【作者】 张继超；

【导师】 余笑寒；

【作者基本信息】 中国科学院研究生院（上海应用物理研究所） ， 核技术及应用， 2013， 博士

【摘要】 纳米材料生物学效应研究是将纳米技术与生物、化学、物理、毒理学与医学等领域的实验技术结合起来，研究纳米尺度物质与生命过程相互作用及其结果的一个新兴科学领域。为了完全评估纳米材料的潜在危险，新的研究方法和手段是解决纳米材料生物学效应问题的关键。同步辐射X射线荧光成像方法，由于具有灵敏度高、无损分析、制样简单、能分析含水样品、能同时探测多种元素、不需要真空条件等优点，非常适合纳米材料生物学效应领域的研究。硬X射线微探针光束线站是上海光源首批建设的七条线站中唯一的一条硬X射线微束线站，提供微束X射线荧光分析、微束X射线吸收精细结构、微束X射线衍射等多种实验方法。虽然能够提供高通量的硬X射线微束，当用户进行细胞样品或其它微米级样品实验研究时，BL15U1线站现有的实验条件已略显不足。例如微区扫描时，样品定位方法不便捷，可见显微镜粗略划定的扫描范围与实际扫描范围有20μm的误差，需要预扫描才能获得精确的微区位置，费时费力。还有，1.5μm×1.5μm的微束已不能满足细胞样品的实验要求，微束对细胞样品的成像结果，只能看到大概的轮廓结构，不能区分细胞内微机构和亚细胞器。因此，发展更快速的样品定位方法、提高X射线束的空间分辨率，既是用户实验过程中对线站的要求，也是线站自身研究方法发展的必然。本论文设计并应用了硬X射线微米探针高精度样品定位系统，实现了样品的微米级快速离线定位，使用户能够快速、准确地定位微区研究对象。将定位系统应用于研究纳米TiO2在小鼠肺部毒性的工作中，研究了肺组织对纳米TiO2的排除机制，及纳米TiO2的入侵对肺组织内K、Ca、Fe、Cu、Zn等原生元素的影响。参与设计和搭建了BL15U1线站基于波带片的亚微米聚焦系统，主要工作为亚微米光斑的调试和实验应用工作，得到亚微米聚焦光，并对亚微米光斑的焦深、聚焦效率、通量、通量密度、元素探测限、细胞实验结果等进行了研究。将亚微米光斑应用到量子点免疫荧光染色后细胞的X射线荧光成像工作中，成功对Hela细胞内的微管蛋白进行了CdSe/ZnS量子点的免疫荧光染色，并取得了染色后细胞亚微米X射线荧光成像工作的初步结果。第一章，首先从纳米材料生物学效应、X射线荧光成像方法、上海光源硬X射线微探针线站三个方面进行了综述，介绍了本论文工作的背景，然后总结提出了本论文的研究内容。第一小节介绍了纳米材料生物学效应的概念、当前面临的挑战和各种研究方法，及X射线荧光成像方法在纳米材料生物学效应研究中的优势；第二小节介绍了X射线荧光物理学基础、同步辐射X射线荧光分析、X射线荧光定量分析方法、及定量分析软件PyMCA和GeoPIXE；第三小节介绍了上海光源硬X射线微探针线站、线站开展的实验方法、及当前线站的优势和不足；最后提出了本论文的研究内容。第二章，设计应用了硬X射线微米探针高精度样品定位系统。该系统由离线样品显微镜系统、在线样品实验系统和高精度定位样品架三部分组成，首次在国内同步辐射装置上实现样品在微米范围内的定位。实验验证了该系统在X方向的平均误差为1.3μm，Z方向的平均误差为2.5μm，系统快速、准确可靠。第三章，应用样品定位系统和微米级的光斑，研究纳米TiO2作用于小鼠肺组织的毒性。研究了肺组织对纳米TiO2的排除机制，及纳米TiO2的入侵对肺组织内K、Ca、Fe、Cu、Zn等原生元素的影响。第四章，介绍了BL15U1基于波带片的硬X射线亚微米聚焦系统。介绍了X射线波带片聚焦的原理、亚微米聚焦系统的设计和构成、聚焦系统的安装与准直、以及聚焦系统的调试和实验。得到亚微米聚焦光，并对亚微米光斑的焦深、聚焦效率、通量、通量密度、元素探测限、细胞实验结果等进行了研究。第五章，将亚微米光斑应用到量子点免疫荧光染色后细胞的X射线荧光成像工作中。介绍了量子点的性能及其在细胞成像中的应用、细胞内量子点的免疫荧光染色、以及量子点染色后细胞的亚微米光斑X射线荧光成像工作。第六章，总结本论文工作，并进行了展望。更多还原

