正常和癌变胃粘膜组织的共焦拉曼光谱初探

目的:分析研究胃正常和癌变粘膜组织的拉曼光谱特征,为拉曼光谱应用于胃癌的临床检测诊断奠定基础。方法:收集胃镜检查中活检的19例正常和12例癌变胃粘膜组织标本,采用785 nm激发光拉曼光谱仪进行拉曼光谱采集。比较分析胃正常和癌变粘膜组织的拉曼光谱特征差异并研究其区分正常和癌变组织的价值。结果:1)特征峰1 098 cm-1、1 444 cm-1、1 555 cm-1、1 660 cm-1等在胃癌组织中发生了移位,平均位移(2.57±1.28)cm-1,以红移为主;2)癌变组织中相对峰强比I1087 cm-1/I1207 cm-1≥1.87,其区别胃癌和正常胃粘膜组织的准确率、灵敏度和特异度分别为87.1%、83.3%、89.5%;3)癌组织中增加了表征蛋白质的特征峰1 262 cm-1、1 586 cm-1,但同时减少了表征蛋白质和脂质特征峰1 172 cm-1。结论:拉曼光谱不仅可以准确区分正常和癌变,而且可以探索癌变相关的分子生化改变。拉曼光谱在胃癌的跟踪发现和检测诊断中具有良好前景。     

皮肤组织显微共聚焦拉曼光谱成像研究

显微共聚焦拉曼光谱成像技术(Confocal Raman Microspectroscopy Imaging,CRMI)能够对样品微区进行精确无损的拉曼光谱分析和光谱图像扫描,提供生物样品的无损高分辨光学信息。本项研究工作,利用CRMI技术实验获取了正常人体离体皮肤组织的拉曼光谱特征,并结合典型特征峰的扫描图像,探讨了脂类、蛋白质等成分在皮肤真皮层的分布特点。实验发现皮肤组织真皮层内胶原蛋白的拉曼特征峰1 248 cm-1强度及其空间分布尤为突出,这一实验结果与组织学中胶原纤维占真皮结缔组织95%的事实相符。实验结果显示,CRMI技术能够全面诠释生物组织内部生化组成与分布信息,在实验描述皮肤组织病理变化的分子生物学机制方面具有广阔的应用前景。     

体外联合使用指纹区及高波数区拉曼光谱诊断胃癌

目的:研究正常胃粘膜和胃癌组织在拉曼光谱指纹区(800-1 800 cm-1)和高波数区(2 800-3 000 cm-1)的光谱特征,并将其联合使用建立胃癌诊断模型。方法:收集38例正常胃粘膜和37例胃癌组织活检标本,采用785 nm激发光拉曼光谱仪进行拉曼光谱采集。比较正常胃粘膜和胃癌组织在指纹区和高波数区的拉曼光谱异同,使用偏最小二乘判别分析(PLS-DA)结合留一法交叉验证建立诊断模型。结果:1)胃癌组织在853 cm~(-1),879cm~(-1),1 003 cm~(-1),1 047 cm~(-1),1 173 cm-1,1 304 cm~(-1),1 319 cm~(-1),1 338 cm~(-1),1 374 cm-1、2 932 cm-1谱峰处与正常胃粘膜的拉曼峰强度差异有统计学意义(P0.05)2)将拉曼光谱指纹区和高波数区联合,利用PLS-DA建立胃癌诊断模型的敏感性为94.59%(35/37),特异性为86.84(33/38),正确率为90.6%(68/75)。结论:正常胃粘膜和胃癌组织在拉曼光谱指纹区和高波数区均有显著差异,将上述两区联合使用建立模型诊断胃癌能取得良好的诊断效果。     

一种酵母细胞生长现象的实时单细胞拉曼光谱观察

用拉曼镊子观察单个即发活性干酵母(Saccharomy cescerevisiae)细胞在2.0%葡萄糖溶液中的活化过程,收集其拉曼光谱。结果发现,在某一批次的产品中,酵母细胞的1364cm-1峰强度随着细胞的活化而显著增加,531cm-1、652cm-1、1053cm-1等源自葡萄糖或葡萄糖基的信号峰也会随细胞的生长而增强,随后增强的还有1432cm-1、1448cm-1、1561cm-1等源自脂类物质的峰,而源自蛋白质及脂类的1000cm-1、1445cm-1、1655cm-1等峰的信号强度基本不变,酵母细胞代谢活跃的标志峰1603cm-1也基本不变。该批次产品中,10次实验有7次观察到上述现象,而在别的批次产品中并没有观察到该现象。用单细胞拉曼光谱实时记录了这一特殊的生长现象。     

褪黑素对癫痫病作用的激光拉曼光谱研究

分别对正常状态,癫痫状态以及癫痫状态注射褪黑素后的Wistar大鼠的大脑皮质上清液样品进行了拉曼光谱分析.研究表明三种样品的拉曼特征峰分别为波数1 654 cm-1、1 623 cm-1及1 632 cm-1.它们有明显的区别.癫痫状态样品光谱在波数1 654 cm-1位置幅值有所减小;注射褪黑素样品光谱在波数1 654 cm-1位置幅值有所增加,证明褪黑素对癫痫病有抑制作用.     

