Discrimination of DNA and RNA distribution in a mammalian cell by scanning transmission soft X-ray microscopy

Abstract

In this study, soft X-ray spectromicroscopy was applied to investigate and analyze the distribution of DNA and RNA in a mammalian cell at the spatial resolution of 400 nm. The relative distribution of DNA and RNA was examined by the SVD (singular value decomposition) method in aXis2000 program using combined full spectra of DNA and RNA at the absorption edge regions of carbon, nitrogen and oxygen. The absorption of nucleic acid was evaluated using 1s-π* transitions in the NEXAFS spectra at the nitrogen K absorption edge and distributed to DNA and RNA according to the relative level obtained above. The study results revealed the usefulness of the SVD method to discriminate closely related molecules such as DNA and RNA.

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)

“…CHO cells at interphase or mitotic (M) phase were prepared as described previously [23,24]. HeLa S3 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics at 37 °C in a humidified 5% CO 2 atmosphere.…”

“…For the observation of CHO and HeLa S3 cells, these cells were plated and grown on a silicon nitride (Si 3 N 4 ) membrane (thickness, 100 nm; purchased from Silson Ltd., Warwickshire, England) and processed using a previously described method [24]. Mitotic CHO cells were treated as previously described [23].…”

“…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. These advantages have been demonstrated by the development of X-ray microscopes [1] with absorption contrast [2,3], followed by phase contrast with coherent diffraction imaging and ptychographic imaging [4,5,6], and include direct observation of intact biological samples under hydrated conditions at a resolution of less than 100 nm with contact microscopy [7,8]; mapping elements in biological samples [9,10]; 3D observation of frozen hydrated specimens [11,12,13,14,15,16,17]; and the mapping of molecular distributions by spectromicroscopy [18,19,20,21,22,23,24]. Spectromicroscopy is a powerful tool for the analysis of local quantities and distribution of biomolecules.…”

“…The characteristic nature of the X-ray absorption spectrum near the absorption edge of each element—called near-edge X-ray absorption fine structure (NEXAFS)—is useful for the separation of constituent molecules in biological specimens because NEXAFS depends on the chemical structures of the molecules. 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. In addition, 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].…”

“…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. In addition, 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]. In the present study, we applied NEXAFS at the carbon, nitrogen, and oxygen K absorption edges to analyze molecular distributions in mammalian cells, apoptotic nuclei, and a chromosome.…”