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المؤلفMerolle, Lucia
المؤلفPascolo, Lorella
المؤلفZupin, Luisa
المؤلفParisse, Pietro
المؤلفBonanni, Valentina
المؤلفGariani, Gianluca
المؤلفKenig, Sasa
المؤلفBedolla, Diana E.
المؤلفCrovella, Sergio
المؤلفRicci, Giuseppe
المؤلفIotti, Stefano
المؤلفMalucelli, Emil
المؤلفKourousias, George
المؤلفGianoncelli, Alessandra
تاريخ الإتاحة2023-03-12T06:38:29Z
تاريخ النشر2023-02-20
اسم المنشورMolecules (Basel, Switzerland)
المعرّفhttp://dx.doi.org/10.3390/molecules28041992
الاقتباسMerolle, L.; Pascolo, L.; Zupin, L.; Parisse, P.; Bonanni, V.; Gariani, G.; Kenig, S.; Bedolla, D.E.; Crovella, S.; Ricci, G.; Iotti, S.; Malucelli, E.; Kourousias, G.; Gianoncelli, A. 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. Molecules 2023, 28, 1992. https://doi.org/10.3390/molecules28041992
معرّف المصادر الموحدhttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85149054233&origin=inward
معرّف المصادر الموحدhttp://hdl.handle.net/10576/40927
الملخصBACKGROUND: 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's 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.
راعي المشروعThis research was partially supported by the Italian Ministry of Health, through the contribution given to the Institute for Maternal and Child Health IRCCS Burlo Garofolo Trieste, Italy (5mille15D1; 5xmille SD03/22). Part of this work has been performed in the frame of the INTEGRA CERIC-ERIC research project.
اللغةen
الناشرMDPI
الموضوعAFM
fixation methods
single cells
XRF
العنوانImpact 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
النوعArticle
رقم العدد4
رقم المجلد28
ESSN1420-3049


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