcell/koaf006\">10.1093/plcell/koaf006</a>","ista":"Gallei MC, Truckenbrodt SM, Kreuzinger C, Inumella S, Vistunou V, Sommer CM, Tavakoli M, Agudelo Duenas N, Vorlaufer J, Jahr W, Randuch M, Johnson AJ, Benková E, Friml J, Danzl JG. 2025. Super-resolution expansion microscopy in plant roots. The Plant Cell. 37(4), koaf006.","chicago":"Gallei, Michelle C, Sven M Truckenbrodt, Caroline Kreuzinger, Syamala Inumella, Vitali Vistunou, Christoph M Sommer, Mojtaba Tavakoli, et al. “Super-Resolution Expansion Microscopy in Plant Roots.” <i>The Plant Cell</i>. Oxford University Press, 2025. <a href=\"https://doi.org/10.1093/plcell/koaf006\">https://doi.org/10.1093/plcell/koaf006</a>.","short":"M.C. Gallei, S.M. Truckenbrodt, C. Kreuzinger, S. Inumella, V. Vistunou, C.M. Sommer, M. Tavakoli, N. Agudelo Duenas, J. Vorlaufer, W. Jahr, M. Randuch, A.J. Johnson, E. Benková, J. Friml, J.G. Danzl, The Plant Cell 37 (2025).","mla":"Gallei, Michelle C., et al. “Super-Resolution Expansion Microscopy in Plant Roots.” <i>The Plant Cell</i>, vol. 37, no. 4, koaf006, Oxford University Press, 2025, doi:<a href=\"https://doi.org/10.1093/plcell/koaf006\">10.1093/plcell/koaf006</a>.","ieee":"M. C. Gallei <i>et al.</i>, “Super-resolution expansion microscopy in plant roots,” <i>The Plant Cell</i>, vol. 37, no. 4. Oxford University Press, 2025."},"related_material":{"record":[{"relation":"earlier_version","status":"public","id":"18689"},{"relation":"research_data","status":"public","id":"18837"}]},"PlanS_conform":"1","article_processing_charge":"Yes (via OA deal)","ddc":["580"],"doi":"10.1093/plcell/koaf006","OA_place":"publisher","OA_type":"hybrid","article_type":"original","tmp":{"short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"status":"public","day":"01","publisher":"Oxford University Press","type":"journal_article","issue":"4","year":"2025","oa_version":"Published Version","date_updated":"2025-10-08T08:43:56Z","project":[{"grant_number":"742985","_id":"261099A6-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"Tracing Evolution of Auxin Transport and Polarity in Plants"},{"grant_number":"665385","_id":"2564DBCA-B435-11E9-9278-68D0E5697425","name":"International IST Doctoral Program","call_identifier":"H2020"},{"_id":"260C2330-B435-11E9-9278-68D0E5697425","grant_number":"754411","call_identifier":"H2020","name":"ISTplus - Postdoctoral Fellowships"},{"grant_number":"ALTF 679-2018","_id":"269B5B22-B435-11E9-9278-68D0E5697425","name":"UltraX - achieving sub-nanometer resolution in light microscopy using iterative X10 microscopy in combination with nanobodies and STED"},{"_id":"6285a163-2b32-11ec-9570-8e204ca2dba5","grant_number":"26137","name":"Studying Organelle Structure and Function at Nanoscale Resolution with Expansion Microscopy"},{"_id":"26AA4EF2-B435-11E9-9278-68D0E5697425","grant_number":"W1232-B24","name":"Molecular Drug Targets","call_identifier":"FWF"}],"has_accepted_license":"1","date_published":"2025-04-01T00:00:00Z","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","quality_controlled":"1","volume":37,"acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"},{"_id":"E-Lib"},{"_id":"M-Shop"}],"abstract":[{"text":"Super-resolution methods provide far better spatial resolution than the optical diffraction limit of about half the wavelength of light (∼200-300 nm). Nevertheless, they have yet to attain widespread use in plants, largely due to plants’ challenging optical properties. Expansion microscopy improves effective resolution by isotropically increasing the physical distances between sample structures while preserving relative spatial arrangements and clearing the sample. However, its application to plants has been hindered by the rigid, mechanically cohesive structure of plant tissues. Here, we report on whole-mount expansion microscopy of thale cress (Arabidopsis thaliana) root tissues (PlantEx), achieving a four-fold resolution increase over conventional microscopy. Our results highlight the microtubule cytoskeleton organization and interaction between molecularly defined cellular constituents. Combining PlantEx with stimulated emission depletion (STED) microscopy, we increase nanoscale resolution and visualize the complex organization of subcellular organelles from intact tissues by example of the densely packed COPI-coated vesicles associated with the Golgi apparatus