Micro X-ray Fluorescence Imaging Method And Its Applications In Biological Effects Of Nano-materials

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.