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.
Highlights
Friedreich’s ataxia (FRDA) is an autosomal recessive disorder with a carrier frequency of 1/100 caused in most cases by a trinucleotide repeat expansion in the FXN gene [1]
Prussian blue staining on control and FRDA fibroblasts
Fibroblasts were taken from FRDA patients at the University Hospital Ghent (UZ Gent, Department of Pediatrics and medical genetics) using skin biopsy
Summary
FRDA is an autosomal recessive disorder with a carrier frequency of 1/100 caused in most cases by a trinucleotide repeat expansion in the FXN gene [1]. It is proposed that an excess in cytosolic rather than mitochondrial iron, often referred to as the ‘labile iron pool’ (LIP), is the causative detrimental factor leading to cell death [14, 20]. Other metals such as copper and zinc were found to be dysregulated in FRDA cells, which has fueled the concept of a more general metal dysmetabolism [9, 21, 22]. The investigation of the spatial distribution of iron and other metals within the subcellular compartments of control and FRDA fibroblasts is of high scientific interest as it may shed light upon the role of metal dysbiosis in FRDA and neurodegenerative diseases in general
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