Biological samples are frequently stained with heavy metals in preparation for examining the macro, micro and ultra-structure using X-ray microtomography and electron microscopy. A single X-ray microtomography scan reveals detailed 3D structure based on staining density, yet it lacks both material composition and functional information. Using a commercially available polychromatic X-ray source, energy integrating detectors and a two-scan configuration labelled by their energy- "High" and "Low", we demonstrate how a specific element, here shown with iron, can be detected from a mixture with other heavy metals. With proper selection of scan configuration, achieving strong overlap of source characteristic emission lines and iron K-edge absorption, iron absorption was enhanced enabling K-edge imaging. Specifically, iron images were obtained by scatter plot material analysis, after selecting specific regions within scatter plots generated from the "High" and "Low" scans. Using this method, we identified iron rich regions associated with an iron staining reaction that marks the nodes of Ranvier along nerve axons within mouse spinal roots, also stained with osmium metal commonly used for electron microscopy.
60kVp with fe filtration.
XRM was performed on a Versa 510, (Carl Zeiss X-ray Microscopy, Pleasanton, CA 94588). The Versa 510 is equipped with a polychromatic tungsten source, tunable in the voltage range 30‐160kVp. Scan configuration: "Low" ‐30kVp peak tube potential without filtration, exposure time 20‐90sec. "High"‐60kVp peak tube potential, filtration 25μm thick iron filter, exposure time 10‐120sec. Common scan conditions: binning 1, source-rotation axis distance 7.5mm, detector-rotation axis distance 5mm, pixel size 0.394μm, optical magnification x20, rotation angle ‐180° to +180°, number of views 3201. Iron filtration used a 0.001"x1"x2" foil placed at the X-ray tube exit slit. The foil was of 3N5 purity and sold by ESPI Metals, Ashland, OR 97520.