and put these into a cellular structural context. Our results show that expansion microscopy can be applied to increase effective imaging resolution in Arabidopsis root specimens. ","lang":"eng"}],"article_number":"koaf006","publication_status":"published","corr_author":"1","language":[{"iso":"eng"}],"external_id":{"pmid":["39792900"],"isi":["001462763100001"]},"department":[{"_id":"EvBe"},{"_id":"JoDa"},{"_id":"JiFr"}],"date_created":"2025-02-05T06:52:06Z","publication_identifier":{"issn":["1040-4651"],"eissn":["1532-298X"]},"oa":1,"file":[{"date_updated":"2025-07-31T07:03:43Z","checksum":"9d3f8218ff37a29f29c48a7bbe831bd3","content_type":"application/pdf","file_size":53904111,"creator":"dernst","file_name":"2025_PlantCell_Gallei.pdf","file_id":"20092","date_created":"2025-07-31T07:03:43Z","access_level":"open_access","relation":"main_file","success":1}],"acknowledgement":"We gratefully acknowledge support by the Scientific Service Units at ISTA, including the Imaging and Optics and Lab Support facilities and the mechanical workshop and Library. We thank Philipp Velicky for STED microscope alignment.\r\nThis project has received funding from the European Research Council under the Horizon 2020 Framework Programme (grant agreement No 742985, J.F.). It has also received funding from the Horizon 2020 Framework Programme under the Marie Skłodowska-Curie Grant Agreement No. 665385 (M.G.). S.T. has received funding as an ISTplus Fellow from the Horizon 2020 Framework Programme under Marie Skłodowska-Curie grant agreement no. 754411 and from EMBO via a Long-Term Fellowship (grant number ALTF 679-2018). M.R.T. received funding from the Austrian Academy of Sciences with DOC fellowship no. 26137. The project has further received funding from the Austrian Science Fund, via grant DK W1232 (M.R.T., N.A.D., and J.G.D). W.J. received a postdoctoral fellowship from the Human Frontier Science Program (LT000557/2018). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.","pmid":1,"ec_funded":1,"intvolume":"        37","isi":1,"scopus_import":"1","file_date_updated":"2025-07-31T07:03:43Z","publication":"The Plant Cell","author":[{"first_name":"Michelle C","id":"35A03822-F248-11E8-B48F-1D18A9856A87","full_name":"Gallei, Michelle C","last_name":"Gallei","orcid":"0000-0003-1286-7368"},{"first_name":"Sven M","id":"45812BD4-F248-11E8-B48F-1D18A9856A87","full_name":"Truckenbrodt, Sven M","last_name":"Truckenbrodt"},{"full_name":"Kreuzinger, Caroline","last_name":"Kreuzinger","id":"382077BA-F248-11E8-B48F-1D18A9856A87","first_name":"Caroline"},{"id":"F8660870-D756-11E9-98C5-34DFE5697425","first_name":"Syamala","full_name":"Inumella, Syamala","last_name":"Inumella","orcid":"0009-0002-5890-120X"},{"full_name":"Vistunou, Vitali","last_name":"Vistunou","first_name":"Vitali","id":"7e146587-8972-11ed-ae7b-d7a32ea86a81"},{"last_name":"Sommer","full_name":"Sommer, Christoph M","first_name":"Christoph M","id":"4DF26D8C-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-1216-9105"},{"orcid":"0000-0002-7667-6854","last_name":"Tavakoli","full_name":"Tavakoli, Mojtaba","id":"3A0A06F4-F248-11E8-B48F-1D18A9856A87","first_name":"Mojtaba"},{"full_name":"Agudelo Duenas, Nathalie","last_name":"Agudelo Duenas","id":"40E7F008-F248-11E8-B48F-1D18A9856A87","first_name":"Nathalie"},{"last_name":"Vorlaufer","full_name":"Vorlaufer, Jakob","first_name":"Jakob","id":"937696FA-C996-11E9-8C7C-CF13E6697425","orcid":"0009-0000-7590-3501"},{"full_name":"Jahr, Wiebke","last_name":"Jahr","first_name":"Wiebke","id":"425C1CE8-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Randuch, Marek","last_name":"Randuch","id":"6ac4636d-15b2-11ec-abd3-fb8df79972ae","first_name":"Marek"},{"orcid":"0000-0002-2739-8843","id":"46A62C3A-F248-11E8-B48F-1D18A9856A87","first_name":"Alexander J","full_name":"Johnson, Alexander J","last_name":"Johnson"},{"last_name":"Benková","full_name":"Benková, Eva","first_name":"Eva","id":"38F4F166-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-8510-9739"},{"orcid":"0000-0002-8302-7596","id":"4159519E-F248-11E8-B48F-1D18A9856A87","first_name":"Jiří","full_name":"Friml, Jiří","last_name":"Friml"},{"orcid":"0000-0001-8559-3973","last_name":"Danzl","full_name":"Danzl, Johann G","id":"42EFD3B6-F248-11E8-B48F-1D18A9856A87","first_name":"Johann G"}]},{"tmp":{"short":"CC BY-NC (4.0)","legal_code_url":"https://creativecommons.org/licenses/by-nc/4.0/legalcode","name":"Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)","image":"/images/cc_by_nc.png"},"status":"public","day":"21","type":"preprint","date_updated":"2026-04-07T12:56:36Z","oa_version":"Preprint","year":"2024","date_published":"2024-02-21T00:00:00Z","project":[{"grant_number":"742985","_id":"261099A6-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","name":"Tracing Evolution of Auxin Transport and Polarity in Plants"},{"call_identifier":"H2020","name":"International IST Doctoral Program","grant_number":"665385","_id":"2564DBCA-B435-11E9-9278-68D0E5697425"},{"_id":"260C2330-B435-11E9-9278-68D0E5697425","grant_number":"754411","name":"ISTplus - Postdoctoral Fellowships","call_identifier":"H2020"},{"name":"Molecular Drug Targets","call_identifier":"FWF","_id":"26AA4EF2-B435-11E9-9278-68D0E5697425","grant_number":"W1232-B24"},{"name":"UltraX - achieving sub-nanometer resolution in light microscopy using iterative X10 microscopy in combination with nanobodies and STED","grant_number":"ALTF 679-2018","_id":"269B5B22-B435-11E9-9278-68D0E5697425"}],"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","month":"02","title":"Super-resolution expansion microscopy in plant roots","_id":"18689","related_material":{"record":[{"relation":"later_version","status":"public","id":"19003"},{"relation":"dissertation_contains","status":"public","id":"18681"}]},"citation":{"mla":"Gallei, Michelle C., et al. “Super-Resolution Expansion Microscopy in Plant Roots.” <i>BioRxiv</i>, doi:<a href=\"https://doi.org/10.1101/2024.02.21.581330\">10.1101/2024.02.21.581330</a>.","short":"M.C. Gallei, S.M. Truckenbrodt, C. Kreuzinger, S. Inumella, V. Vistunou, C.M. Sommer, M. Tavakoli, N. Agudelo Duenas, J. Vorlaufer, W. Jahr, M. Randuch, A.J. Johnson, E. Benková, J. Friml, J.G. Danzl, BioRxiv (n.d.).","ieee":"M. C. Gallei <i>et al.</i>, “Super-resolution expansion microscopy in plant roots,” <i>bioRxiv</i>. .","ama":"Gallei MC, Truckenbrodt SM, Kreuzinger C, et al. Super-resolution expansion microscopy in plant roots. <i>bioRxiv</i>. doi:<a href=\"https://doi.org/10.1101/2024.02.21.581330\">10.1101/2024.02.21.581330</a>","apa":"Gallei, M. C., Truckenbrodt, S. M., Kreuzinger, C., Inumella, S., Vistunou, V., Sommer, C. M., … Danzl, J. G. (n.d.). Super-resolution expansion microscopy in plant roots. <i>bioRxiv</i>. <a href=\"https://doi.org/10.1101/2024.02.21.581330\">https://doi.org/10.1101/2024.02.21.581330</a>","chicago":"Gallei, Michelle C, Sven M Truckenbrodt, Caroline Kreuzinger, Syamala Inumella, Vitali Vistunou, Christoph M Sommer, Mojtaba Tavakoli, et al. “Super-Resolution Expansion Microscopy in Plant Roots.” <i>BioRxiv</i>, n.d. <a href=\"https://doi.org/10.1101/2024.02.21.581330\">https://doi.org/10.1101/2024.02.21.581330</a>.","ista":"Gallei MC, Truckenbrodt SM, Kreuzinger C, Inumella S, Vistunou V, Sommer CM, Tavakoli M, Agudelo Duenas N, Vorlaufer J, Jahr W, Randuch M, Johnson AJ, Benková E, Friml J, Danzl JG. Super-resolution expansion microscopy in plant roots. bioRxiv, <a href=\"https://doi.org/10.1101/2024.02.21.581330\">10.1101/2024.02.21.581330</a>."},"article_processing_charge":"No","doi":"10.1101/2024.02.21.581330","OA_place":"repository","oa":1,"acknowledgement":"We gratefully acknowledge support by the Scientific Service Units at ISTA, including the Imaging and Optics and Lab Support facilities and the mechanical workshop and Library. We thank Philipp Velicky for STED microscope alignment.\r\n\r\nThis project has received funding from the Austrian Science Fund (FWF): I 3630-B25 (J.G.D) and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 742985, J.F.). It has also received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 665385. S.T. has received funding as an ISTplus Fellow from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie grant agreement no. 754411 and from an EMBO Long-Term Fellowship (grant number ALTF 679-2018). It has further received funding from the Austrian Science Fund (FWF) grant DK W1232 (M.T, N.A-D., J.G.D). W.J. received funding via a Human Frontier Science Program postdoctoral fellowship LT000557/2018.