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. 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.
authors: Department of Physics & Astronomy, Weinberg College of Arts and Sciences, Evanston, Illinois, U.S.A.
authors: Qiaoling
authors: Q
authors: Jin
authors: Department of Radiation Oncology, Northwestern University, Chicago, Illinois, U.S.A.
authors: Tatjana
authors: T
authors: Paunesku
authors: Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, U.S.A.
authors: Barry
authors: B
authors: Lai
authors: Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, U.S.A.
authors: Sophie-Charlotte
authors: SC
authors: Gleber
authors: Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, U.S.A.
authors: S I
authors: SI
authors: Chen
authors: Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, U.S.A.
authors: Lydia
authors: L
authors: Finney
authors: Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, U.S.A.
authors: David
authors: D
authors: Vine
authors: Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, U.S.A.
authors: Stefan
authors: S
authors: Vogt
authors: Department of Radiation Oncology, Northwestern University, Chicago, Illinois, U.S.A.
authors: Gayle
authors: G
authors: Woloschak
authors: Department of Physics & Astronomy, Weinberg College of Arts and Sciences, Evanston, Illinois, U.S.A.|Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois, U.S.A.
authors: Chris
authors: C
authors: Jacobsen
mesh_terms: D000818:Animals; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D041681:NIH 3T3 Cells; D013052:Spectrometry, X-Ray Emission / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*; D014131:Trace Elements / Q000032:analysis*
chemical_list: D014131:Trace Elements
keywords: Biological; cryomicroscopy; specimen preparation; x-ray microanalysis; x-ray microscopy