{"volume":24,"intvolume":"        24","year":"2023","file_date_updated":"2023-02-20T07:09:27Z","isi":1,"acknowledgement":"This work has been supported by funding of the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG], CRC 166, Project B06 to M.H. and A.-L.S., FOR 3004 SYNABS P1 to M.H.) and by the Interdisciplinary Clinical Research Center (IZKF) Würzburg (Z-3/69 to M.M.P., N-229 to M.H. and A.-L.S.). A.M. is funded by the University of Leipzig Clinician Scientist Program.","ddc":["570"],"publication":"International Journal of Molecular Sciences","author":[{"full_name":"Mrestani, Achmed","first_name":"Achmed","last_name":"Mrestani"},{"last_name":"Lichter","first_name":"Katharina","full_name":"Lichter, Katharina","id":"39302e62-fcfc-11ec-8196-8b01447dbd3d"},{"first_name":"Anna Leena","last_name":"Sirén","full_name":"Sirén, Anna Leena"},{"full_name":"Heckmann, Manfred","last_name":"Heckmann","first_name":"Manfred"},{"full_name":"Paul, Mila M.","first_name":"Mila M.","last_name":"Paul"},{"full_name":"Pauli, Martin","last_name":"Pauli","first_name":"Martin"}],"has_accepted_license":"1","doi":"10.3390/ijms24032128","publication_identifier":{"eissn":["1422-0067"]},"external_id":{"isi":["000930324700001"]},"article_type":"original","citation":{"short":"A. Mrestani, K. Lichter, A.L. Sirén, M. Heckmann, M.M. Paul, M. Pauli, International Journal of Molecular Sciences 24 (2023).","ama":"Mrestani A, Lichter K, Sirén AL, Heckmann M, Paul MM, Pauli M. Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation. <i>International Journal of Molecular Sciences</i>. 2023;24(3). doi:<a href=\"https://doi.org/10.3390/ijms24032128\">10.3390/ijms24032128</a>","apa":"Mrestani, A., Lichter, K., Sirén, A. L., Heckmann, M., Paul, M. M., &#38; Pauli, M. (2023). Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation. <i>International Journal of Molecular Sciences</i>. MDPI. <a href=\"https://doi.org/10.3390/ijms24032128\">https://doi.org/10.3390/ijms24032128</a>","chicago":"Mrestani, Achmed, Katharina Lichter, Anna Leena Sirén, Manfred Heckmann, Mila M. Paul, and Martin Pauli. “Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila Melanogaster after Rapid Cryofixation.” <i>International Journal of Molecular Sciences</i>. MDPI, 2023. <a href=\"https://doi.org/10.3390/ijms24032128\">https://doi.org/10.3390/ijms24032128</a>.","ista":"Mrestani A, Lichter K, Sirén AL, Heckmann M, Paul MM, Pauli M. 2023. Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation. International Journal of Molecular Sciences. 24(3), 2128.","ieee":"A. Mrestani, K. Lichter, A. L. Sirén, M. Heckmann, M. M. Paul, and M. Pauli, “Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation,” <i>International Journal of Molecular Sciences</i>, vol. 24, no. 3. MDPI, 2023.","mla":"Mrestani, Achmed, et al. “Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila Melanogaster after Rapid Cryofixation.” <i>International Journal of Molecular Sciences</i>, vol. 24, no. 3, 2128, MDPI, 2023, doi:<a href=\"https://doi.org/10.3390/ijms24032128\">10.3390/ijms24032128</a>."},"article_number":"2128","_id":"12567","language":[{"iso":"eng"}],"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","date_created":"2023-02-19T23:00:56Z","oa_version":"Published Version","oa":1,"scopus_import":"1","publisher":"MDPI","status":"public","quality_controlled":"1","title":"Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation","day":"21","type":"journal_article","file":[{"date_updated":"2023-02-20T07:09:27Z","date_created":"2023-02-20T07:09:27Z","file_size":2823025,"file_id":"12569","success":1,"relation":"main_file","checksum":"69a35dcd3e0249f902ab881b06ee2e58","file_name":"2023_IJMS_Mrestani.pdf","creator":"dernst","access_level":"open_access","content_type":"application/pdf"}],"date_updated":"2023-08-01T13:16:36Z","tmp":{"image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"month":"01","article_processing_charge":"No","issue":"3","date_published":"2023-01-21T00:00:00Z","publication_status":"published","abstract":[{"lang":"eng","text":"Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations."}],"department":[{"_id":"PeJo"}]}