\r\n\r\nThe funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1101/2024.02.21.581330"}],"ec_funded":1,"publication":"bioRxiv","author":[{"first_name":"Michelle C","id":"35A03822-F248-11E8-B48F-1D18A9856A87","last_name":"Gallei","full_name":"Gallei, Michelle C","orcid":"0000-0003-1286-7368"},{"last_name":"Truckenbrodt","full_name":"Truckenbrodt, Sven M","id":"45812BD4-F248-11E8-B48F-1D18A9856A87","first_name":"Sven M"},{"last_name":"Kreuzinger","full_name":"Kreuzinger, Caroline","id":"382077BA-F248-11E8-B48F-1D18A9856A87","first_name":"Caroline"},{"orcid":"0009-0002-5890-120X","first_name":"Syamala","id":"F8660870-D756-11E9-98C5-34DFE5697425","last_name":"Inumella","full_name":"Inumella, Syamala"},{"last_name":"Vistunou","full_name":"Vistunou, Vitali","id":"7e146587-8972-11ed-ae7b-d7a32ea86a81","first_name":"Vitali"},{"orcid":"0000-0003-1216-9105","full_name":"Sommer, Christoph M","last_name":"Sommer","first_name":"Christoph M","id":"4DF26D8C-F248-11E8-B48F-1D18A9856A87"},{"orcid":"0000-0002-7667-6854","full_name":"Tavakoli, Mojtaba","last_name":"Tavakoli","first_name":"Mojtaba","id":"3A0A06F4-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Agudelo Duenas, Nathalie","last_name":"Agudelo Duenas","id":"40E7F008-F248-11E8-B48F-1D18A9856A87","first_name":"Nathalie"},{"orcid":"0009-0000-7590-3501","full_name":"Vorlaufer, Jakob","last_name":"Vorlaufer","id":"937696FA-C996-11E9-8C7C-CF13E6697425","first_name":"Jakob"},{"orcid":"0000-0003-0201-2315","first_name":"Wiebke","id":"425C1CE8-F248-11E8-B48F-1D18A9856A87","full_name":"Jahr, Wiebke","last_name":"Jahr"},{"last_name":"Randuch","full_name":"Randuch, Marek","first_name":"Marek","id":"6ac4636d-15b2-11ec-abd3-fb8df79972ae"},{"orcid":"0000-0002-2739-8843","first_name":"Alexander J","id":"46A62C3A-F248-11E8-B48F-1D18A9856A87","last_name":"Johnson","full_name":"Johnson, Alexander J"},{"id":"38F4F166-F248-11E8-B48F-1D18A9856A87","first_name":"Eva","full_name":"Benková, Eva","last_name":"Benková","orcid":"0000-0002-8510-9739"},{"full_name":"Friml, Jiří","last_name":"Friml","id":"4159519E-F248-11E8-B48F-1D18A9856A87","first_name":"Jiří","orcid":"0000-0002-8302-7596"},{"last_name":"Danzl","full_name":"Danzl, Johann G","first_name":"Johann G","id":"42EFD3B6-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-8559-3973"}],"acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"},{"_id":"M-Shop"},{"_id":"E-Lib"}],"abstract":[{"lang":"eng","text":"Multiplexed fluorescence microscopy imaging is widely used in biomedical applications. However, simultaneous imaging of multiple fluorophores can result in spectral leaks and overlapping, which greatly degrades image quality and subsequent analysis. Existing popular spectral unmixing methods are mainly based on computational intensive linear models and the performance is heavily dependent on the reference spectra, which may greatly preclude its further applications. In this paper, we propose a deep learning-based blindly spectral unmixing method, termed AutoUnmix, to imitate the physical spectral mixing process. A tranfer learning framework is further devised to allow our AutoUnmix adapting to a variety of imaging systems without retraining the network. Our proposed method has demonstrated real-time unmixing capabilities, surpassing existing methods by up to 100-fold in terms of unmixing speed. We further validate the reconstruction performance on both synthetic datasets and biological samples. The unmixing results of AutoUnmix achieve a highest SSIM of 0.99 in both three- and four-color imaging, with nearly up to 20% higher than other popular unmixing methods. Due to the desirable property of data independency and superior blind unmixing performance, we believe AutoUnmix is a powerful tool to study the interaction process of different organelles labeled by multiple fluorophores."}],"publication_status":"draft","corr_author":"1","language":[{"iso":"eng"}],"date_created":"2024-12-19T12:28:00Z","department":[{"_id":"EvBe"},{"_id":"JoDa"},{"_id":"JiFr"}]},{"type":"book_chapter","day":"01","publisher":"Elsevier","status":"public","page":"33-56","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","year":"2021","oa_version":"None","date_updated":"2024-10-09T20:59:36Z","date_published":"2021-01-01T00:00:00Z","citation":{"ieee":"S. M. Truckenbrodt and S. O. Rizzoli, “Simple multi-color super-resolution by X10 microscopy,” in <i>Methods in Cell Biology</i>, vol. 161, Elsevier, 2021, pp. 33–56.","mla":"Truckenbrodt, Sven M., and Silvio O. Rizzoli. “Simple Multi-Color Super-Resolution by X10 