木霉生物吸附重金属铬机理的研究

利用木霉（Trichoderma lhd）菌体作为吸附剂,对水体中的六价铬进行生物吸附,借助傅立叶红外变换光谱和拉曼光谱对六价铬的生物吸附机理进行了探讨。实验条件优化结果表明,温度28 ℃以及酸性环境条件（pH 1）有利于Cr （VI） 的生物吸附,12小时内,Cr （VI） 的生物吸附去除效率达99 %。吸附机理实验结果分析表明,相比于对照实验,2350 cm^-1吸收峰的出现为吸附剂表面质子化的氨基如＞NH2^＋, NH^＋, ＞C=NH^＋―等基团吸附Cr （VI）所致。拉曼光谱中吸收峰2097 cm^-1强度显著增强进一步表明,Cr （VI）的生物吸附是吸附剂表面氨基基团在起作用。     

卵巢癌患者血清的拉曼光谱研究初探

目的:探究拉曼光谱技术应用于卵巢癌研究的可能性。方法:收集卵巢癌患者血清及健康人血清各20例,用激光共聚焦显微拉曼光谱仪进行检测。结果:两组血清的平均拉曼光谱形态和谱峰基本相似,但在约1010、1158、1283、1520、1646、2307和2661cm-17个拉曼频移附近,卵巢癌患者血清的拉曼光谱谱峰强度明显低于健康对照组,而在其余大部分波段,卵巢癌患者血清的拉曼光谱强度均高于健康对照组。结论:拉曼光谱技术可以初步区分卵巢癌及健康人血清,值得进一步研究和探讨其临床应用价值。     

动脉粥样硬化斑块的微区拉曼光谱检测

为探讨动脉粥样硬化斑块的微区拉曼光谱特征,以球囊损伤日本长耳白兔右侧颈总动脉后予以高脂饮食喂养,在实验过程中监测体重和血脂变化情况。3个月后,以中国斑点蝰蛇毒和组胺加以触发使斑块破裂,将动物处死并查找动脉硬化斑块,动脉组织经大体病理分类后,进行微区拉曼光谱及病理检测。结果显示:动脉粥样硬化斑块的拉曼光谱图在1450及1660cm-1处均有明显的胆固醇等脂质特征峰。特征峰曲线下相对面积统计结果表明:明显动脉粥样硬化斑块的谱峰下相对面积(5.80×10-3±3.51×10-3)显著高于轻度动脉粥样硬化组织(2.01×10-3±1.49×10-3)及正常动脉组织(1.01×10-3±0.94×10-3),P<0.05。正常动脉组织拉曼光谱曲线较光滑,无明显特征峰。血栓形成处拉曼光谱图荧光背底较强,未见特征谱峰。该研究结果证明微区拉曼光谱可以对动脉粥样硬化斑块的胆固醇等脂质含量进行特异性定量检测,表明微区拉曼光谱是评估动脉粥样硬化程度及斑块稳定性的可行方法。     

姜黄薄壁细胞原位拉曼光谱研究

用拉曼光谱原位分析姜黄橙黄色及黄色薄壁细胞中的物质。德宏、广西及西双版纳姜黄三种样品橙黄色细胞的拉曼光谱非常相似,较强峰出现在1 632/1 633/1 637、1 599/1 601/1 605、1 184/1 186/1 190 cm-1,中等强度的峰出现在1 529/1 528/1 534、1 425/1 426/1 430、1 306/1 308/1 311、1 235/1 235/1 239、1 167/1 168/1 173、1 122/1 125/1 130、967/969/975 cm-1。三种姜黄薄壁细胞中出现的强峰、次强峰位置、峰型都一致,说明三种姜黄橙色薄壁细胞中物质的主要成分相同。与姜黄素(curcumin)拉曼光谱的主要的19条谱线比较,在姜黄橙色细胞拉曼谱的17条谱峰中有16条与之对应。而黄色薄壁细胞中大部分谱线与支链淀粉的谱峰有较好的对应关系。用密度泛函理论计算了姜黄素的拉曼光谱,并对谱线进行了初步的归属。     