【Abstract】 Biological effects of nanomaterials is a new scientific field studying theinteraction of nanoscale materials and life processes, combining nanotechnology withthe experimental techniques in biology, chemistry, physics, medicine and toxicology.In order to fully assess the potential dangers of nanomaterials, new research methodsand techniques are the key of solving the problem of biological effects ofnanomaterials. Because of its high sensitivity, non-destructive analysis, simple samplepreparation, analyzing aqueous samples, detecting multiple elements simultaneously,needless vacuum conditions, etc, synchrotron radiation X-ray fluorescence imagingmethod is an ideal tool for the research of biological effects of nanomaterials.BL15U1hard X-ray microprobe beamline is the only hard X-ray microfocusbeamline among the first seven beamline in Shanghai Synchrotron Radiation Facitity(SSRF), providing multiple experimental methods, including micro X-rayfluorescence analysis, micro X-ray absorption fine structure, micro X-ray diffraction.Though it provides high flux hard X-ray beam, the existing experimental conditionsof BL15U1beamline have been slightly less, when users do research into cells orother microscale samples. For example, when doing micro-scanning, the samplepositioning method is not convenient. There is an error of20μm between the roughscan scope delineated by the visible light microscope and the actual scan scope. Apre-scan is needed to get the exact micro location, which spends lots of research time.In addition, the microbeam of1.5μm×1.5μm can not satisfy the requirements of cellsample experiments. From the cell sample imaging results using microbeam, we onlyget the outline of the structure in cells, instead of clear microstructure and subcellularorganelles. Therefore, developing a rapid sample positioning method, and improvingthe spatial resolution of X-ray beam, are not only the requirements of doingexperiment for users, but also the necessary trend of research methods developing forbeamline.In this paper, a new kind of hard X-ray microprobe precision sample positioningsystem is designed and applied to achieve fast offline sample positioning, helpingusers to locate micro studying objects quickly and accurately. The positioning system was used to study pulmonary toxicity of nanoscale titanium dioxide in mice. Theexclusion mechanism of nano TiO2from lung tissue, and the effects of the nativeelements (K, Ca, Fe, Cu, Zn) in lung tissue because of the exposing of nano TiO2,were studied. I took part in the design and construction of the BL15U1beamlinesubmicron focusing system based on zone plates. The main work of mine isdebugging and experiment of submicron X-ray spot. A submicron spot was gotten.Many features of submicron spot were studied, such as depth of focus, focusingefficiency, flux, flux density, detection limit, cell experiments and so on. Thesubmicron spot was applied to image the cells after quantum dotsimmunofluorescence staining, using X-ray fluorescence. The tubulin of Hela cells wasimmunofluorescence stained successfully by CdSe/ZnS quantum dots. Thepreliminary results of research of submicron X-ray fluorescence image to stained cellswere gotten.In the first chapter, we reviewed the biological effects of namomaterials, X-rayfluorescence imaging method, and the hard X-ray microprobe beamline station inSSRF, described the background of the work. In the first section, we introduced theconcept of biological effects of nanomaterials, current challenges and researchmethods, the advantages of X-ray fluorescence imaging method to study thebiological effects of nanomaterials. In the second section, we introduced the physicsprinciple of X-ray fluorescence, synchrotron radiation X-ray fluorescence analysis,quantitative X-ray fluorescence analysis, and quantitative analysis software (PyMCA,GeoPIXE). In the third section, we introduced the hard X-ray microprobe beamlinestation, the experimental methods provided by BL15U1, current advantages anddisadvantages. At last, we presented the research content.In the second chapter, a new kind of hard X-ray microprobe precision samplepositioning system is designed and applied. The positioning system is composed ofthree parts: off-line sample microscope system, on-line sample experiment system,high-precision positioning sample holder. It’s the first time in the domesticsynchrotron radiation devices to achieve sample offline positioning in micron scalequickly. The experiment results showed that the average errors of X-axis and Z-axiswere1.3μm and2.5μm respectively, using the positioning method. The sample offlinepositioning system is fast, accurate, reliable.In the third chapter, the positioning system and micro X-ray spot were used to study pulmonary toxicity of nanoscale titanium dioxide in mice. The exclusionmechanism of nano TiO2from lung tissue, and the effects of the native elements (K,Ca, Fe, Cu, Zn) in lung tissue because of the exposing of nano TiO2, were studied.In the fourth chapter, we introduced the BL15U1hard X-ray submicron focussystem based on zone plates. X-ray focusing principle of zone plates, design andcomposition of submicron focusing system, collimating and installation of submicronfocusing system, debugging and experiment of focusing system were introduced. Asubmicron spot was gotten. Many features of submicron spot were studied, such asdepth of focus, focusing efficiency, flux, flux density, detection limit, cell experimentsand so on.In the fourth chapter, the submicron X-ray spot was applied to image thequantum dots immunofluorescence staining cells using X-ray fluorescence. Theproperties of quantum dots and their application in cell imaging, intracellularimmunofluorescence staining of quantum dots, the submicron spot X-ray fluorescenceimaging of the quantum dot staining cells were introduced.In the sixth chapter, we summarized the work and prospected.更多还原