單細(xì)胞水平同位素拉曼散射分析

1、前言 細(xì)胞是生物體結(jié)構(gòu)和功能的基本單位，環(huán)境生態(tài)中的所有物質(zhì)轉(zhuǎn)化和生命活動(dòng)現(xiàn)象，只有基于細(xì)胞的分析才能得到本質(zhì)上的闡述，而細(xì)胞的生命驅(qū)動(dòng)力是細(xì)胞代謝，解析細(xì)胞代謝的過(guò)程，展現(xiàn)其瞬時(shí)動(dòng)態(tài)圖像，是一切生物轉(zhuǎn)化相關(guān)過(guò)程研究在方法學(xué)上必須最終解決的問(wèn)題，其關(guān)系到生態(tài)學(xué)，環(huán)境學(xué)，生物學(xué)，生物醫(yī)學(xué)等眾多相關(guān)領(lǐng)域，具有重大意義。第1頁(yè)/共35頁(yè) 單細(xì)胞同位素拉曼散射是一種非破壞性的，非細(xì)胞培養(yǎng)的，集共聚焦顯微、拉曼非彈性散射及其與同位素標(biāo)記探針技術(shù)結(jié)合為一體的分析技術(shù)。共聚焦顯微可以實(shí)現(xiàn)分析對(duì)象在細(xì)胞或亞細(xì)胞尺度上的測(cè)度，而拉曼非彈性散可以貫穿細(xì)胞，包含有幾乎所有細(xì)胞儲(chǔ)藏大分子指紋的信息，加之水介質(zhì)的拉曼背

2、景信號(hào)小，可實(shí)現(xiàn)對(duì)細(xì)胞的整體分析，若同時(shí)采用同位素探針技術(shù)，可以將功能過(guò)程示蹤與生物系統(tǒng)發(fā)育信息相聯(lián)系，進(jìn)行功能與分類的耦合分析，以及元素生態(tài)循環(huán)和細(xì)胞代謝等系統(tǒng)生物學(xué)研究。第2頁(yè)/共35頁(yè) 1.激光共聚焦顯微成像技術(shù) 以激光作為光源，激光器發(fā)出的激光通過(guò)照明針孔形成點(diǎn)光源， 經(jīng)過(guò)透鏡、分光鏡形成平行光后，再由物透鏡聚焦在樣品上，對(duì)樣品內(nèi)聚焦平面上的每一點(diǎn)進(jìn)行掃描，樣品激發(fā)的光信號(hào)，通過(guò)分光鏡分光，再經(jīng)像透鏡聚焦，到達(dá)探測(cè)針孔處，被后續(xù)的光電倍增管檢測(cè)，并在顯示器上成像，得到所需的熒光圖像，而非聚焦光線被探測(cè)針孔光欄所阻擋，因而不能不能被檢測(cè)。這種雙共軛成像方式稱為共聚焦。第3頁(yè)/共35頁(yè)基本

3、原理 利用放置在光源后的照明針孔(P1)和放置在檢測(cè)器前的探測(cè)針孔(P2)實(shí)現(xiàn)點(diǎn)照明和點(diǎn)探測(cè)，激光經(jīng)過(guò)照明針孔形成點(diǎn)光源，由物鏡聚焦在樣品焦面的某個(gè)物點(diǎn)上，只有該點(diǎn)所發(fā)射的熒光成像在探測(cè)針孔上，該點(diǎn)以外的任何位置發(fā)射光線被探測(cè)器阻擋，不能到達(dá)PMT探測(cè)器，從而提高了成像效果。照明針孔和探測(cè)針孔共軛聚焦，所有被探測(cè)點(diǎn)均在物平面為共焦的平面。第4頁(yè)/共35頁(yè)技術(shù)指標(biāo)p 分辨率人眼：0.2mm；光學(xué)顯微鏡：0.25m；電子顯微鏡：0.2nm；共聚焦顯微鏡：0.18 m。p 較之傳統(tǒng)顯微鏡1）抑制圖像的模糊，成像更清楚；2）具有更高的軸向分辨率，可連續(xù)層相成像；3）增加側(cè)向分辨率。d2alconven

4、tionconfocaldd第5頁(yè)/共35頁(yè)2.曼非彈性散射光譜p 自發(fā)拉曼光譜 入射光被其誘導(dǎo)的分子極化場(chǎng)所散射而頻率發(fā)生變化的光譜，在每條入射光頻率V0的兩側(cè)對(duì)稱地分布著頻率為V0Vi的譜線，其中V相應(yīng)于分子的某一振動(dòng)躍遷頻率，長(zhǎng)波一側(cè)的譜線稱為紅伴線或斯托克斯線，短波一側(cè)的譜線稱為紫伴線或反斯托克斯線。第6頁(yè)/共35頁(yè)p 譜線特征 1）與入射光的頻率無(wú)關(guān)，而由散射物質(zhì)分子的振（轉(zhuǎn)）能級(jí)所決定，因此有些譜線是與紅外吸收譜線相一致。 2）在以波數(shù)為變量的拉曼光譜圖上，斯托克斯線和反斯托克斯線對(duì)稱地分布在瑞利散射線兩側(cè)，上述兩種情況下分別相應(yīng)于得到或失去了一個(gè)分子聲子的振動(dòng)能量。 3）一般情況

5、下，斯托克斯線比反斯托克斯線的強(qiáng)度大，因?yàn)橐罁?jù)Boltzmann分布，處于振動(dòng)基態(tài)上的粒子數(shù)遠(yuǎn)大于處于振動(dòng)激發(fā)態(tài)上的粒子數(shù)。第7頁(yè)/共35頁(yè)p 分析特征1）利用待測(cè)樣品分子的特征拉曼譜線作為檢測(cè)或灰度對(duì)比度成像，無(wú)需引入外源標(biāo)記物（同位素由底物引入，認(rèn)為是內(nèi)原的），因此是非侵入式的測(cè)量；2）水的拉曼背景信號(hào)較小，因此可在水環(huán)境下測(cè)量；3）一般采用近紅外光激發(fā)，對(duì)細(xì)胞組織的光損傷較小，能夠穿透較厚的生物樣品，有利于長(zhǎng)時(shí)間采集樣本和進(jìn)行樣品內(nèi)部測(cè)量；4）不利因素是自發(fā)斯托克斯的信號(hào)較弱，只有106-108個(gè)光子，影響探測(cè)靈敏度的提高。第8頁(yè)/共35頁(yè)p測(cè)量裝置第9頁(yè)/共35頁(yè)p增強(qiáng)拉曼光譜 自發(fā)拉

6、曼光譜的主要不足是信號(hào)太弱，若一味提高激發(fā)光的強(qiáng)度，可能會(huì)損傷細(xì)胞，同時(shí)采樣時(shí)間太長(zhǎng)也不利于動(dòng)態(tài)過(guò)程的測(cè)定，目前提高信噪比的兩種基本辦法是：表面增強(qiáng)拉曼散射和相干反斯托克斯光譜兩種分析策略。第10頁(yè)/共35頁(yè) 1）表面增強(qiáng)拉拉曼光譜 將樣品與膠質(zhì)金屬顆粒如銀、金或銅混合，或吸附在這些金屬片的粗糙表面上嗎，發(fā)現(xiàn)光譜的強(qiáng)度可提高103-106倍。一般認(rèn)為具有一定表面粗糙度的類自由電子金屬基底的存在，使得入射光在表面局域的電磁場(chǎng)得到加強(qiáng)，而拉曼散射強(qiáng)度與分子誘導(dǎo)極化化率平方成正比，因此極大地增強(qiáng)了表面吸附分子的拉曼效應(yīng)。第11頁(yè)/共35頁(yè) 2）共振反斯托克斯拉曼光譜 該過(guò)程是一種三階非線性光學(xué)過(guò)程，

7、通常采用兩束中心頻率不同的激光脈沖，分別作為抽運(yùn)光（L）和斯托克斯光（S）來(lái)激發(fā)樣品分子，當(dāng)兩束激光的頻率差與分子某一振動(dòng)模式的固有振動(dòng)頻率一致時(shí)（R=L-S）， 分子的固有振動(dòng)模式得到共振增強(qiáng)，并在第三束探測(cè)光（P）的作用下產(chǎn)生反斯托克斯信號(hào)（AS）， 其能級(jí)圖如圖所示，分為兩步，第一步，抽運(yùn)光和斯托克斯光脈沖同時(shí)到達(dá)樣品使分子共振激發(fā)，在湮沒(méi)一個(gè)抽運(yùn)光光子的同時(shí)產(chǎn)生一個(gè)斯托克斯光子和一個(gè)相干聲子；第二步, 產(chǎn)生的相干聲子隨后與探測(cè)光光子作用, 反射反斯托克斯光子。第12頁(yè)/共35頁(yè) 共振反斯托克斯拉曼光譜顯著地提高了信號(hào)強(qiáng)度，同時(shí)由于信號(hào)相對(duì)藍(lán)移，可以很好的與激發(fā)光分開(kāi)，而過(guò)程存在的固有非

8、共振背景噪聲，可以利于兩者的偏振狀態(tài)、退相時(shí)間，以及空間域的差異進(jìn)行相應(yīng)抑制。第13頁(yè)/共35頁(yè) 3.同位素探針-拉曼光譜 同位素探針是指通過(guò)底物完成對(duì)生物標(biāo)志物的標(biāo)記，帶有重同位素的生物標(biāo)志物，其拉曼特征譜線因同位效應(yīng)將發(fā)生紅移動(dòng)，采用光譜對(duì)比分析很容易鑒別出來(lái)，進(jìn)而確定標(biāo)志物，并與系統(tǒng)生物功能成分相聯(lián)系，甚至由標(biāo)記結(jié)合率確定新合成生物的比例及其動(dòng)態(tài)過(guò)程。 理論解釋 拉曼譜是入射光與分子相互作用，誘導(dǎo)其共價(jià)鍵的電子云形變產(chǎn)生極化場(chǎng)，進(jìn)而相干疊加而形成散射光，散射光頻率相對(duì)于入射光丟失和增加一個(gè)相應(yīng)分子鍵的振動(dòng)頻率。按照經(jīng)典的分子鍵的諧振子模型， 第14頁(yè)/共35頁(yè) 分子鍵的振動(dòng)頻率可以表示為

9、 此處，c為光速(ms-1),k為雙原子鍵的作用常數(shù)（Nm-1）,是體系的約化質(zhì)量， =m1m2/(m1+m2),可見(jiàn)振動(dòng)頻率反比于約化質(zhì)量的平方根，當(dāng)原子被其重同位素原子取代后，約化質(zhì)量增加，而振動(dòng)頻率減小，因此特征譜線發(fā)生紅移，以C-H鍵的氘代為例，C-D的頻率相對(duì)變化為0.73，漂移帶的相對(duì)強(qiáng)度則與重同位素的結(jié)合率成正比，依此可以計(jì)算底物的結(jié)合率。/kc21第15頁(yè)/共35頁(yè)Shifts of bands in SCRS caused by incorporation of different stable isotopes, Fully 13C-labeled E. coli (red

10、) and unlabeled E. coli (blue). Available online at 第16頁(yè)/共35頁(yè)SERS spectra of single E. coli DH5a cells with different levels of 15N incorporation when growing i n v a r i o u s c o n c e n t r a t i o n s o f 1 5N - N H4C l . T h e detection limit is 10% 15N incorporation (P 0.05). (a) A sharp and c

11、lear Raman band at 728 cm- 1 ( a d e n i n e - l i k e c o m p o u n d ) s h i f t e d d u e t o 1 5N i n c o r p o r a t i o n i n t o c e l l u l a r b i o m a s s . ( b ) Relationship between the 1 5N ratio in single cells and Raman shift. 第17頁(yè)/共35頁(yè)Tef.single cell stable isotope probing in microb

12、iology using Raman microspectroscopy Yun Wang1, Wei E Huang2, Li Cui3 and Michael Wagner. Available online at 第18頁(yè)/共35頁(yè) 4.單細(xì)胞同位素拉曼譜的應(yīng)用 單細(xì)胞同位素拉曼分析一種具有高通量、非破壞，細(xì)胞全化學(xué)譜系的（包括蛋白質(zhì)，核酸，碳水化合物，脂類 和色素等細(xì)胞儲(chǔ)藏分子），非細(xì)胞培養(yǎng)的，單細(xì)胞水平上的分析方法，可用于細(xì)胞代謝、細(xì)胞活性分類、及儲(chǔ)藏物質(zhì)周轉(zhuǎn)等生態(tài)學(xué)和生物學(xué)研究。第19頁(yè)/共35頁(yè)一般分析線路第20頁(yè)/共35頁(yè) Overview of the single cell

13、 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

14、. 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 accordin

15、g to the intensities of Raman spectral bands. The Raman imaging distinguishes the actively CO2 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

16、 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

17、(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 Ra

18、man micro-spectroscopy system to achieve RACS.第21頁(yè)/共35頁(yè) 1）微生態(tài)生理分析 單細(xì)胞同位素拉曼分析和熒光原位雜交技術(shù)相結(jié)合，可用于生態(tài)群落中微生物的鑒別與生理功能角色的分析。 參加：Hydrocarbon and Lipid Microbiology, 2016 Springer-Single Cell Microbial Ecophysiology with Raman-FISH.第22頁(yè)/共35頁(yè)Fig. 1 Schematic diagram of a Raman microspectrometer. Microbial cells

19、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 fr

20、equencies are filtered with a notch filter,before being diffracted on a grating and dispersed onto a cooled CCD camera, generating the Raman spectrum第23頁(yè)/共35頁(yè)Fig. 2 Outline of the major steps involved in a Raman-FISH experiment第24頁(yè)/共35頁(yè) 2）細(xì)胞代謝分析 拉曼光譜含有細(xì)胞中所有儲(chǔ)藏分子的特征譜，而同位素效應(yīng)會(huì)使相應(yīng)譜線發(fā)生紅移（重同位素結(jié)合率10%時(shí)），因此結(jié)合

