Effect of sample preparation techniques upon single cell chemical imaging: A practical comparison between synchrotron radiation based X-ray fluorescence (SR-XRF) and Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS).

Analytical capabilities of Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS) and Synchrotron Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for nanochemical imaging of polymorphonuclear human neutrophils (PMNs). PMNs were high pressure frozen (HPF), cryo-substituted, embedded in Spurr's resin and cut in thin sections (500 nm and 2 µm for both techniques resp.) Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted and imbedded cells should be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells - close to their native state - remains the golden standard. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives for nanochemical imaging of single cells at the nanoscale.

Nanoscopic X-ray imaging and quantification of the iron cellular architecture within single fibroblasts of Friedreich's ataxia patients.

Friedreich's ataxia (FRDA) is a neurodegenerative disease characterized by an increase in intracytoplasmic iron concentration. Here the nanoscale iron distribution within single fibroblasts from FRDA patients was investigated using synchrotron-radiation-based nanoscopic X-ray fluorescence and X-ray in-line holography at the ID16A nano-imaging beamline of the ESRF. This unique probe was deployed to uncover the iron cellular two-dimensional architecture of freeze-dried FRDA fibroblasts. An unsurpassed absolute detection capability of 180 iron atoms within a 30 nm × 50 nm nanoscopic X-ray beam footprint was obtained using state-of-the-art X-ray focusing optics and a large-solid-angle detection system. Various micrometre-sized iron-rich organelles could be revealed for the first time, tentatively identified as endoplasmic reticulum, mitochondria and lysosomes. Also a multitude of nanoscopic iron hot-spots were observed in the cytosol, interpreted as chaperoned iron within the fibroblast's labile iron pool. These observations enable new hypotheses on the storage and trafficking of iron in the cell and ultimately to a better understanding of iron-storage diseases such as Friedreich's ataxia.

Three-Dimensional Reconstruction of the Tissue-Specific Multielemental Distribution within Ceriodaphnia dubia via Multimodal Registration Using Laser Ablation ICP-Mass Spectrometry and X-ray Spectroscopic Techniques.

In this work, the three-dimensional elemental distribution profile within the freshwater crustacean Ceriodaphnia dubia was constructed at a spatial resolution down to 5 µm via a data fusion approach employing state-of-the-art laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) and laboratory-based absorption microcomputed tomography (µ-CT). C. dubia was exposed to elevated Cu, Ni, and Zn concentrations, chemically fixed, dehydrated, stained, and embedded, prior to µ-CT analysis. Subsequently, the sample was cut into 5 µm thin sections that were subjected to LA-ICP-TOFMS imaging. Multimodal image registration was performed to spatially align the 2D LA-ICP-TOFMS images relative to the corresponding slices of the 3D µ-CT reconstruction. Mass channels corresponding to the isotopes of a single element were merged to improve the signal-to-noise ratios within the elemental images. In order to aid the visual interpretation of the data, LA-ICP-TOFMS data were projected onto the µ-CT voxels representing tissue. Additionally, the image resolution and elemental sensitivity were compared to those obtained with synchrotron radiation based 3D confocal µ-X-ray fluorescence imaging upon a chemically fixed and air-dried C. dubia specimen.

Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based X-Ray Fluorescence.

High pressure frozen (HPF), cryo-substituted microtome sections of 2 µm thickness containing human neutrophils (white blood cells) were analyzed using synchrotron radiation based X-ray fluorescence (SR nano-XRF) at a spatial resolution of 50 nm. Besides neutrophils from a control culture, we also analyzed neutrophils stimulated for 1-2 h with phorbol myristate acetate (PMA), a substance inducing the formation of so-called Neutrophil Extracellular Traps (or NETs), a defense system again pathogens possibly involving proteins with metal chelating properties. In order to gain insight in metal transport during this process, precise local evaluation of elemental content was performed reaching limits of detection (LODs) of 1 ppb. Mean weight fractions within entire neutrophils, their nuclei and cytoplasms were determined for the three main elements P, S and Cl, but also for the 12 following trace elements: K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Sr and Pb. Statistical analysis, including linear regression provided objective analysis and a measure for concentration changes. The nearly linear Ca and Cl concentration changes in neutrophils could be explained by already known phenomena such as the induction of Ca channels and the uptake of Cl under activation of NET forming neutrophils. Linear concentration changes were also found for P, S, K, Mn, Fe, Co and Se. The observed linear concentration increase for Mn could be related to scavenging of this metal from the pathogen by means of the neutrophil protein calprotectin, whereas the concentration increase of Se may be related to its antioxidant function protecting neutrophils from the reactive oxygen species they produce against pathogens. We emphasize synchrotron radiation based nanoscopic X-ray fluorescence as an enabling analytical technique to study changing (trace) element concentrations throughout cellular processes, provided accurate sample preparation and data-analysis.

Study of the distribution of actinides in human tissues using synchrotron radiation micro X-ray fluorescence spectrometry.

This study aims at evaluating the capabilities of synchrotron radiation micro X-ray fluorescence spectrometry (SR micro-XRF) for qualitative and semi-quantitative elemental mapping of the distribution of actinides in human tissues originating from individuals with documented occupational exposure. The investigated lymph node tissues were provided by the United States Transuranium and Uranium Registries (USTUR) and were analyzed following appropriate sample pre-treatment. Semi-quantitative results were obtained via calibration by external standards and demonstrated that the uranium concentration level in the detected actinide hot spots reaches more than 100 µg/g. For the plutonium hot spots, concentration levels up to 31 µg/g were found. As illustrated by this case study on these unique samples, SR micro-XRF has a high potential for this type of elemental bio-imaging owing to its high sensitivity, high spatial resolution, and non-destructive character.