{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T18:01:20Z","timestamp":1787508080625,"version":"build-2736575974"},"reference-count":34,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2023,2,20]],"date-time":"2023-02-20T00:00:00Z","timestamp":1676851200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of Health","award":["5mille15D1"],"award-info":[{"award-number":["5mille15D1"]}]},{"name":"Italian Ministry of Health","award":["5xmille SD03\/22"],"award-info":[{"award-number":["5xmille SD03\/22"]}]},{"name":"Institute for Maternal and Child Health IRCCS Burlo Garofolo Trieste, Italy","award":["5mille15D1"],"award-info":[{"award-number":["5mille15D1"]}]},{"name":"Institute for Maternal and Child Health IRCCS Burlo Garofolo Trieste, Italy","award":["5xmille SD03\/22"],"award-info":[{"award-number":["5xmille SD03\/22"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Background: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment\u2019s success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. Methods: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol\/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. Results: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. Conclusion: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy.<\/jats:p>","DOI":"10.3390\/molecules28041992","type":"journal-article","created":{"date-parts":[[2023,2,20]],"date-time":"2023-02-20T06:37:59Z","timestamp":1676875079000},"page":"1992","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Impact of Sample Preparation Methods on Single-Cell X-ray Microscopy and Light Elemental Analysis Evaluated by Combined Low Energy X-ray Fluorescence, STXM and AFM"],"prefix":"10.3390","volume":"28","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5712-4272","authenticated-orcid":false,"given":"Lucia","family":"Merolle","sequence":"first","affiliation":[{"name":"AUSL-IRCCS di Reggio Emilia, Transfusion Medicine Unit, 42123 Reggio Emilia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1044-7263","authenticated-orcid":false,"given":"Lorella","family":"Pascolo","sequence":"additional","affiliation":[{"name":"IRCCS Burlo Garofolo, Institute for Maternal and Child Health, 34137 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5886-9129","authenticated-orcid":false,"given":"Luisa","family":"Zupin","sequence":"additional","affiliation":[{"name":"IRCCS Burlo Garofolo, Institute for Maternal and Child Health, 34137 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pietro","family":"Parisse","sequence":"additional","affiliation":[{"name":"Consiglio Nazionale delle Ricerche, Istituto Officina dei Materiali, 34149 Trieste, Italy"},{"name":"Elettra\u2014Sincrotrone Trieste S.C.p.A., Basovizza, 34149 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8346-0069","authenticated-orcid":false,"given":"Valentina","family":"Bonanni","sequence":"additional","affiliation":[{"name":"Elettra\u2014Sincrotrone Trieste S.C.p.A., Basovizza, 34149 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gianluca","family":"Gariani","sequence":"additional","affiliation":[{"name":"Elettra\u2014Sincrotrone Trieste S.C.p.A., Basovizza, 34149 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4272-3058","authenticated-orcid":false,"given":"Sasa","family":"Kenig","sequence":"additional","affiliation":[{"name":"Faculty of Health Sciences, University of Primorska, Polje 42, 6310 Izola, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1902-1517","authenticated-orcid":false,"given":"Diana E.","family":"Bedolla","sequence":"additional","affiliation":[{"name":"Elettra\u2014Sincrotrone Trieste S.C.p.A., Basovizza, 34149 Trieste, Italy"},{"name":"Area Science Park, Padriciano 99, 34149 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8493-1168","authenticated-orcid":false,"given":"Sergio","family":"Crovella","sequence":"additional","affiliation":[{"name":"Biological Science Program, Department of Biological and Environmental Science, College of Arts and Sciences, Qatar University, Doha 2713, Qatar"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8031-1102","authenticated-orcid":false,"given":"Giuseppe","family":"Ricci","sequence":"additional","affiliation":[{"name":"IRCCS Burlo Garofolo, Institute for Maternal and Child Health, 34137 Trieste, Italy"},{"name":"Department of Medical, Surgical, and Health Sciences, University of Trieste, 34149 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1426-6400","authenticated-orcid":false,"given":"Stefano","family":"Iotti","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy"},{"name":"National Institute of Biostructures and Biosystems, 00136 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1077-0098","authenticated-orcid":false,"given":"Emil","family":"Malucelli","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1243-7168","authenticated-orcid":false,"given":"George","family":"Kourousias","sequence":"additional","affiliation":[{"name":"Elettra\u2014Sincrotrone Trieste S.C.p.A., Basovizza, 34149 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2457-3618","authenticated-orcid":false,"given":"Alessandra","family":"Gianoncelli","sequence":"additional","affiliation":[{"name":"Elettra\u2014Sincrotrone Trieste S.C.p.A., Basovizza, 34149 Trieste, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1063\/1.2947651","article-title":"Applications of Synchrotron Radiation: Micro Beams in Cell Micro Biology and Medicine","volume":"61","author":"Vogt","year":"2008","journal-title":"Phys. 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