The Bionanoprobe has been developed to study trace elements in frozen-hydrated biological systems with sub-100 nm spatial resolution. Here its performance is demonstrated and first results reported.
X-ray fluorescence (XRF) microscopy is one of the most sensitive imaging techniques to study trace elemental distributions in biological specimens. A typical setup for XRF imaging is presented in Fig.1. By using spatially coherent, highly brilliant X-rays as the excitation source and advanced X-ray optics, current synchrotron-based XRF microprobes at the Advanced Photon Source (APS) at Argonne National Laboratory reproducibly achieve a 150 nm spatial resolution on biomaterials. Conventional sample preparation approaches for XRF imaging usually require dehydration, and may include chemical fixation. These methods limit the ability to preserve the 'natural' element distribution at high spatial resolution, and thus may not be sufficient as the spatial resolution of a XRF probe reaches sub-100 nm. Additionally, even dry samples could accumulate radiation damage during repeated imaging and ultimately cause resolution degradation [1]. As a comparison, preserving samples in their frozen-hydrated states and imaging them under cryogenic conditions not only allows studying in the most-close-to-nature states but also significantly increases the radiation tolerance of hydrated samples [2].With the aim of studying frozen-hydrated biomaterials at sub-100 nm resolution, we have been developing a Bionanoprobe (BNP). It is a sample scanning X-ray nanoprobe with cryo-capabilities and robotic sample changer, and is the first microscope of its kind. The BNP has recently been assembled at Xradia Inc. and is currently under commissioning at Life Science Collaborative Access Team (LS-CAT) at the APS. As shown in Fig.2, most components are mounted inside a vacuum chamber and decoupled from the chamber by using a kinematic plate. Utilizing multiple zone plates, the BNP can be operated in an energy range from 5-20 keV, with a spatial resolution down to 30 nm for fluorescence mapping and 50 nm for spectroscopy. The laser interferometer system maintains an accurate beam focus during scans. The cryo sample handling module and sample loading robot enable samples to be well preserved around 100 K in the BNP.By using the BNP, the first images of a frozen-hydrated whole cell have been acquired from a plunge-frozen rat fibroblast sample (Fig.3). Both phase contrast transmission signal and fluorescence signal were obtained simultaneously showing the structure and elemental distributions within the cell in detail, which could not be resolved by conventional XRF. In addition to 2D scans, the BNP also enables semi-automated tomography by using a rotational sample stage and associated algorithm. In summary, the preliminary results from the BNP have demonstrated its unique capability in probing frozen-hydrated biological specimens at high resolution. Many scientific areas, 962
Correlative microscopy allows us to identify sites-of-interest across different length scales and perform both structural and chemical analysis by combining the use of light, electrons and x-ray photons. It becomes an essential method to address questions for both biological and materials sciences. We have presented here example studies as well as the challenges of using synchrotronbased x-ray fluorescence microscopy (SXFM) as a complementary tool to either light microscopy (LM) or transmission electron microscopy (TEM) for trace element analysis.Trace elements are essential for the existence of life. It is estimated that one-third of all known proteins contain metal cofactors, and the majority of these function as essential metalloenzymes. For example, Mn has vital roles in enzyme reactions, however excessive Mn can induce neurological diseases [1]. In a recent study using rat primary midbrain neurons, we have found that αSynulein (αSyn) alters the level of intracellular Mn, which may contribute to chronic neurodegeneration [2]. By using SXRF in conjunction with the GFP fluorescence, we were able to measure the intercellular elements in GFP-positive cells, i.e. cells that were overexpressing αSyn (Figure 1). In order to minimize structural/elemental alternation and reduce radiation damage under x-ray exposure, the samples were cryopreserved. The measurements were carried out in cryogenic conditions at both a light microscope and the Bionanoprobe, i.e. an SXFM instrument dedicated to cryogenic studies at sub-100 nm resolution at the Advanced Photon Source [3]. In a different study, Mn was used to enhance magnetic resonance imaging of brain to trace neuronal connections [4]. In order to elucidate the mechanism of Mn uptake, rat hippocampal slices of 80 nm were imaged using a TEM first and then measured at the BNP with a photon energy above Mn k-edge. The intercellular Mn distribution was then correlated with the TEM ultrastructure [5]. SXFM delivers unprecedented elemental sensitivity, which well complements the higher spatial resolution offered by TEM.Trace elements also play important roles in materials science. For example, addition of elements in trace amount to Al alloys, i.e. microalloying, is an effective way to improve their mechanical and anticorrosive properties [6]. Our present work has been focusing on a novel microalloyed 5xxx Al alloy with a nominal composition of Al-6Mg-0.9Mn-0.07Zr-0.2Er (wt.%). The microstructure and elemental distributions have been characterized using both SXFM beamlines and an analytical TEM. While the TEM was able to detect Er-and Mn-enriched precipitates of sub-10 nm in size, SXFM was used to survey relatively large and thick regions (Figure 2). Further quantitative analysis and correlation will be carried out.
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