Synchrotron-based X-ray Fluorescence Microscopy as a Complementary Tool to Light Microscopy/Electron Microscopy for Multi-scale and Multi-modality Analysis

Abstract

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.

Metal ion fluxes controlling amphibian fertilization

“…X-ray Fluorescence Microscopy (XFM) 32 methods indicated that cortical vesicles in mouse eggs contain on average one million zinc ions each 3 and we employed this approach to test for the presence of analogous zinc- and manganese-enriched sites in small sections at the cortices of Xenopus eggs and embryos. To preserve transition metal contents in our samples we used a modified form of Timm’s silver staining.…”