光镊拉曼光谱分析酿酒活性干酵母的活化与生长

应用光镊拉曼光谱新技术(LTRS)对酿酒活性干酵母复水活化与生长进行动态观察, 探索从分子光谱角度窥视胞内糖类、核酸、蛋白等生物大分子的变化过程, 及葡萄糖消耗和乙醇生成的动态过程。结果显示, 酿酒活性干酵母复水活化后, 第6小时和9小时, 即酵母对数生长中期及乙醇产生前期, 是调控酵母细胞生理变化的2个重要的时间点。核酸类物质在细胞活化后迅速增加, RNA在第6小时达到最大值; 而蛋白质和脂类物质从第6小时开始快速增加, 在第9小时达 到最大值, 而后呈下降趋势; 胞内乙醇则是在9 h开始出现, 在9     

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。     

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。     

毛姜花油细胞原位拉曼光谱研究

为避免复杂的样品的制备及提取过程,最大限度避免精油活性成分变化,常温下,用拉曼光谱原位分析毛姜花油细胞中精油。样品切片后置于共聚焦显微拉曼光谱仪下,用10倍物镜可观察到油细胞。油细胞精油的拉曼光谱与1,8-桉油精拉曼光谱非常相似。以毛姜花油细胞/1,8-桉油精的拉曼峰为序,较强峰出现在2928/2 921、647/652 cm~(-1),次强峰出现在540/545、808/813、915/920、926/930、1 012/1 016、1 075/1 080、1 270/1273、1 427/1 432 cm~(-1)。在油细胞中出现的强峰、次强峰与1,8-桉油精的拉曼峰一致,说明毛姜花油细胞中油的主要成分为1,8-桉油精。毛姜花油细胞的25条拉曼峰都与1,8-桉油精的拉曼峰有很好的对应关系。     

表面增强拉曼散射技术无标记检测乳腺癌细胞来源外泌体

外泌体含有释放细胞的特异性物质,反映了释放细胞的组成成分,成为液体活检的重要物质。为了鉴别正常乳腺上皮细胞和乳腺癌细胞来源的外泌体,本研究采用表面增强拉曼(SERS)技术检测乳腺癌细胞MCF-7和人正常乳腺上皮细胞MCF-10A来源的外泌体,并且对比两种细胞外泌体的SERS光谱,筛选出相应的特征拉曼光谱。结果发现在800~1800 cm-1区域内,相对MCF-10A细胞,MCF-7来源的外泌体中归属于核酸的拉曼峰相对强度明显增高,且部分脂类峰强有所降低或部分特征峰消失,同时发现MCF-7还表现出其自己独特的拉曼谱型。此结果表明SERS技术可高灵敏度检测不同细胞来源的外泌体细微分子变化,可区分肿瘤细胞来源的外泌体与正常细胞来源的外泌体。SERS技术可作为癌症的早期检测和诊断的一种快速、无标记和无损的方法。     

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。     

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。     

基于傅里叶变换红外光谱的葡萄褐斑病研究

利用傅里叶变换红外(Fourier Transform Infrared,FTIR)光谱研究了葡萄褐斑病病叶和正常叶片。测试分析了大褐斑病叶病斑处和非病斑处、小褐斑病叶病斑处和非病斑处、未染病正常叶片的红外光谱,结果显示:葡萄叶片光谱主要由碳水化合物、蛋白质、脂类的吸收带组成。褐斑病叶片光谱与正常叶片红外光谱变化主要在1 800-1 500 cm-1范围脂类、蛋白质酰胺的吸收区和1 200-900 cm-1范围多糖的吸收区。用1 800-1 500 cm-1范围拟合峰的峰面积比A1657/A1517考察蛋白质相对含量变化,A1738/A11517考察脂类相对含量变化,用吸光度比I1066/I1441考察多糖相对含量变化,结果显示患褐斑病的葡萄叶中脂类、蛋白质、糖类等营养物质含量降低。褐斑病和正常葡萄叶红外光谱的二阶导数光谱结合聚类分析方法,利用1 800-1 500 cm-1与1 200-900 cm-1范围数据进行聚类分析,结果将正常叶片与患褐斑病叶片准确区分开。证明褐斑病叶片红外光谱在1 800-1 500 cm-1与1 200-900 cm-1范围为敏感谱带。     

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.     

二甲基亚砜对离体猪皮肤组织的拉曼光谱的影响

拉曼光谱技术作为鉴定生物分子种类最有力的分析工具之一,具有快速、简单、无损、准确等优点.目前,拉曼光谱已被国内外学者广泛开展了在人体组织的应用研究,但由于生物组织具有高散射性,因此限制了拉曼光谱对其的检测深度.本文主要采用光透明剂--二甲基亚砜对组织拉曼光谱的影响进行研究,对离体猪皮组织的不同深度（100 μm,200 μm,300 μm,400 μm）拉曼光谱强度随处理前、后不同时间（0 min,10 min,20 min,30 min,60 min）的变化进行对比.结果发现,不管是经二甲基亚砜处理前还是后,都出现随着距猪皮组织表面的深度加深,其拉曼光谱强度都不断减少;同时发现各层猪皮组织随着处理后时间的加长,其特征峰的强度不断加强,信噪比逐渐提高,在处理后的60 min效果最好;而且出现了在经二甲亚砜处理前看不见的峰（1 126 cm-1和1 426 cm-1）.结果表明：5%DMSO对组织的处理能增强其拉曼光谱的强度,同时也能使拉曼光谱仪的信噪比提高,并且使组织谱图中特征峰也得到相应的增加.     

