单细胞水平同位素拉曼散射分析

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2016-12-29,#,单细胞同位素拉曼散射分析,中国农业大学,齐孟文,前言,细胞是生物体结构和功能的基本单位，环境生态中的所有物质转化和生命活动现象，只有基于细胞的分析才能得到本质上的阐述，而细胞的生命驱动力是细胞代谢，解析细胞代谢的过程，展现其瞬时动态图像，是一切生物转化相关过程研究在方法学上必须最终解决的问题，其关系到生态学，环境学，生物学，生物医学等众多相关领域，具有重大意义。,单细胞同位素拉曼散射是一种非破坏性的，非细胞培养的，集共聚焦显微、拉曼非弹性散射及其与同位素标记探针技术结合为一体的分析技术。共聚焦显微可以实现分析对象在细胞或亚细胞尺度上的测度，而拉曼非弹性散可以贯穿细胞，包含有几乎所有细胞储藏大分子指纹的信息，加之水介质的拉曼背景信号小，可实现对细胞的整体分析，若同时采用同位素探针技术,，,可以,将,功能过程示踪与生物系统发育信息相联系，进行功能与,分类,的,耦合分析,，以及元素生态循环和细胞代谢等系统生物学研究,。,1.,激光共聚焦显微成像技术,以,激光作为光源，激光器发出的激光通过照明针孔形成点光源，,经过透镜,、分光镜形成平行光后,，再由物透镜,聚焦在样品上,，对,样品内聚焦平面上的每一点进行,扫描，样品激发的光信号，通过分光镜分光，再经像透镜聚焦，到达探测,针孔处，被后续的光电倍增管,检测，,并在显示器上成像，得到所需的荧光图像，而非聚焦,光线被探测,针孔光,栏所阻挡，,因而,不能,不,能被检测。,这种双共轭成像方式称为共聚焦,。,基本原理,利用,放置在光源后的照明针孔,(P,1,),和放置在检测器前的探测针孔,(P,2,),实现点照明和点,探测，激光,经过照明针孔形成点光源，由物镜聚焦在样品焦面的,某个物点,上，只有该点所,发射的,荧光成像在探测针孔上，该点以外的,任何位置发射,光线被探测器阻挡，不能到达,PMT,探测器，从而提高了成像效果。照明针孔和探测,针孔共轭聚焦，所有被,探测,点均在物平面,为,共焦的平面,。,技术指标,分辨率,人,眼：,0.2mm,；光学显微镜：,0.25,m,；电子显微镜：,0.2nm,；共聚焦,显微镜：,0.18,m,。,较之传统显微镜,1,）,抑制图像的模糊，成像更清楚；,2,）具有更高的轴向分辨率，可连续层相成像；,3,）增加侧向分辨率。,2.,曼非弹性散射光谱,自发拉曼光谱,入射光被其诱导的分子极化场所散射而频率发生变化的光谱，在每条入射光频率,V,0,的两侧对称地分布着频率为,V,0,V,i,的谱线，其中,V,相应于分子的某一振动跃迁频率，长波一侧的谱线称为红伴线或斯托克斯线，短波一侧的谱线称为紫伴线或反斯托克斯线。,谱线特征,1,）与入射光的频率无关，而由散射物质分子的振（转）能级所决定，因此有些谱线是与红外吸收谱线相一致。,2,）,在以波数为变量的拉曼光谱图上，斯托克斯线和反斯托克斯线对称地分布在瑞利散射线两侧，上述两种情况下分别相应于得到或失去了一个分子声子的振动能量。,3,）一般情况下，斯托克斯线比反斯托克斯线的强度大，因为依据,Boltzmann,分布，处于振动基态上的粒子数远大于处于振动激发态上的粒子数。,分析特征,1,）,利用待测样品分子的,特征拉曼谱线作为检测或灰度,对比度,成像，无需引入外源标记物（同位素由底物引入，认为是内原的），因此是非侵入式的测量；,2,）水的拉曼背景信号较小，因此可在水环境下测量；,3,）一般采用,近红外,光,激发,，,对细胞组织的光损伤较小,，,能够穿透较厚的生物样,品，有利于,长时间,采集样本和进行样品内部测量；,4,）不利因素是自发斯托克斯的信号较弱，只有,10,6,-10,8,个光子，影响探测灵敏度的提高。,测量装置,增强拉曼光谱,自发拉曼光谱的主要不足是信号太弱，若一味提高激发光的强度，可能会损伤细胞，同时采样时间太长也不利于动态过程的测定，目前提高信噪比的两种基本办法是：,表面增强拉曼散射,和相干反斯托克斯光谱两种分析策略。,1,）表面增强拉拉曼光谱,将样品与胶质金属颗粒如银、金或铜混合，或吸附在这些金属片的粗糙表面上吗，发现光谱的强度可提高,10,3,-10,6,倍。一般认为具有一定表面粗糙度的类自由电子金属基底的存在，使得入射光在表面局域的电磁场得到加强，而拉曼散射强度与分子诱导极化化率平方成正比，因此极大地增强了表面吸附分子的拉曼效应。,2,）,共振反斯托克斯拉曼光谱,该,过程是一种三阶非线性光学过程,，,通常采用两束中心频率不同的激光脉冲,，,分别作为抽运光,（,L,）,和斯托克斯光,（,S,）,来激发样品分子,，,当两束激光的频,率,差与分子,某一振动模式的,固有振动频率一致时,（,R,=,L,-,S,），,分子的固有振动模式得到共振增强,，,并在,第三,束探测光,（,P,）,的作用下产生反斯托克斯信号,（,AS,），,其能级图如图所示,，分为两步，,第一步,，,抽运光和斯托克斯光脉冲同时到达样品,使,分子共振激发,，,在湮没一个抽运光光子的同时产生一个斯托克斯光子和一个相干声子,；,第二,步,产生的相干声子随后与探测光光子作用,反射,反斯托克斯光子,。,共振反斯托克斯拉曼光谱显著地提高了信号强度，同时由于信号相对蓝移，可以很好的与激发光分开，而过程存在的固有,非共振背景,噪声，可以利于两者的偏振状态、退相时间，以及空间域的差异进行相应抑制。,3.,同位素探针,-,拉曼光谱,同位素探针是指通过底物完成对生物标志物的标记，带有重同位素的生物标志物，其拉曼特征谱线因同位效应将发生红移动，采用光谱对比分析很容易鉴别出来，进而确定标志物，并与系统生物功能成分相联系，甚至由标记结合率确定新合成生物的比例及其动态过程。,理论解释,拉曼谱是入射光与分子相互作用，诱导其共价键的电子云形变产生极化场，进而相干叠加而形成散射光，散射光频率相对于入射光丢失和增加一个相应分子键的振动频率。按照经典的分子键的谐振子模型，,分子键的振动频率可以表示为,此处，,c,为光速,(ms,-1,),k,为双原子键的作用常数（,Nm,-1,）,是体系的约化质量，,=m,1,m,2,/(m,1,+m,2,),可见振动频率反比于约化质量的平方根，当原子被其重同位素原子取代后，约化质量增加，而振动频率减小，因此特征谱线发生红移，以,C-H,键的氘代为例，,C-D,的频率相对变化为,0.73,，漂移带的相对强度则与重同位素的结合率成正比，依此可以计算底物的结合率。,Shifts of bands in SCRS caused by incorporation of different stable isotopes, Fully 13C-labeled E. coli (red) and unlabeled E. coli (blue). Available online at ,SERS spectra of single E. coli DH5a cells with different levels of,15,N incorporation when growing in various concentrations of,15,N-NH,4,Cl. The detection limit is 10%,15,N incorporation (P 0.05). (a) A sharp and clear Raman band at 728 cm,-1,(adenine-like compound) shifted due to,15,N incorporation into cellular biomass. (b) Relationship between the,15,N ratio in single cells and Raman shift.,Tef.single cell stable isotope probing in microbiology using Raman microspectroscopy Yun Wang1, Wei E Huang2, Li Cui3 and Michael Wagner. Available online at ,4.,单细胞同位素拉曼谱的应用,单细胞同位素拉曼分析,一种,具有高通量,、非破坏，细胞全化学谱系的（包括,蛋白质，核酸，碳水化合物，脂类 和色素,等细胞储藏分子），非细胞培养的，单细胞水平上的分析方法，可用于细胞代谢、细胞活性分类、及储藏物质周转等生态学和生物学研究。,一般分析线路,Overview of the single cell bio-analysis technology. Single cell bio-analysis can be generally classified as indirect or direct measurements. A typical indirect measurement is fluorescence based which is sensitive but requires external labelling. Single cell Raman spectroscopy (SCRS) is a noninvasive and label-free technology. SCRS detects biomolecule vibrations from a single cell which serves as a cellular intrinsic fingerprint that reflects the cell phenotype and physiological state. Raman-FISH is a combination of fluorescence and Raman technology . Raman imaging and mapping produce Raman pseudo-colour images according to the intensities of Raman spectral bands. The Raman imaging distinguishes the actively CO,2,fixing cells (red) and the inert cells (green) . Raman activated cell sorting (RACS) is able to separate cells according to their Raman spectra. The two buffer channels will converge cells (fluorescent dye replaces the cells to show the cell flow path) from a central channel and pass them through the Raman measurement window one by one (a). Because of the laminar flow and the special geometric design of the chip, cells will usually remain in the white flow (c) that channels them to the waste chamber (a) unless an optical force, for example, a 1064 nm infrared fibre laser (b) selectively pushes the cells of interest to another parallel flow, shown as the red-dye stained flow (c), that brings the cells the collection chamber. Microfluidic device based cell sorters are readily compatible with a Raman micro-spectroscopy system to achieve RACS.,1,）微生态生理分析,单细胞同位素拉曼分析和荧光原位杂交技术相结合，可用于生态群落中微生物的鉴别与生理功能角色的分析。