同步辐射元素成像技术

同步辐射元素成像技术在环境科学 研究中的应用,元素成像：样品内元素的空间分布同步辐射：电子运动的方向发生变化时在切线方向产生电磁辐射 1947年, 在电子同步加速器上首次观察到，命名为 synchrotron radiation,同步辐射的特点,高亮度：同步辐射的亮度比最强的X光管特征线亮度强万倍以上。用X光机拍摄一幅晶体缺陷照片，通常需要7-15天的感光时间，而利用同步辐射光源只需要几秒。 宽波谱：覆盖了红外、可见、紫外和X光波段。利用单色器可以随意选择所需要的波长，进行单色光的实验。 高准直：同步辐射的发射度极小，准直性可以与激光相媲美。,BSRF,BSRF：直线加速器200米储存环周长240米，电子能量-2 GeV,SSRF 储存环周长432米，光子能量为0.140 keV ，电子能量3.5GeV,Spring-8 大储存环直径457米，周长约1436米，电子能量3.5GeV 。直线加速器部分长140米,同步辐射的发展,第一代同步辐射光源. 在核物理/粒子物理研究的空挡，利用同步加速器所发射的同步光进行科学研究,称为寄生方式。北京高能所同步辐射装置(BSRF) 。 第二代同步辐射光源. 第一代同步辐射光源已不能满足研究需求，建立了用专门的装置产生同步光,例如合肥同步辐射装置。 第三代同步辐射光源. 科学家发现在储存环中加入插入件可以使同步辐射的亮度再提高千倍以上，得到的同步辐射主要来自插入件。上海光源（SSRF）。,元素成像原理,An electron in the inner shell (e.g. K shell) is ejected from the atom by an external primary excitation x-ray, creating a vacancy. An electron from outer shell jumps in to fill the vacancy. In the process, it emits a characteristic x-ray unique to this element and in turn, produces a vacancy in the outer shell.,元素成像原理,荧光产额 较低的原子序数Z的元素，荧光产额较低 原则上， 可测元素Z4 实际工作中 Z11 （空气吸收）光线能量,同步辐射元素成像设备,光学部分,成像设备,Detection limit: 10-18 g， 103 atoms Nat Phys,2006, 2:101104lateral resolution: 50 nm Anal Chem, 2003, 75:38063816,样品测试部分,信号处理部分,XRF spectra,Typical energy dispersive XRF spectrum for a multi-element standardE: qualitative analysis peak eara: quautification,生物样品中微量元素的空间分布,吸收、转运、蓄积、代谢生理生化功能作用机制实例,1 mm,Schematic drawing of anatomical structures of an adult hermaphrodite C. elegans,Elemental distribution in C. elegans which was fed by E. coli OP50+ PBS (A), E. coli OP50 + nano Cu (B), and E. coli OP50 +Cu2+ (C).,JAAS, 2008, 23, 11211124,模式动物 2D,3.55.0 m at BL-4A in, KEK,Cyclotella meneghiniana account for 20% of global carbon fixation. facilitate the transport of photosynthetically fixed CO2 from the surface ocean to deep water , 10m 制样 细胞悬液滴加在电镜栅网上，自然风干定量 XRF standards NIST 1832 ,1833,Quantitative 3D elemental microtomography of Cyclotella meneghiniana at 400-nm resolution PNAS, 2010，107，15676-15680,XRF microprobe at beamline 2-ID-E of the Advanced Photon Source Following monochromation the X-ray beam was focused to a spot of approximately 270nm diameter using a Fresnel zone-plate objective. Fluorescence spectra were recorded at 150nm intervals as the specimen was raster scanned through the focus of the X-ray beam to obtain 2D projection images. The flux transmitted by the sample was monitored using both an ion chamber and a configured detector. A rotation series was obtained by acquiring such images with the specimen oriented at 24 different angles with respect to a vertical rotation axis.,Quantitative 3D elemental microtomography of Cyclotella meneghiniana at 400-nm resolution PNAS, 2010，107，15676-15680,240,000 single-point 24 projections 36 h (0.1 S/pixel),Quantitative 3D elemental microtomography of Cyclotella meneghiniana at 400-nm resolution PNAS, 2010, 107, 15676-15680,Cyclotella meneghiniana中元素 的3D 分布P, K, Ca 分布的细胞器官类似，少量Mn存在于同一区域Si 主要分布在细胞壁，Fe、Mn在细胞壁呈环状分布Cl, Cu, Zn，S分布于沿硅藻内轴向分布的柱状区域内元素总含量相差3个数量级 8g for Si ， 2ng for Mn.,各元素分布图叠加PNAS, 2010, 107, 15676-15680,沿硅藻轴向分布的柱状细胞结构为细胞质，主要含Cl, Cu, Zn, 和 S，这些元素在细胞膜内侧也有一薄层分布三个球状细胞器主要含K, P和Ca，也有少量 Mn, Cu和Ni; 其中两个与柱状结构连接，一个位于硅藻瓣端,C. meneghinianas三维结构的元素分布细节PNAS, 2010, 107, 15676-15680,(AC) Si (灰)， Fe (橙) ， Mn (红)在硅藻纵断面上的 分布. A 三元素分布细节，在含Si的硅藻细胞壁上还含有Fe和Mn. 二者在细胞壁特定部位呈环状分布。 Mn 结合于硅藻瓣面和环带交界的富硅环状结构上。 B Si和Mn分布，在细胞下部一个硅化较轻的环状区正好与Fe环状结构之一重合。该区域正是细胞伸展区，靠近硅藻细胞膜上壳和较小的新形成的子壳之间的连接区。C 表示 Mn和 Fe的分布, 在瓣与环带链接区有一个Fe、Mn共存区 。(DF) P (蓝), S (黄), Cl (黄绿), 和Zn (紫)在硅藻纵向切片上的分布以及他们间的联系。两个主要的含磷结构，S和Cl共存于中央独立的柱状结构以及紧邻细胞膜下细胞质内的局部区域，含磷细胞器与柱状结构链接，大的液泡空腔，Zn在柱状结构内富集，Zn与S、Cl相关。(G) 硅藻的横断截面表明柱状结构和空泡，两个含磷结构与柱状结构相连。,实测值和重建数据（36.0）,实测值体现了硅藻的真实结构，Si, Fe, 和 Mn界定了硅藻的细胞膜，Ca, K, 和 P 在三个球状细胞器内， Zn, Cu, S 分布于细胞质.