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.     

Deciphering the finger Prints of Brain Cancer Astrocytoma in comparison to Astrocytes by using near infrared Raman Spectroscopy

To explore the biochemical differences between brain cancer cells Astrocytoma and normal cells Astrocyte, we investigated the Raman spectra of single cell from these two cell types and analyzed the difference in spectra and intensity. Raman spectrum shows the banding pattern of different compounds as detected by the laser. Raman intensity measures the intensity of these individual bands. The Raman spectra of brain cancer cells was similar to those of normal cells, but the Raman intensity of cancer cells was much higher than that of normal cells. The Raman spectra of brain cancer Astrocytoma shows that the structural changes of cancer cells happen so that many biological functions of these cells are lost. The results indicate that Raman spectra can offer the experimental basis for the cancer diagnosis and treatment.     

Raman spectroscopy of interferon-induced 2',5'-linked oligoadenylates

Raman spectra of model compounds and of 2',5'-oligoadenylates in D2O were utilized to assign the Raman bands of 2',5'-oligoadenylates. The Raman spectra of A2'pA2'pA, pA2'pA2'pA, and pppA2'pA2'pA contained features that were similar to those of adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate, respectively. When AMP and pA2'pA2'pA were titrated from pH 2 to 9, the normalized Raman intensity of their ionized (980 cm-1) and protonated (1080 cm-1) phosphate bands revealed similar pKa's for the 5'-monophosphates. The Raman spectrum of pA2'pA2'pA was altered slightly by elevations in temperature, but not in a manner supporting the postulate that 2-5A possesses intermolecular base stacking. Major differences in the Raman spectrum of 2',5'- and 3',5'-oligoadenylates were observed in the 600-1200-cm-1 portion of the spectrum that arises predominately from ribose and phosphate vibrational modes. Phosphodiester backbone modes in A3'pA3'pA and pA3'pA3'pA produced a broad band at 802 cm-1 with a shoulder at 820 cm-1, whereas all 2',5'-oligoadenylates contained a major phosphodiester band at 823 cm-1 with a shoulder at 802 cm-1. The backbone mode of pppA2'pA2'pA contained the sharpest band at 823 cm-1, suggesting that the phosphodiester backbone may be more restrained in the biologically active, 5'-triphosphorylated molecule. The Raman band assignments for 2',5'-oligoadenylates provide a foundation for using Raman spectroscopy to explore the mechanism of binding of 2',5'-oligoadenylates to proteins.     

Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy

The structure, transformation, and bioactivity of single living Schizosaccharomyces pombe cells at the molecular level have been studied in vivo by time- and space-resolved Raman spectroscopy. A time resolution of 100 s and a space resolution of 250 nm have been achieved with the use of a confocal Raman microspectrometer. The space-resolved Raman spectra of living S. pombe cells at different cell cycle stages were recorded in an effort to elucidate the molecular compositions of organelles, including nuclei, cytoplasm, mitochondria, and septa. The time- and space-resolved measurement of the central part of a dividing yeast cell showed continuous spectral evolution from that of the nucleus to those of the cytoplasm and mitochondria and finally to that of the septum, in accordance with the transformation during the cell cycle. A strong Raman band was observed at 1602 cm(-)(1) only when cells were under good nutrient conditions. The effect of a respiration inhibitor, KCN, on a living yeast cell was studied by measuring the Raman spectra of its mitochondria. A sudden disappearance of the 1602 cm(-)(1) band followed by the change in the shape and intensity of the phospholipid bands was observed, indicating a strong relationship between the cell activity and the intensity of this band. We therefore call this band "the Raman spectroscopic signature of life". The Raman mapping of a living yeast cell was also carried out. Not only the distributions of molecular species but also those of active mitochondria in the cell were successfully visualized in vivo.     

二氧化硅与脂质体相互作用的激光拉曼光谱研究

脂质体在SiO_2作用下1127cm~(-1)与1093cm~(-1)强度比和2883cm~(-1)与2847cm~(-1)强度比均降低,715cm~(-1)谱带强度也降低.并且频率有2—3cm~(-1)的位移,峰形变宽.表明磷脂以其极性头部与SiO_2结合,形成稳定的复合体.磷脂头部的作用和脂质体的变形引起烃链构象变化.TiO_2不引起拉曼光谱的变化,与脂质体的作用甚微.     

Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.     

The Resonance Raman Spectra of β-Carotene Molecules in the Photosystem Ⅱ Reaction Center D1/D2/Cyt b-559 Complex

The resonance Raman spectrum of β-carotene in photosystem Ