Measurement of soft X-ray absorption spectra of biomolecules in biological environments

由于XANES （x射线吸收近边结构）剖面中的吸收峰具有分子特异性，因此可以用于化学作图。然而，对于生物标本的分子成像，应采用在与生物标本相似的环境中测量的XANES。本研究介绍了XANES在软x射线区域的水和细胞环境中的测量。水溶液中生物分子的测定含硫氨基酸、三肽和蛋白质的xanes采用真空专用样品室在S-K吸收边测定。通过峰移来区分含sh分子和含ss分子。此外，这种变化并不取决于标本的分子大小。基于这些结果，我们展示了(1)估计蛋白质中55的含量和(2)检测氧化应激样品中谷胱甘肽水平的可能性。哺乳动物细胞和细胞器的测定测定了CHO细胞、分离细胞核和线粒体的干燥颗粒的xanes。在细胞、细胞核和线粒体的N-K吸收边缘发现了与蛋白质和DNA相对应的XANES峰。在Ca-L和S-K吸收边也得到了类似的结果。这些结果表明，获得的生物分子XANES峰可以应用于细胞内的分子成像。此外，这一结论还需要对其他吸收边进行检验。利用电子变焦管接触显微镜在S-K吸收边缘测量了细胞核和细胞质区域的局部吸收光谱。两者的吸收边结构清晰可见。区域。这个结果之后，XANES将对小的局部区域进行测量，这将提供有关细胞中元素化学状态的信息。

It has been proposed that absorption peaks in XANES (X-ray Absorption Near Edge Structure) profiles can be used for chemical mapping due to their molecular specific nature. However for molecular imaging of biological specimens, XANES which was measured in environments similar to biological specimens should be applied. This study presents XANES measurements in water and cellular environments in the soft X-ray region.1. Measurement for biomolecules in water solutionXANES of sulfur-containing amino acids, tripeptides and proteins were measured at the S-K absorption edge using specimen chamber specially made for the use in vacuum. SH-containing molecules were discriminated from SS-containing molecules by the peak shift. In addition the shift did not depend on the molecular size of the specimens. Based on these results, we showed the possibility to the application of (1) estimation of the 55 content in proteins and (2) detection of glutathione level in specimen exposed to oxidative stress.2. Measurement for mammalian cells and cellular organellesXANES was measured for dried pellets of CHO cells, fractionated nucleus and mitochondria. At the N-K absorption edge XANES peaks corresponding to those of proteins and DNA were found for cells, nucleus and mitochondria. Similar results were obtained at the Ca-L and S-K absorption edges. These results suggest that XANES peaks obtained for biomolecules can be applied to the molecular imaging in a cell. Moreover this conclusion should be tested for other absorption edges.3. Measurement for local regions in a cellAbsorption spectra of nuclear and cytoplasmic regions were measured at the S-K absorption edge using the contact microscopy with an electronic zooming tube. The absorption edge structure was clearly observed in both. areas. This result will be followed by the XANES measurement of small local regions, which provides the information about the chemical states of elements in a cell.