,参加：,Hydrocarbon and Lipid Microbiology, 2016 Springer-Single Cell Microbial Ecophysiology with Raman-FISH.,Fig. 1 Schematic diagram of a Raman microspectrometer. Microbial cells are visualised with visible and light on a Raman inert slide such as calcium fluoride (CaF2). A monochromatic laser source is directed down the microscope objective and focussed onto a target such as a single cell. Raman-scattered light is collected back through the same objective. Unwanted light frequencies are filtered with a notch filter,before being diffracted on a grating and dispersed onto a cooled CCD camera, generating the Raman spectrum,Fig. 2 Outline of the major steps involved in a Raman-FISH experiment,2,）细胞代谢分析,拉曼光谱含有细胞中所有储藏分子的特征谱，而同位素效应会使相应谱线发生红移（重同位素结合率,10%,时,），因此结合同位素标记，可方便识别活性细胞，对单细胞代谢过程进行原位分析。,参见：,Hydrocarbon and Lipid Microbiology, 2016 Springer- Single-Cell Metabolomics.,Fig. 1 Single-cell Raman spectra from single bacterial cells with and without,13,C,15,N and,2,H incorporation(532 nm laser, acquisition time: 0.5 s). For,13,C-incorporation, the strong and distinguishable bands are cytosine, uracil ring stretching at 784 cm,-1, breathing aromatic ring of phenylalanine at 1,003 cm,-1,and protein amideI band around 1,664 cm1. For,15,N-incorporation, the main bands shifted are nucleic acids at 728 and 784 cm,-1,. For 2H-incorporation, the Raman band 2,174 cm,-1,is CD shifted from CH at 2,937 cm,-1,whencells grow in a medium with 50% D2O. The relative intensity of 2,174 cm,-1,indicates general metabolicactivity of a single cell .,3,）细胞生物学研究,确定生物活性细胞种群，例如藻类光合作用细胞。因为其中,类胡萝卜素，是光合作用细胞,的,光合俘获天线，,又,是细胞中类别最多的化合物之一，可以作为光合细胞的标志化合物，,利用,结合,13,CO,2,后,拉曼谱线红移的,特性,，可以把光合作用细胞与,CO,2,固定活性相,关联，,以确定和分离光合作用细胞,。,参见：,Rapid resonance Raman microspectroscopy to,probe carbon dioxide fixation by single cells in microbial communities The ISME Journal (2012) 6, 875885,Synechocystis sp. PCC 6803 grown in BG11 medium supplemented with 10.8%,13,C-NaHCO,3,mixed with S. elongatus PCC 7942 grown in BG11mediumsupplemented with,12,C-NaHCO,3,. Two Raman images were generated based on the,1,and,2,bands. The,13,C-incorporated single cells are displayed in red and the,12,C cells green. Some,13,C-incorporated cells with lower signal strength are displayed in yellow. The SCRR spectra are coloured to correspond to the,13,C-labelled cells and,12,C cellsindicated by red and green circles, respectively.,4,）细胞生物学分类,根据单细胞同位素拉曼谱的数据，利用主量分析有效减少特征谱变量空间的维数后，由规范变量分析,PC-CVA,可对生物活性细胞进行分类，分类后的细胞又可以挑选出来，利用生物技术方法进一步进行下游分析。