De Samber Björn, Meul Eline, Laforce Brecht, De Paepe Boel, Smet Joél, De Bruyne Michiel, De Rycke Riet, Bohic Sylvain, Cloetens Peter, Van Coster Rudy, Vandenabeele Peter, Vanden Berghe Tom
Department of Analytical Chemistry, Ghent University, Ghent, Belgium.
VIB Center for Inflammation Research; Ghent, Belgium.
PLoS One. 2018 Jan 17;13(1):e0190495. doi: 10.1371/journal.pone.0190495. eCollection 2018.
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.
基于同步辐射的纳米级X射线荧光（SR纳米XRF）分析能够以完全定量的方式可视化单细胞内痕量元素的分布。然而，空气中的XRF分析需要化学固定，这会改变细胞的化学成分。在此，我们首次描述了在位于法国格勒诺布尔欧洲同步辐射装置（ESRF）的ID16A-NI“纳米成像”终端站上，对低温冷冻（-150°C）的成纤维细胞进行的纳米级XRF分析。成纤维细胞取自对照和弗里德赖希共济失调（FRDA）患者的皮肤活检样本。FRDA是一种常染色体隐性疾病，其关键特征是铁代谢失调。通过X射线基本参数（FP）方法，包括对沉积在成纤维细胞上的冰层进行吸收校正，获得了整个冷冻人成纤维细胞中P、S、Cl、K、Ca、Fe和Zn的背景校正质量分数元素图。尽管在玻璃态冰基质中存在衍射微晶且有轻微的样品辐射损伤效应，但在对照和成纤维细胞的细胞质中均发现了质量分数相似的富铁热点簇。有趣的是，在FRDA成纤维细胞的细胞质中未发现平均铁浓度有显著差异，但锌浓度显著降低。这一发现可能突出了FRDA患者来源的细胞中除铁之外的金属失调情况。总之，尽管目前与非低温模式相比检测限略有提高，但低温样品条件下基于SR的纳米级XRF在很大程度上消除了关于化学样品保存的争论，并为接近其天然状态的单细胞痕量元素成像提供了一种独特工具，具有20 nm的卓越空间分辨率。