饮用水中硫化物的表面增强拉曼光谱检测方法

基于表面增强拉曼光谱(SERS)技术,建立了一种对饮用水中硫化物快速筛查的方法。以自制纳米/胶体金为表面增强基底,并利用紫外-可见吸收光谱法和透射电镜(TEM)对该基底进行表征。待检样本经简单前处理,其中所含性质活泼的硫化物能与N,N-二乙基对苯二胺(DPD)反应生成亚甲基蓝类化合物,该化合物经衍生后,在低功率(100 m W)和短积分时间(500 ms)条件下直接测试其拉曼光谱,并通过与硫化物衍生物的标准拉曼图谱比对进行定性分析。硫化物衍生物的SERS化学信号大约在453 cm-1和458 cm-1两处,分别对应于亚甲基蓝化合物中C—N—C的不同振动模式。本方法能够准确筛查饮用水中硫化物,整个测试过程少于10 min,检测限为0. 02mg/L,能够满足国家标准的限量要求。该方法简单、快速,结果准确,可以作为一种高灵敏的快速筛查手段。     

Intensity changes of base and backbone Raman modes in the B to Z transition of poly(dG-dm5C)

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm5C). The transition midpoint was about 37 degrees C for a solvent containing 20 mM Mg2+. A 10-fold change in Mg2+ concentration altered the transition midpoint by at least 60 degrees C. Raman spectra of the B and Z forms of poly(dG-dm5C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm-1 guanine mode and 835 cm-1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm-1, 805 and 819 cm-1. Several bands unique to poly(dG-dm5C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

Structural analysis and comparison of cobrotoxin and cardiotoxins by near-IR Fourier transform Raman spectroscopy.

Venom toxins were isolated from Formosan cobra (Naja naja atra) by cation-exchange chromatography. The near-IR FT-Raman analytical method has been applied to the characterization and classification of the toxin components in their lyophilized forms. Structural analysis and comparison of various purified toxin fractions were made with respect to their amino acid compositions and near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structure of cobra toxins including cobrotoxin and various cardiotoxins is mainly anti-parallel beta-pleated sheet as judged by the Raman signals at 1238 cm-1 (amide III) and 1671 cm-1 (amide I). It is also found that the relative Raman signal intensities of Tyr, Phe, Trp and Met residues in purified toxins correlate very well with the structural data obtained from amino acid analysis. The advantage and improvement of applying the near-IR FT-Raman spectroscopy to the unambiguous classification and comparison of venom toxins are evident and the discrepancies with previous Raman studies on these venom toxins are also revealed and discussed.     

Using Raman Spectroscopy in Studying the Effect of Propylene Glycol,Oleic Acid,and Their Combination on the Rat Skin

The permeability enhancement effect of oleic acid (OA) and propylene glycol (PG) as well as their (1:1 v/v) combined mixture was studied using rat skin. The percutaneous drug administration is a challenge and an opportunity for drug delivery. To date, there is limited research that illustrates the mechanism of penetration enhancers and their combinations on the skin. This project aims to explore the skin diffusion and penetration enhancement of PG, OA, and a combination of PG-OA (1:1 v/v) on rat skin and to identify the potential synergistic effect of the two enhancers utilizing Raman spectroscopy. Dissected dorsal skin was treated with either PG or OA or their combination for predetermined time intervals after which the Raman spectra of the treated skin were collected with the enhancer. A spectrum of the wiped and the washed skin were also collected. The skin integrity was tested before and after exposure to PG. The skin histology proved that the skin integrity has been maintained during experiments and the results indicated that OA disrupted rat skin lipid as evident by changes in the lipid peak. The results also showed that PG and OA improved the diffusion of each other and created faster, yet reversible changes of the skin peaks. In conclusion, Raman spectroscopy is a potential tool for ex vivo skin diffusion studies. We also concluded that PG and OA have potential synergistic reversible effect on the skin.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light (800 μmol photons/m2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSll RC as determined by HPLC after light treatment were as follows: with increasing illumination time chlorophyll (Chi) a and β-carotene (β-car)content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illumination, the initial bleaching occurred maximally at 680 nm but that with increasing illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 min light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination.After illumination, the fluorescence emission intensity changed and the fluorescence maximum blue shifted,showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm-1 (υ1), 1 159 cm-1 (υ2), 1 006 cm-1 (υ3), 966 cm-1 (υ4) for 488.0 nm excitation and 1 525 cm-1 (υ1), 1 159 cm-1 (υ2), 1 007 cm-1 (υ3), 968 cm-1 (υ4) for 514.5 nm excitation.It was confirmed that two spectroscopically different β-car molecules exist in the PSII RC. After light treatment for 20 min, band positions and bandwidths were unchanged. This indicates that carotenoid configuration is not the parameter that regulates photoprotection in the PSII RC.     