Microscopy.” <i>Methods in Cell Biology</i>, vol. 161, Elsevier, 2021, pp. 33–56, doi:<a href=\"https://doi.org/10.1016/bs.mcb.2020.04.016\">10.1016/bs.mcb.2020.04.016</a>.","short":"S.M. Truckenbrodt, S.O. Rizzoli, in:, Methods in Cell Biology, Elsevier, 2021, pp. 33–56.","chicago":"Truckenbrodt, Sven M, and Silvio O. Rizzoli. “Simple Multi-Color Super-Resolution by X10 Microscopy.” In <i>Methods in Cell Biology</i>, 161:33–56. Elsevier, 2021. <a href=\"https://doi.org/10.1016/bs.mcb.2020.04.016\">https://doi.org/10.1016/bs.mcb.2020.04.016</a>.","ista":"Truckenbrodt SM, Rizzoli SO. 2021.Simple multi-color super-resolution by X10 microscopy. In: Methods in Cell Biology. vol. 161, 33–56.","ama":"Truckenbrodt SM, Rizzoli SO. Simple multi-color super-resolution by X10 microscopy. In: <i>Methods in Cell Biology</i>. Vol 161. Elsevier; 2021:33-56. doi:<a href=\"https://doi.org/10.1016/bs.mcb.2020.04.016\">10.1016/bs.mcb.2020.04.016</a>","apa":"Truckenbrodt, S. M., &#38; Rizzoli, S. O. (2021). Simple multi-color super-resolution by X10 microscopy. In <i>Methods in Cell Biology</i> (Vol. 161, pp. 33–56). Elsevier. <a href=\"https://doi.org/10.1016/bs.mcb.2020.04.016\">https://doi.org/10.1016/bs.mcb.2020.04.016</a>"},"article_processing_charge":"No","title":"Simple multi-color super-resolution by X10 microscopy","month":"01","_id":"7941","doi":"10.1016/bs.mcb.2020.04.016","publication_identifier":{"isbn":["978012820807-6"],"issn":["0091-679X"]},"pmid":1,"intvolume":"       161","publication":"Methods in Cell Biology","author":[{"id":"45812BD4-F248-11E8-B48F-1D18A9856A87","first_name":"Sven M","full_name":"Truckenbrodt, Sven M","last_name":"Truckenbrodt"},{"full_name":"Rizzoli, Silvio O.","last_name":"Rizzoli","first_name":"Silvio O."}],"scopus_import":"1","abstract":[{"lang":"eng","text":"Expansion microscopy is a recently developed super-resolution imaging technique, which provides an alternative to optics-based methods such as deterministic approaches (e.g. STED) or stochastic approaches (e.g. PALM/STORM). The idea behind expansion microscopy is to embed the biological sample in a swellable gel, and then to expand it isotropically, thereby increasing the distance between the fluorophores. This approach breaks the diffraction barrier by simply separating the emission point-spread-functions of the fluorophores. The resolution attainable in expansion microscopy is thus directly dependent on the separation that can be achieved, i.e. on the expansion factor. The original implementation of the technique achieved an expansion factor of fourfold, for a resolution of 70–80 nm. The subsequently developed X10 method achieves an expansion factor of 10-fold, for a resolution of 25–30 nm. This technique can be implemented with minimal technical requirements on any standard fluorescence microscope, and is more easily applied for multi-color imaging than either deterministic or stochastic super-resolution approaches. This renders X10 expansion microscopy a highly promising tool for new biological discoveries, as discussed here, and as demonstrated by several recent applications."}],"quality_controlled":"1","volume":161,"language":[{"iso":"eng"}],"corr_author":"1","department":[{"_id":"JoDa"}],"external_id":{"pmid":["33478696"]},"date_created":"2020-06-07T22:00:55Z","publication_status":"published"},{"oa":1,"pmid":1,"file":[{"file_size":84478958,"date_created":"2021-06-29T14:41:46Z","file_id":"9619","file_name":"181031_Truckenbrodt_ExM_NatProtoc.docx","creator":"kschuh","date_updated":"2021-06-29T14:41:46Z","checksum":"7efb9951e7ddf3e3dcc2fb92b859c623","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","success":1,"relation":"main_file","access_level":"open_access"}],"intvolume":"        14","isi":1,"ec_funded":1,"scopus_import":"1","file_date_updated":"2021-06-29T14:41:46Z","publication":"Nature Protocols","author":[{"id":"45812BD4-F248-11E8-B48F-1D18A9856A87","first_name":"Sven M","full_name":"Truckenbrodt, Sven M","last_name":"Truckenbrodt"},{"orcid":"0000-0003-1216-9105","id":"4DF26D8C-F248-11E8-B48F-1D18A9856A87","first_name":"Christoph M","last_name":"Sommer","full_name":"Sommer, Christoph M"},{"first_name":"Silvio O","last_name":"Rizzoli","full_name":"Rizzoli, Silvio O"},{"orcid":"0000-0001-8559-3973","full_name":"Danzl, Johann G","last_name":"Danzl","first_name":"Johann