Abstract
Soft X-ray microscopy is ideally suited to visualizing and quantifying biological cells. Specimens, including eukaryotic cells, are imaged intact, unstained and fully hydrated, and therefore visualized in a near-native state. The contrast in soft X-ray microscopy is generated by the differential attenuation of X-rays by the molecules in the specimen—water is relatively transmissive to this type of illumination compared to carbon and nitrogen. The attenuation of X-rays by the specimen follows the Beer–Lambert law, and therefore both linear and a quantitative measure of thickness and chemical species present at each point in the cell. In this chapter, we will describe the procedures and computational methods that lead to 50 nm (or better) tomographic reconstructions of cells using soft X-ray microscope data, and the subsequent segmentation and analysis of these volumetric reconstructions. In addition to being a high-fidelity imaging modality, soft X-ray tomography is relatively high-throughput; a complete tomographic data set can be collected in a matter of minutes. This new modality is being applied to imaging cells that range from small prokaryotes to stem cells obtained from mammalian tissues.
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Acknowledgments
XM-2 and associated research is funded by the Department of Energy Office of Biological and Environmental Research Grant DE-AC02-05CH11231, and the NIH National Center for Research Resources Grant RR019664.
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Parkinson, D.Y., Epperly, L.R., McDermott, G., Le Gros, M.A., Boudreau, R.M., Larabell, C.A. (2013). Nanoimaging Cells Using Soft X-Ray Tomography. In: Sousa, A., Kruhlak, M. (eds) Nanoimaging. Methods in Molecular Biology, vol 950. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-137-0_25
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DOI: https://doi.org/10.1007/978-1-62703-137-0_25
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