Quantitative Distribution of DNA, RNA, Histone and Proteins Other than Histone in Mammalian Cells, Nuclei and a Chromosome at High Resolution Observed by Scanning Transmission Soft X-Ray Microscopy (STXM).

Kunio Shinohara,Takeo Ejima,Takuji Ohigashi,Shigenobu Toné,Atsushi Ito

doi:10.3390/cells8020164

Kunio Shinohara, Takeo Ejima + Show 3 more

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https://doi.org/10.3390/cells8020164

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Journal: Cells	Publication Date: Feb 16, 2019
Citations: 8	License type: CC BY 4.0

Affiliation: Tokai University, Tohoku University, Tokyo Denki University

Abstract

Soft X-ray microscopy was applied to study the quantitative distribution of DNA, RNA, histone, and proteins other than histone (represented by BSA) in mammalian cells, apoptotic nuclei, and a chromosome at spatial resolutions of 100 to 400 nm. The relative distribution of closely related molecules, such as DNA and RNA, was discriminated by the singular value decomposition (SVD) method using aXis2000 software. Quantities of nucleic acids and proteins were evaluated using characteristic absorption properties due to the 1s–π * transition of N=C in nucleic acids and amide in proteins, respectively, in the absorption spectra at the nitrogen K absorption edge. The results showed that DNA and histone were located in the nucleus. By contrast, RNA was clearly discriminated and found mainly in the cytoplasm. Interestingly, in a chromosome image, DNA and histone were found in the center, surrounded by RNA and proteins other than histone. The amount of DNA in the chromosome was estimated to be 0.73 pg, and the content of RNA, histone, and proteins other than histone, relative to DNA, was 0.48, 0.28, and 4.04, respectively. The method we present in this study could be a powerful approach for the quantitative molecular mapping of biological samples at high resolution.

Highlights

In principle, X-ray microscopy has the following advantages for the observation of biological samples over other microscopic methods: higher resolution than optical microscopy with respect to the diffraction limit; good absorption contrast in hydrated conditions with soft X-rays in an energy range, the so-called water window; better transmittance than electron microscopy; and the discrimination of biological molecules by spectromicroscopy, the combination of microscopy and spectroscopy using absorption of fine structures in biomolecules according to the energies of carbon, nitrogen, and oxygen absorption edges
near-edge X-ray absorption fine structure (NEXAFS) at the nitrogen K absorption edge shows clear separation of the absorption spectra of nucleic acids and proteins, and it has been applied to identify these molecules in images of a mammalian cell and a chromosome observed with a scanning transmission soft X-ray microscope (STXM) [23,24], a microscopy technique in which samples are moving on the focused soft X-ray beam
The images of DNA and RNA in the absorption images of the energy stack file of the sample were extracted by the singular value decomposition (SVD) method on aXis2000 using the spectra of DNA and RNA, each of which was measured at the photon energy regions of carbon, nitrogen, and oxygen K absorption edges and combined

Summary

Introduction

X-ray microscopy has the following advantages for the observation of biological samples over other microscopic methods: higher resolution than optical microscopy with respect to the diffraction limit; good absorption contrast in hydrated conditions with soft X-rays in an energy range, the so-called water window; better transmittance than electron microscopy; and the discrimination of biological molecules by spectromicroscopy, the combination of microscopy and spectroscopy using absorption of fine structures in biomolecules according to the energies of carbon, nitrogen, and oxygen absorption edges. Combined NEXAFS at the K absorption edges of carbon, nitrogen, and oxygen was found to be useful for differentiating closely related molecules, such as DNA and RNA [24]

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