,参见：,Raman Activated Cell Ejection for Isolation of,Single Cells,，,Anal. Chem. 2013, 85, 1069710701,Figure 1. Bacterial species differentiation by PCCVA based on their SCRS. Fifteen SCRS data (black) were used for training and five SCRS (red) for testing to validate the discrimination,.,5,）分子代谢研究,通过标记氨基酸对分子代谢进行示踪，并利用特征拉曼谱线的对比度成像，对细胞活性分子进行伪色彩定位成像。,参见：,Noninvasive Imaging of Protein Metabolic Labeling,in Single Human Cells Using Stable Isotopes and Raman,Microscopy,Figure .,Raman imaging of a HeLa cell incubated for 28 h with Phe-,d,5,.,(A) Hierarchical cluster analysis image with five clusters.(B-F) Univariate images of (B) nucleotides (770-790 cm-,1,), (C) phospholipids (700-730 cm,-1,), (D) Phe-d,5,(950-965 cm-1), (E) Pheh,5,(995-1005 cm,-1,), and (F) Phe-d,5,/Phe-h,5,ratios (950-965 cm,-1,region divided by the 995-1005 cm,-1,region). Image acquisition parameters: excitation power 70 mW, exposure time 1 s/pixel, step size 0.47,m/pixel, image size 15 15,m,2,.,6,）,D,2,O,标记及拉曼分析,利用来自,D,2,O,的,D,标记细胞成分，如脂类和蛋白质等，由于其与环境的同位素交换并不影响分子特征带的红移信号，所以同样可以方便地用于活性细胞的识别和分类研究。,参加：,Tracking heavy water (D2O) incorporation for,identifying and sorting active microbial cells,www.pnas.org/cgi/doi/10.1073/pnas.1420406112,Fig. 1. E. coli incorporation of D from heavy water during growth as detected by Raman microspectroscopy and NanoSIMS. (A) Raman spectra of single E. coli cells grown to stationary phase in media with heavy water (0%, 2.5%, 5%, 10%, 15%, 20%, 30%, 50%, and 100% D,2,O of growth water). Ten to 21 single-cell spectra were used to produce mean spectra. It should be noted that identical peak shifts were observed in E. coli cells that were grown in the presence of deuterated glucose instead of heavy water (SI Appendix, Fig. S11). AU, arbitrary unit. (B) Difference between mean spectra of E. coli cells from D,2,O-containing media and cells grown without D,2,O. Colors are the same as in A. (C) Quantification of D incorporation in individual E. coli cells from the same experiment as detected by NanoSIMS and shown as the isotope fraction D/(H + D) given as at%. All isotope fraction images are on the same scale (0100 at%).,31,P signal intensity distribution is displayed to indicate the location of cellular biomass. (D) Comparison of D content in single cells measured by NanoSIMS with respect to the D,2,O percentage of growth water. Box plots show the quartiles for each population of cells. The detection limit, defined as the mean + 3 SD of unlabeled cells, is shown in gray (0.17 at%). A linear regression between D,2,O concentration and cellular D is shown in red (R,2,= 0.84).,