G","id":"42EFD3B6-F248-11E8-B48F-1D18A9856A87"}],"abstract":[{"text":"Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60–80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Here, we provide a step-by-step protocol for X10 expansion microscopy. A typical experiment consists of seven sequential stages: (i) immunostaining, (ii) anchoring, (iii) polymerization, (iv) homogenization, (v) expansion, (vi) imaging, and (vii) validation. The protocol presented here includes recommendations for optimization, pitfalls and their solutions, and detailed guidelines that should increase reproducibility. Although our protocol focuses on X10 expansion microscopy, we detail which of these suggestions are also applicable to classic fourfold expansion microscopy. We exemplify our protocol using primary hippocampal neurons from rats, but our approach can be used with other primary cells or cultured cell lines of interest. This protocol will enable any researcher with basic experience in immunostainings and access to an epifluorescence microscope to perform super-resolution microscopy with X10. The procedure takes 3 d and requires ~5 h of actively handling the sample for labeling and expansion, and another ~3 h for imaging and analysis.","lang":"eng"}],"quality_controlled":"1","volume":14,"language":[{"iso":"eng"}],"date_created":"2019-02-24T22:59:20Z","department":[{"_id":"JoDa"},{"_id":"Bio"}],"external_id":{"pmid":["30778205"],"isi":["000459890700008"]},"publication_status":"published","issue":"3","type":"journal_article","day":"01","publisher":"Nature Publishing Group","status":"public","page":"832–863","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","date_updated":"2025-04-14T07:44:00Z","oa_version":"Submitted Version","year":"2019","project":[{"_id":"260C2330-B435-11E9-9278-68D0E5697425","grant_number":"754411","call_identifier":"H2020","name":"ISTplus - Postdoctoral Fellowships"},{"_id":"265CB4D0-B435-11E9-9278-68D0E5697425","grant_number":"I03600","name":"Optical control of synaptic function via adhesion molecules","call_identifier":"FWF"}],"date_published":"2019-03-01T00:00:00Z","has_accepted_license":"1","citation":{"ieee":"S. M. Truckenbrodt, C. M. Sommer, S. O. Rizzoli, and J. G. Danzl, “A practical guide to optimization in X10 expansion microscopy,” <i>Nature Protocols</i>, vol. 14, no. 3. Nature Publishing Group, pp. 832–863, 2019.","short":"S.M. Truckenbrodt, C.M. Sommer, S.O. Rizzoli, J.G. Danzl, Nature Protocols 14 (2019) 832–863.","mla":"Truckenbrodt, Sven M., et al. “A Practical Guide to Optimization in X10 Expansion Microscopy.” <i>Nature Protocols</i>, vol. 14, no. 3, Nature Publishing Group, 2019, pp. 832–863, doi:<a href=\"https://doi.org/10.1038/s41596-018-0117-3\">10.1038/s41596-018-0117-3</a>.","ista":"Truckenbrodt SM, Sommer CM, Rizzoli SO, Danzl JG. 2019. A practical guide to optimization in X10 expansion microscopy. Nature Protocols. 14(3), 832–863.","chicago":"Truckenbrodt, Sven M, Christoph M Sommer, Silvio O Rizzoli, and Johann G Danzl. “A Practical Guide to Optimization in X10 Expansion Microscopy.” <i>Nature Protocols</i>. Nature Publishing Group, 2019. <a href=\"https://doi.org/10.1038/s41596-018-0117-3\">https://doi.org/10.1038/s41596-018-0117-3</a>.","apa":"Truckenbrodt, S. M., Sommer, C. M., Rizzoli, S. O., &#38; Danzl, J. G. (2019). A practical guide to optimization in X10 expansion microscopy. <i>Nature Protocols</i>. Nature Publishing Group. <a href=\"https://doi.org/10.1038/s41596-018-0117-3\">https://doi.org/10.1038/s41596-018-0117-3</a>","ama":"Truckenbrodt SM, Sommer CM, Rizzoli SO, Danzl JG. A practical guide to optimization in X10 expansion microscopy. <i>Nature Protocols</i>. 2019;14(3):832–863. doi:<a href=\"https://doi.org/10.1038/s41596-018-0117-3\">10.1038/s41596-018-0117-3</a>"},"article_processing_charge":"No","month":"03","title":"A practical guide to optimization in X10 expansion microscopy","_id":"6052","article_type":"original","doi":"10.1038/s41596-018-0117-3","ddc":["570"]},{"file":[{"relation":"main_file","access_level":"open_access","content_type":"application/pdf","checksum":"a540feb6c9af6aefc78de531461a8835","date_updated":"2020-07-14T12:44:56Z","file_id":"5710","date_created":"2018-12-17T14:17:29Z","file_name":"2018_EMBO_Truckenbrodt.pdf","creator":"dernst","file_size":2846470}],"acknowledgement":"We thank Reinhard Jahn for providing a plasmid for