Nanoscopic X-ray fluorescence imaging and quantification of intracellular key-elements in cryofrozen Friedreich's ataxia fibroblasts

PLoS One. 2018 Jan 17;13(1):e0190495. doi: 10.1371/journal.pone.0190495. eCollection 2018.

Abstract

Synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) analysis can visualize trace level elemental distribution in a fully quantitative manner within single cells. However, in-air XRF analysis requires chemical fixation modifying the cell's chemical composition. Here, we describe first nanoscopic XRF analysis upon cryogenically frozen (-150°C) fibroblasts at the ID16A-NI 'Nano-imaging' end-station located at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France). Fibroblast cells were obtained from skin biopsies from control and Friedreich's ataxia (FRDA) patients. FRDA is an autosomal recessive disorder with dysregulation of iron metabolism as a key feature. By means of the X-ray Fundamental Parameter (FP) method, including absorption correction of the ice layer deposited onto the fibroblasts, background-corrected mass fraction elemental maps of P, S, Cl, K, Ca, Fe and Zn of entire cryofrozen human fibroblasts were obtained. Despite the presence of diffracting microcrystals in the vitreous ice matrix and minor sample radiation damage effects, clusters of iron-rich hot-spots with similar mass fractions were found in the cytoplasm of both control and FRDA fibroblasts. Interestingly, no significant difference in the mean iron concentration was found in the cytoplasm of FRDA fibroblasts, but a significant decrease in zinc concentration. This finding might underscore metal dysregulation, beyond iron, in cells derived from FRDA patients. In conclusion, although currently having slightly increased limits of detection (LODs) compared to non-cryogenic mode, SR based nanoscopic XRF under cryogenic sample conditions largely obliterates the debate on chemical sample preservation and provides a unique tool for trace level elemental imaging in single cells close to their native state with a superior spatial resolution of 20 nm.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryopreservation
  • Fibroblasts / metabolism
  • Friedreich Ataxia / metabolism*
  • Friedreich Ataxia / pathology
  • Humans
  • Limit of Detection
  • Nanotechnology
  • Reference Standards
  • Spectrometry, X-Ray Emission / methods*

Associated data

  • figshare/bdda8108e9ca1153d187

Grants and funding

Björn De Samber acknowledges his postdoctoral research grant from FWO Vlaanderen (www.fwo.be, application no. 12B3313N) and Brecht Laforce was funded by a PhD grant from the Flemish Institute for the Promotion of Scientific and Technological Research in Industry (IWT Flanders, Belgium). Research in the Vandenabeele lab is supported by grants from the Vlaams Instituut voor Biotechnologie (VIB), from Ghent University (MRP, GROUP-ID consortium), grants from the 'Foundation against Cancer' (2012-188 and FAF-F/2016/865), grants from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO) (FWO G.0875.11, FWO G.0A45.12N, FWO G.0787.13N, FWO G.0C37.14N, FWO G.0E04.16N), grants from the Flemish Government (Methusalem BOF09/01M00709 and BOF16/MET_V/007), a grant from the Belgian science policy office (BELSPO)(IAP 7/32). Tom Vanden Berghe has an assistant academic staff position at Ghent University.