A Raman spectroscopic study on the interaction of an ion-channel protein with a phospholipid in a model membrane system (gramicidin A/L-alpha-lysophosphatidylcholine)

The interaction of the ion channel polypeptide gramicidin A with the L-alpha-lysophosphatidylcholine micelles in a membrane state association (approximative molar ratio 1:9) was investigated by Raman spectroscopy. Studies were carried out over the spectral ranges of 700-1700 cm-1 and 2800-3100 cm-1 at 10 degrees C. The Raman spectrum of L-alpha-lysophosphatidylcholine micelles indicated a disordered structure of the lipid acyl chains by the high intensities of the gauche conformation vibrations. Changing from the micellar phase to the membrane state of association with gramicidin A, the intensities of all-trans stretching modes increased whereas the intensities of gauche conformation vibrations decreased, reflecting the emergence of ordered lipid chains. Hydrophobic interactions between the acyl chains and the polypeptide side chain residues were demonstrated. The absence of modifications in intensities of the very strong tryptophan vibrations in the complex spectrum indicated that, if the tryptophan-stacking interactions suggested by some authors exist, they are very weak ones.     

Iron-histidine stretching Raman line and enzymic activities of bovine and bacterial cytochrome c oxidases

Resonance Raman spectra of the reduced form of cytochrome c oxidase isolated from bovine heart and the thermophilic bacterium PS3 were investigated in relation to their H+-pumping- and cytochrome-c-oxidizing activities, which were varied by incubating the enzyme at raised temperatures or at alkaline pH at room temperature. For both the bovine and PS3 enzymes, the intensity of the iron-histidine stretching Raman line of the ferrous a3 heme (214 cm-1) exhibited an incubation-temperature-dependent change, which fell between the similar curves of the H+-pumping and cytochrome-c-oxidizing activities. The intensities of the formyl CH=O stretching Raman line of the ferrous a3 heme (1665 cm-1) as well as of other lines were insensitive to the heat treatment. The iron-histidine stretching Raman line of both enzymes showed pH-dependent intensity change which was nearly parallel with the pH dependence of cytochrome-c-oxidizing activity. Therefore, deprotonation affecting the 214 cm-1 Raman line is responsible for the decrease of activity. This limited alkaline treatment to the PS3 enzyme was reversible and the recovered enzyme exhibited Raman intensities and enzymic activities similar to the native one. However, the neutralized, bovine enzyme with a similar intensity of the 214 cm-1 line showed increased cytochrome-c-oxidizing activity and null H+-pumping activity.     

Contribution of the resonance Raman spectroscopy to the identification of Z DNA

Poly(dG-dC).poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition. With 257 nm excitation wavelength the 1580 cm-1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm-1 and at 1193 cm-1 and the bands of the cytosines at 780 cm-1, 1242 cm-1 and 1268 cm-1. By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.     

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.     

Direct observation of spectral differences between normal and basal cell carcinoma (BCC) tissues using confocal Raman microscopy

Raman spectroscopy has strong potential for providing noninvasive dermatological diagnosis of skin cancer. In this study, confocal Raman microscopy was applied to the dermatological diagnosis for one of the most common skin cancers, basal cell carcinoma (BCC). BCC tissues were obtained from 10 BCC patients using a routine biopsy and used for confocal Raman measurements. Autofluorescence signals from tissues, which interfere with the Raman signals, were greatly reduced using a confocal slit adjustment. Distinct Raman band differences between normal and BCC tissues for the amide I mode and the PO2- symmetric stretching mode showed that this technique has strong potential for use as a dermatological diagnostic tool without the need for statistical treatment of spectral data. It was also possible to precisely differentiate BCC tissue from surrounding noncancerous tissue using the confocal Raman depth profiling technique. We propose that confocal Raman microscopy provides a novel method for dermatological diagnosis since direct observations of spectral differences between normal and BCC tissues are possible.     

The study of microviscosity of plasma membranes of Nitella cells during rest and excitation

Changes in the microviscosity of excitable membranes was investigated using resonance Raman spectroscopy of carotenoids. The Raman resonance spectra of carotenoids in Nitella cells were excited by 514.5 nm line of an argon ion laser. The bands at 1525 cm-1, 1160 cm-1 and 1008 cm-1 were observed and they were assigned to C=C, C-C and C-CH vibrations, respectively. The rhythmic excitation of cell reduced the intensity and increased the ratios of intensity of major carotenoid bands with no noticeable shift in the position of peaks. The Arrhenius plot of relative intensity ratios of 1525 cm-1 and 1160 cm-1 bands versus reciprocal temperature showed a change of the slope in the range of 13-18 degrees C. This indicates a membrane phase transitions in which a reorientation of carotenoids species takes place. The interpretation was supported by parallel microcalorimetric and EPR measurements. The decrease of microviscosity with increasing temperature is probably caused by changes in polyene chain conformation. It is suggested that membrane microviscosity during NH4(+)-stimulated rhythmic excitation of algal cells increases, and membrane-associated carotenoids act as microviscosity-sensitive "potential sensor" for the channel.