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Title: Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

Abstract

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailedmore » comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Authors:
 [1];  [2];  [3];  [3];  [3];  [3];  [3];  [3];  [2];  [4]
  1. Weinberg College of Arts and Sciences, Evanston, IL (United States). Dept. of Physics & Astronomy
  2. Northwestern Univ., Chicago, IL (United States). Dept. of Radiation Oncology
  3. Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
  4. Weinberg College of Arts and Sciences, Evanston, IL (United States). Dept. of Physics & Astronomy; Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Institutes of Health (NIH)
OSTI Identifier:
1336508
Alternate Identifier(s):
OSTI ID: 1390261; OSTI ID: 1464537
Grant/Contract Number:  
AC02-06CH11357; R01 GM104530
Resource Type:
Journal Article: Published Article
Journal Name:
Journal of Microscopy
Additional Journal Information:
Journal Volume: 265; Journal Issue: 1; Journal ID: ISSN 0022-2720
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; Biological; Cryomicroscopy; X-ray microscopy; specimen preparation; x-ray microanalysis

Citation Formats

Jin, Qiaoling, Paunesku, Tatjana, Lai, Barry, Gleber, Sophie-Charlotte, Chen, Si, Finney, Lydia, Vine, David, Vogt, Stefan, Woloschak, Gayle, and Jacobsen, Chris. Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy. United States: N. p., 2016. Web. doi:10.1111/jmi.12466.
Jin, Qiaoling, Paunesku, Tatjana, Lai, Barry, Gleber, Sophie-Charlotte, Chen, Si, Finney, Lydia, Vine, David, Vogt, Stefan, Woloschak, Gayle, & Jacobsen, Chris. Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy. United States. doi:10.1111/jmi.12466.
Jin, Qiaoling, Paunesku, Tatjana, Lai, Barry, Gleber, Sophie-Charlotte, Chen, Si, Finney, Lydia, Vine, David, Vogt, Stefan, Woloschak, Gayle, and Jacobsen, Chris. Wed . "Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy". United States. doi:10.1111/jmi.12466.
@article{osti_1336508,
title = {Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy},
author = {Jin, Qiaoling and Paunesku, Tatjana and Lai, Barry and Gleber, Sophie-Charlotte and Chen, Si and Finney, Lydia and Vine, David and Vogt, Stefan and Woloschak, Gayle and Jacobsen, Chris},
abstractNote = {Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.},
doi = {10.1111/jmi.12466},
journal = {Journal of Microscopy},
issn = {0022-2720},
number = 1,
volume = 265,
place = {United States},
year = {2016},
month = {8}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1111/jmi.12466

Citation Metrics:
Cited by: 10 works
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Works referenced in this record:

Advances in ultrarapid freezing for the preservation of cellular ultrastructure
journal, January 1986

  • Gilkey, John C.; Staehelin, L. Andrew
  • Journal of Electron Microscopy Technique, Vol. 3, Issue 2, p. 177-210
  • DOI: 10.1002/jemt.1060030206