YFP-SNAP25. We thank Erwin Neher for help with the development of the mathematical model of the synaptic vesicle life cycle. We thank Martin Meschkat, Andreas Höbartner, Annedore Punge, and Peer Hoopmann for help with the experiments. We thank Burkhard Rammner for providing the illustrations of synaptic vesicle and protein dynamics. We thank Manuel Maidorn, Martin Helm, and Katharina N. Richter for critically reading the manuscript. S.T. was supported by an Excellence Stipend of the Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (GGNB). E.F.F. is a recipient of long-term fellowships from the European Molecular Biology Organization (ALTF_797-2012) and from the Human Frontier Science Program (HFSP_LT000830/2013). The work was supported by grants to S.O.R. from the European Research Council (ERC-2013-CoG NeuroMolAnatomy) and from the Deutsche Forschungsgemeinschaft (Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, SFB1190/P09, SFB889/A05, and SFB1286/A03, and DFG RI 1967 7/1). The nanoSIMS instrument was funded by the German Federal Ministry of Education and Research (03F0626A).","pmid":1,"oa":1,"publication_identifier":{"issn":["0261-4189"]},"author":[{"id":"45812BD4-F248-11E8-B48F-1D18A9856A87","first_name":"Sven M","last_name":"Truckenbrodt","full_name":"Truckenbrodt, Sven M"},{"last_name":"Viplav","full_name":"Viplav, Abhiyan","first_name":"Abhiyan"},{"full_name":"Jähne, Sebsatian","last_name":"Jähne","first_name":"Sebsatian"},{"first_name":"Angela","last_name":"Vogts","full_name":"Vogts, Angela"},{"full_name":"Denker, Annette","last_name":"Denker","first_name":"Annette"},{"full_name":"Wildhagen, Hanna","last_name":"Wildhagen","first_name":"Hanna"},{"full_name":"Fornasiero, Eugenio","last_name":"Fornasiero","first_name":"Eugenio"},{"first_name":"Silvio","full_name":"Rizzoli, Silvio","last_name":"Rizzoli"}],"file_date_updated":"2020-07-14T12:44:56Z","publication":"The EMBO Journal","scopus_import":"1","intvolume":"        37","isi":1,"abstract":[{"text":"Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non-recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.","lang":"eng"}],"article_number":"e98044","volume":37,"quality_controlled":"1","department":[{"_id":"JoDa"}],"external_id":{"isi":["000440416900005"],"pmid":["29950309"]},"date_created":"2018-12-11T11:44:52Z","language":[{"iso":"eng"}],"corr_author":"1","publication_status":"published","type":"journal_article","day":"01","publisher":"Wiley","issue":"15","status":"public","tmp":{"short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","has_accepted_license":"1","date_published":"2018-08-01T00:00:00Z","oa_version":"Published Version","year":"2018","date_updated":"2024-10-09T20:58:32Z","article_processing_charge":"No","citation":{"mla":"Truckenbrodt, Sven M., et al. “Newly Produced Synaptic Vesicle Proteins Are Preferentially Used in Synaptic Transmission.” <i>The EMBO Journal</i>, vol. 37, no. 15, e98044, Wiley, 2018, doi:<a href=\"https://doi.org/10.15252/embj.201798044\">10.15252/embj.201798044</a>.","short":"S.M. Truckenbrodt, A. Viplav, S. Jähne, A. Vogts, A. Denker, H. Wildhagen, E. Fornasiero, S. Rizzoli, The EMBO Journal 37 (2018).","ieee":"S. M. Truckenbrodt <i>et al.</i>, “Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission,” <i>The EMBO Journal</i>, vol. 37, no. 15. Wiley, 2018.","ama":"Truckenbrodt SM, Viplav A, Jähne S, et al. Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission. <i>The EMBO Journal</i>. 2018;37(15). doi:<a href=\"https://doi.org/10.15252/embj.201798044\">10.15252/embj.201798044</a>","apa":"Truckenbrodt, S. M., Viplav, A., Jähne, S., Vogts, A., Denker, A., Wildhagen, H., … Rizzoli, S. (2018). Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission. <i>The EMBO Journal</i>. Wiley. <a href=\"https://doi.org/10.15252/embj.201798044\">https://doi.org/10.15252/embj.201798044</a>","chicago":"Truckenbrodt, Sven M, Abhiyan Viplav, Sebsatian Jähne, Angela Vogts, Annette Denker, Hanna Wildhagen, Eugenio Fornasiero, and Silvio Rizzoli. “Newly Produced Synaptic Vesicle Proteins Are Preferentially Used in Synaptic Transmission.” <i>The EMBO Journal</i>. Wiley, 2018. <a href=\"https://doi.org/10.15252/embj.201798044\">https://doi.org/10.15252/embj.201798044</a>.","ista":"Truckenbrodt SM, Viplav A, Jähne S, Vogts A, Denker A, Wildhagen H, Fornasiero E, Rizzoli S. 2018. Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission. The EMBO Journal. 37(15), e98044."},"_id":"145","title":"Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission","month":"08","doi":"10.15252/embj.201798044","publist_id":"7778","article_type":"original","ddc":["570"]},{"isi":1,"intvolume":"        19","file_date_updated":"2020-07-14T12:47:32Z","scopus_import":"1","publication":"EMBO reports","author":[{"first_name":"Sven M","id":"45812BD4-F248-11E8-B48F-1D18A9856A87","full_name":"Truckenbrodt, Sven M","last_name":"Truckenbrodt"},{"last_name":"Maidorn","full_name":"Maidorn, Manuel","first_name":"Manuel"},{"full_name":"Crzan, Dagmar","last_name":"Crzan","first_name":"Dagmar"},{"full_name":"Wildhagen, Hanna","last_name":"Wildhagen","first_name":"Hanna"},{"full_name":"Kabatas, Selda","last_name":"Kabatas","first_name":"Selda"},{"first_name":"Silvio O","last_name":"Rizzoli","full_name":"Rizzoli, Silvio O"}],"oa":1,"publication_identifier":{"issn":["1469-221X"],"eissn":["1469-3178"]},"file":[{"access_level":"open_access","relation":"main_file","file_size":2005572,"creator":"kschuh","file_name":"2018_embo_Truckenbrodt.pdf","file_id":"6500","date_created":"2019-05-28T13:17:19Z","date_updated":"2020-07-14T12:47:32Z","content_type":"application/pdf","checksum":"6ec90abc637f09cca3a7b6424d7e7a26"}],"publication_status":"published","language":[{"iso":"eng"}],"department":[{"_id":"JoDa"}],"external_id":{"isi":["000443682200009"]},"date_created":"2019-05-28T13:16:08Z","quality_controlled":"1","volume":19,"abstract":[{"lang":"eng","text":"Expansion microscopy is a recently introduced imaging technique that achieves super‐resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20–30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000‐fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25–30 nm on conventional epifluorescence microscopes. X10 provides multi‐color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high‐quality super‐resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge."}],"article_number":"e45836","oa_version":"Published Version","year":"2018","date_updated":"2024-12-11T11:48:40Z","date_published":"2018-09-01T00:00:00Z","has_accepted_license":"1","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","tmp":{"short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"status":"public","day":"01","publisher":"Embo Press","type":"journal_article","issue":"9","ddc":["580"],"doi":"10.15252/embr.201845836","title":"X10 expansion microscopy enables 25‐nm resolution on conventional microscopes","month":"09","_id":"6499","citation":{"short":"S.M. Truckenbrodt, M. Maidorn, D. Crzan, H. Wildhagen, S. Kabatas, S.O. Rizzoli, EMBO Reports 19 (2018).","mla":"Truckenbrodt, Sven M., et al. “X10 Expansion Microscopy Enables 25‐nm Resolution on Conventional Microscopes.” <i>EMBO Reports</i>, vol. 19, no. 9, e45836, Embo Press, 2018, doi:<a href=\"https://doi.org/10.15252/embr.201845836\">10.15252/embr.201845836</a>.","ieee":"S. M. Truckenbrodt, M. Maidorn, D. Crzan, H. Wildhagen, S. Kabatas, and S. O. Rizzoli, “X10 expansion microscopy enables 25‐nm resolution on conventional microscopes,” <i>EMBO reports</i>, vol. 19, no. 9. Embo Press, 2018.","apa":"Truckenbrodt, S. M., Maidorn, M., Crzan, D., Wildhagen, H., Kabatas, S., &#38; Rizzoli, S. O. (2018). X10 expansion microscopy enables 25‐nm resolution on conventional microscopes. <i>EMBO Reports</i>. Embo Press. <a href=\"https://doi.org/10.15252/embr.201845836\">https://doi.org/10.15252/embr.201845836</a>","ama":"Truckenbrodt SM, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO. X10 expansion microscopy enables 25‐nm resolution on conventional microscopes. <i>EMBO reports</i>. 2018;19(9). doi:<a href=\"https://doi.org/10.15252/embr.201845836\">10.15252/embr.201845836</a>","ista":"Truckenbrodt SM, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO. 2018. X10 expansion microscopy enables 25‐nm resolution on conventional microscopes. EMBO reports. 19(9), e45836.","chicago":"Truckenbrodt, Sven M, Manuel Maidorn, Dagmar Crzan, Hanna Wildhagen, Selda Kabatas, and Silvio O Rizzoli. “X10 Expansion Microscopy Enables 25‐nm Resolution on Conventional Microscopes.” <i>EMBO Reports</i>. Embo Press, 2018. <a href=\"https://doi.org/10.15252/embr.201845836\">https://doi.org/10.15252/embr.201845836</a>."},"article_processing_charge":"No"}]