21、同位素標(biāo)記，可方便識(shí)別活性細(xì)胞，對(duì)單細(xì)胞代謝過(guò)程進(jìn)行原位分析。 參見(jiàn)：Hydrocarbon and Lipid Microbiology, 2016 Springer- Single-Cell Metabolomics.第25頁(yè)/共35頁(yè)Fig. 1 Single-cell Raman spectra from single bacterial cells with and without 1 3C, 1 5N and 2H incorporation(532 nm laser, acquisition time: 0.5 s). For 1 3C-incorporation, the st

22、rong 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 1 5N-incorporation, the main bands shifted are nucleic acids at 728 and 784 cm- 1. For 2H-incorporation, the Raman b

23、and 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 .第26頁(yè)/共35頁(yè) 3）細(xì)胞生物學(xué)研究 確定生物活性細(xì)胞種群，例如藻類光合作用細(xì)胞。因?yàn)槠渲蓄惡}卜素，是光合作用細(xì)胞的光合俘獲天線，又是細(xì)胞中類別最多的化合物之一，可以作為光合細(xì)胞的標(biāo)志化合物，利用結(jié)合13CO2后拉曼譜線紅移

24、的特性，可以把光合作用細(xì)胞與CO2固定活性相關(guān)聯(lián)，以確定和分離光合作用細(xì)胞。 參見(jiàn)： Rapid resonance Raman microspectroscopy to probe carbon dioxide fixation by single cells in microbial communities The ISME Journal (2012) 6, 875885第27頁(yè)/共35頁(yè)Synechocystis sp. PCC 6803 grown in BG11 medium supplemented with 10.8% 13C-NaHCO3mixed with S. elong

25、atus PCC 7942 grown in BG11mediumsupplemented with 12C-NaHCO3. Two Raman images were generated based on the 1 and 2 bands. The 13C-incorporated single cells are displayed in red and the 12C cells green. Some 13C-incorporated cells with lower signal strength are displayed in yellow. The SCRR spectra

26、are coloured to correspond to the 13C-labelled cells and 12C cellsindicated by red and green circles, respectively.第28頁(yè)/共35頁(yè) 4）細(xì)胞生物學(xué)分類 根據(jù)單細(xì)胞同位素拉曼譜的數(shù)據(jù)，利用主量分析有效減少特征譜變量空間的維數(shù)后，由規(guī)范變量分析PC-CVA可對(duì)生物活性細(xì)胞進(jìn)行分類，分類后的細(xì)胞又可以挑選出來(lái)，利用生物技術(shù)方法進(jìn)一步進(jìn)行下游分析。 參見(jiàn)： Raman Activated Cell Ejection for Isolation of Single Cells， Anal

27、. Chem. 2013, 85, 1069710701第29頁(yè)/共35頁(yè)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.第30頁(yè)/共35頁(yè) 5）分子代謝研究 通過(guò)標(biāo)記氨基酸對(duì)分子代謝進(jìn)行示蹤，并利用特征拉曼譜線的對(duì)比度成像，對(duì)細(xì)胞活性分子進(jìn)行偽色彩定位成像。 參見(jiàn)：Noni

28、nvasive Imaging of Protein Metabolic Labeling in Single Human Cells Using Stable Isotopes and Raman Microscopy第31頁(yè)/共35頁(yè)Figure . Raman imaging of a HeLa cell incubated for 28 h with Phe-d5. (A) Hierarchical cluster analysis image with five clusters.(B-F) Univariate images of (B) nucleotides (770-790

29、cm-1), (C) phospholipids (700-730 cm-1), (D) Phe-d5 (950-965 cm-1), (E) Pheh5 (995-1005 cm-1), and (F) Phe-d5/Phe-h5 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

30、15 m2.第32頁(yè)/共35頁(yè) 6）D2O標(biāo)記及拉曼分析 利用來(lái)自D2O的D標(biāo)記細(xì)胞成分，如脂類和蛋白質(zhì)等，由于其與環(huán)境的同位素交換并不影響分子特征帶的紅移信號(hào)，所以同樣可以方便地用于活性細(xì)胞的識(shí)別和分類研究。 參加：Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells /cgi/doi/10.1073/pnas.1420406112第33頁(yè)/共35頁(yè)第34頁(yè)/共35頁(yè) Fig. 1. E. coli incorporation of D f

31、rom 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% D2O of growth water). Ten to 21 single-cell spectra were used to produce m

32、ean 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 D2O-containing media and cells

33、 grown without D2O. 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%). 31P 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 D2O percentage of growth water. Box plots show the quartiles for each population of cells. The detection limit