重建数据与实测值无明显差别，重建效果好。大多数情况下，实测值和重建值之间的差别在2-3像素之内，据此估计成像分辨率在400nm左右。 PNAS, 2010, 107, 15676-15680,XAS 成像,Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern,蜈蚣草Pteris vittata,Spot size 15 or 5 m diameter focused by tapered metal monocapillaries鲜活组织，室温，潮湿定量：0.2L 5 mM Arsenate滴于滤纸,Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern,Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern,As(glutathione)3 11869.8 eVArsenite 11871.4 eVArsenate 11874.8 eV,EST, 2006, 40, 5010-5014,Localizing the Biochemical Transformations Of Arsenate in a Hyperaccumulating Fern,XAS 成像,EST, 2006, 40, 5010-5014,XAS 成像,Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern,方法评价,非破坏性 制样简单 避免污染 后续研究灵敏度高 多元素分析 高通量 元素间的相互作用空间分辨率,Biological,Samples,SR,45,Si(Li) detector,Amplifier,MCA,Computer,Slit,Ionization,chamber,Sample moving,direction,IEF,separation,SIGOUT,Reconstruction,Protein,extraction,Detection of metalloproteins with SRXRF after electrophoresis,Gao Y, Anal. Chem. Acta, 2003, 485(1): 131-137Gao Y, Analyst, 2002, 127, 1400-1404 Gao Y, JAAS, 2007, 22, 856866,Detection of mercury-, arsenic-, and selenium-containing proteins in fish liver from a mercury polluted area of Guizhou Province, China with SRXRF after IEF,Se /As /Hg often co-exist in the same band, suggesting that The bands may be corresponding to the antagonistic effect of Se against the toxicity of Hg and As, Hg and As may be involved in the same metabolic processes.,Li LN, J Toxicol Env Health, A, 2008, 71: 12661269,Imaging Metals in Proteins after native Electrophoresis,XRF map of a one-dimensional native-PAGE blot of multiple metalloproteins. (Upper right) Photograph of blot. (Upper left) Zinc map shows zinc present in carbonic anhydrase (CA). (Lower left) Iron map shows iron present in hemoglobin (Hb). (Lower right) Copper map shows copper present in tyrosinase (Tyr), as well as a contaminant in carbonic anhydrase. Maps displayed are quantified, with threshold values in g/cm2.,ACS CHEM BIOL,2010,5,577-587,Detection of Se-containing proteins in selenium-enriched yeast with SRXRF after 2-dimensional gel electrophoresis,About 30 Se-containing proteins can be distinguished by Comparison between the two Figs, whitch were marked with ellipses in Fig.,Metallome of Nile tilapia tissue identified by SRXRF after 2D-PAGE separation,Gel obtained for the liver samples with the protein spots which were detected calcium, iron and zinc by SRXRF highlighted with a circle.,Three-dimensional protein spots of liver sample contained calcium, iron and zinc, which highlighted with a circle.,Talanta,2010,82,1052-1056,准在线联用技术非破坏性、基底影响小同时给出蛋白质表达水平、等电点、分子量、所含金属种类、含量等信息（价态、肽质指纹谱、序列）多元素分析，可用于研究不同元素之间的协同、拮抗等生物效应研究,方法评价,X-ray absorption spectroscopy,I = I0e -d，线性吸收系数f(Z, , )XAS:透射光强度随入射光波长的变化曲线,X射线吸收谱,E010,E0+10-50,E0+50-1000,XANES:X射线吸收近边结构： 元素种类、电子构型（价态）、对称性NEXAFS:近边X射线吸收精细结构： 键角、键长EXAFS:扩展X射线吸收精细结构：配位原子种类、数目、距离,EXAFS优点,不依赖晶体结构，因此可用于大量的非晶态材料的研究不受其他元素的干扰，对不同的元素的原子，可由吸收边位置不同，而得以分别研究可测配位原子的种类、个数、间距等浓度很低的样品，百万分之几的元素也能分析样品制备比较简单，数据收集时间短,XAS measurement after SRXRF imaging,Protein map (a) and Cr map (b) for Cr compounds binding to albumin-depleted bovine serum (BS) proteins. The main protein bands at 65 and 130 kDa correspond to monomeric and dimeric forms of BSA, respectively, total protein content in each lane is 10 g. Lane 1: BS. Lane 4: GSH (5.0 mM), Lane 2,3: Cr(III), Cr(IV) (1.0 mM)+BS. Lane 5,6:Cr(IV), Cr(III)-(1.0 mM) + GSH (5.0 mM). Lane 7: Cr(III)-propionate, 1.0 mM). All Cr compounds were incubated with serum proteins (1.0 mg protein per mL in 20 mM HEPES buffer, pH 7.4) for 2 h at 310 K prior to the separation by SDS-PAGE.,ACS CHEM BIOL,2010,5,577-587,硒对植物体内汞的生物学行为及效应的影响,植物是食物链的基础，降低植物重金属的含量或改变其形态是防止重金属危害人体健康的有