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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
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 researcher...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9962160/ https://www.ncbi.nlm.nih.gov/pubmed/36838979 http://dx.doi.org/10.3390/molecules28041992 |
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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 |
author_facet | 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 |
author_sort | Merolle, Lucia |
collection | PubMed |
description | 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/ C(3)H(6)O 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. |
format | Online Article Text |
id | pubmed-9962160 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-99621602023-02-26 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 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 Molecules Article 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/ C(3)H(6)O 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. MDPI 2023-02-20 /pmc/articles/PMC9962160/ /pubmed/36838979 http://dx.doi.org/10.3390/molecules28041992 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article 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 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 |
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 |
title_full | 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 |
title_fullStr | 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 |
title_full_unstemmed | 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 |
title_short | 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 |
title_sort | 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 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9962160/ https://www.ncbi.nlm.nih.gov/pubmed/36838979 http://dx.doi.org/10.3390/molecules28041992 |
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