Nanoscale Examination of Biological Tissues Using X-ray Spectromicroscopy

Abstract

Establishing the composition and distribution of chemical compounds within biological materials is fundamental to understanding the chemistry that drives life. Additionally, region-specific changes in chemical homeostasis have been linked to disease states [1]. Traditional techniques used to explore these relationships rely on the use of fixatives and dyes, which can significantly alter the native chemistry of the sample material [2]. Here we describe methodology for the preparation and examination of biological sample materials, including human tissues, using the X-ray Spectromicroscopy technique Scanning Transmission X-ray Microscopy (STXM). Through STXM we show the distribution and speciation of organic and inorganic materials within biological samples at a nanoscale resolution. Importantly this approach does not require the use of dyes, aldehyde fixatives or contrast agents and therefore offers an unprecedented insight into the native chemistry of biological samples. Human brain tissues were ethanol dehydrated and embedded in a resin comprised of equimolar trimethylolpropane triglycidyl ether: 4,4'-methylenebis (2-methylcyclohexylamine). Semi-thin (typically 200-500 nm) sections from embedded samples were cut using a non-metallic blade, before being mounted onto a support. STXM was performed at the carbon K-edge, oxygen K-edge, calcium L-edge and iron Ledge. To map distributions of chemical species, paired images were taken at the energy corresponding to a peak feature of interest and an off-peak energy a few eV away from this feature. The off-peak image is then subtracted from the peak image to give a difference map. This process is demonstrated in Figure 1, for a 200 nm thick section of human substantia nigra tissue imaged at the carbon K-edge. Here the peak image (288.3 eV; left) corresponds to the 1s to π* transition for amide groups (proteins), whereas the offpeak image (290 eV; middle) corresponds to the principal carbon K-edge absorption feature for the resin (see also [1]). This image processing allows artifacts and background resin absorption features to be removed, revealing the true tissue structure. From these difference maps, tissue structure is shown to be well preserved over the 160 µm 2 area presented. The high resolution map taken at ca. 40 nm resolution (right; inset) shows cellular structures, also displaying subcellular organelles.Further speciation maps and a carbon K-edge absorption spectrum from a 200 nm thick section of human amygdala tissue are shown in Figure 2. X-ray absorption spectra were obtained from a series of images taken at energies spanning the carbon K-edge edge. The resulting spectrum from tissue area A1 ( Figure 2f; green spectrum) was consistent with a synthetic peptide standard (Figure 2f; black spectrum), indicating the tissue to have been preserved through the embedding process. Further dense areas of tissue (as evidenced by enhanced contrast in Figure 2a) were found to be loaded with calcium (Figure 2b), carbonates ( Figure 2c) and iron (Figure 2d). This demonstra...

Label‐Free Nanoimaging of Neuromelanin in the Brain by Soft X‐ray Spectromicroscopy

“…Forschungsartikel carbon K-edge,a llowing metal speciation to be correlated with protein distribution. This has been previously demonstrated to good effect in detailed characterizations of amyloid-b/iron aggregates formed in vitro, [20] ex vivo human brain tissue, [19] and amyloid plaque cores extracted from Alzheimersdisease brain tissue. [21] In addition, the ability to generate image contrast artificially by exploiting specific spectral features,u niquely allows the technique to be used to map selectively structures for which histological stains do not exist.…”

“…Thin sections used for STXM (100–200 nm) permit spectra to be collected at the carbon K ‐edge, allowing metal speciation to be correlated with protein distribution. This has been previously demonstrated to good effect in detailed characterizations of amyloid‐β/iron aggregates formed in vitro, ex vivo human brain tissue, and amyloid plaque cores extracted from Alzheimer's disease brain tissue …”

Label‐Free Nanoimaging of Neuromelanin in the Brain by Soft X‐ray Spectromicroscopy

“…Thescope for characterization of both organic and inorganic tissue components using STXM over these length scales has been previously demonstrated. [19,21] Hence,i ti si mportant to consider also the potential for distinguishing NM from other intracellular constituents,p articularly those with affinity for metal ions.F igure 4i ndicates that x-ray absorption spectroscopy (XAS) at the carbon K-edge may indeed be used to differentiate NM from the iron storage protein, ferritin, and the dominant constituent of Lewy bodies,a lpha-synuclein. Given the relative ease with which soft x-ray techniques can switch between organic and inorganic (metal) edges,S TXM could be further applied to examine NM-bound metals independent of ferritin-/synuclein-bound metals within the same cell.…”

“…[17] Thin sections used for STXM (100-200 nm) permit spectra to be collected at the carbon K-edge,a llowing metal speciation to be correlated with protein distribution. This has been previously demonstrated to good effect in detailed characterizations of amyloid-b/iron aggregates formed in vitro, [20] ex vivo human brain tissue, [19] and amyloid plaque cores extracted from Alzheimersdisease brain tissue. [21] In addition, the ability to generate image contrast artificially by exploiting specific spectral features,u niquely allows the technique to be used to map selectively structures for which histological stains do not exist.…”

“…[18] Scanning transmission x-ray microscopy (STXM) is asynchrotron-based spectromicroscopy technique that combines excellent chemical sensitivity with high-resolution microscopy.Usually employed in the soft x-ray range (circa 0.1-1 keV) for biological samples,t his approach permits access to both organic and inorganic (metal) absorption edges without the necessity for artificial staining (typically required for light or electron microscopy). [17,19] Soft x-ray microscopy is capable of spanning the water window (280-540 eV range), in which there is an order of magnitude difference between absorption coefficients for water and protein.…”