Spectroscopic Speciation of Sulfur in Whole Cells

描述（由申请人提供）：我们建议开发硫K边X射线吸收近边光谱，以便它可以用于监测和提高我们对生命系统中硫代谢的理解，特别是在哺乳动物细胞中。目前还没有生物系统中硫的有效光谱探针。X射线吸收光谱法将检测样品中、固体中、水溶液中或任何其他形式的硫的所有化学形式，并且不同形式的硫显示出显著不同的光谱。因此，在有利的条件下，我们可以定量区分生物样品中不同形式的硫。不需要预处理，并且该技术至少是潜在的非破坏性的。我们提出的工作的最终目标是提供健康和患病组织中代谢状态的体内探针。拟议的工作可分为两大类：1。批量测量，其中使用大的X射线束来询问样品以提供平均硫形态。我们建议研究活的哺乳动物细胞培养物的硫生物化学，特别是极化上皮细胞培养物对渗透压休克的反应，细胞分化过程中的变化，细胞凋亡，以及与细胞机械刺激相关的反应。化学特异性成像，我们建议开发硫K边X射线吸收光谱成像作为研究完整活细胞中不同化学形式硫的定位的工具。在这项技术中，X射线微聚焦光学器件用于产生非常小的光束，并使用精心选择的入射X射线能量对样品进行光栅扫描，以区分特定类别的化合物。我们的初步研究将集中在植物细胞上，因为它们的尺寸很大，硫含量很高。随着该技术的分辨率和实验方法的提高，我们将把该方法应用于更具挑战性的系统，最终目标是实现可应用于哺乳动物细胞的1 pm分辨率探针。

DESCRIPTION (provided by applicant): We propose to develop sulfur K-edge X-ray absorption near-edge spectroscopy so that it can be used to monitor and to improve our understanding of sulfur metabolism in living systems, and especially in mammalian cells. There are currently no effective spectroscopic probes of sulfur in biological systems. X-ray absorption spectroscopy will detect all chemical forms of sulfur in a sample, in solid, in aqueous solution or in any other form, and different forms of sulfur display strikingly different spectra. Under favorable conditions we can therefore quantitatively discriminate between different forms of sulfur in biological samples. No pre-treatment is needed and the technique is at least potentially non-destructive. The ultimate goal of the work we propose is to provide an in vivo probe of metabolic status in healthy and diseased tissue. The proposed work can be divided into two major categories: 1. Bulk measurements, where a large X-ray beam is used to interrogate samples to provide the average sulfur speciation. We propose to study the sulfur biochemistry of living mammalian cell cultures, and specifically the responses of polarized epithelial cultures to osmotic shock, changes during cell differentiation, apoptosis, and responses associated with mechanical stimulation of the cells.2. Chemically-specific imaging, where we propose to develop sulfur K-edge X-ray absorption spectroscopy imaging as a tool for investigating the localization of different chemical forms of sulfur in intact living cells. In this technique X-ray micro-focusing optics are used to produce a very small beam, and the sample is raster scanned using carefully chosen incident X-ray energies to discriminate specific categories of compounds.Our initial studies will focus on plant cells, because of their large size and high sulfur content. As the resolution of the technique and the experimental methodology improve, we will apply the method to more challenging systems, with the ultimate goal of a 1 pm resolution probe that can be applied to mammalian cells.