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    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

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    Single_Cells_Xray (4.507Mb)
    Date
    2023-02-20
    Author
    Merolle, Lucia
    Pascolo, Lorella
    Zupin, Luisa
    Parisse, Pietro
    Bonanni, Valentina
    Gariani, Gianluca
    Kenig, Sasa
    Bedolla, Diana E.
    Crovella, Sergio
    Ricci, Giuseppe
    Iotti, Stefano
    Malucelli, Emil
    Kourousias, George
    Gianoncelli, Alessandra
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    Abstract
    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'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.
    URI
    https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85149054233&origin=inward
    DOI/handle
    http://dx.doi.org/10.3390/molecules28041992
    http://hdl.handle.net/10576/40927
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    • Biological & Environmental Sciences [‎931‎ items ]

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