Iron-specific Signal Separation from within Heavy Metal Stained Biological Samples Using X-Ray Microtomography with Polychromatic Source and Energy-Integrating Detectors

Tsvi Katchalski,Ranjan Ramachandra,Thomas J Deerinck,Albert R Lawrence,Nobuko Fujikawa,Matthias G Haberl,Mark H Ellisman,Tom Case,Keun-Young Kim,Steven Peltier,Guillaume A Castillon,Eric A Bushong,Mason R Mackey

doi:10.1038/s41598-018-25099-z

Tsvi Katchalski, Ranjan Ramachandra + Show 11 more

Open Access

https://doi.org/10.1038/s41598-018-25099-z

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Abstract

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.

Highlights

Reconstructed images will display overall attenuation correlated with specimen density without the ability to discriminate variations in material composition or the ability to detect a specific marker
In the work presented here we used XRM to detect stained iron regions of micron and sub-micron scale in a biological sample stained with other heavy metals typically used for electron microscopy staining, such as osmium
The second scan is performed after X-pray filtration which absorbs strongly the characteristic emission lines relative to the higher energy continuum part of the spectrum causing a reduction in iron absorption

Summary

Introduction

Reconstructed images will display overall attenuation correlated with specimen density without the ability to discriminate variations in material composition or the ability to detect a specific marker. Other methods include dual energy as used in medical imaging, where the different contributions of photoelectric and Compton scatter to the total absorption are used (e.g., to separate calcium or bone from iodine[19]) In those cases the minimum X-ray energy of substantial power due to filtration is considerably above any low Z-element K-edge energy. We initially demonstrated results on a phantom, theoretically explored optimal scan configuration and applied this information to identify iron rich regions associated with the nodes of Ranvier in samples of mouse spinal root stained with iron and osmium These results could potentially allow following iron specific biological markers such as ferritin[23] and introduce functionality into XRM measurements

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Results

Conclusion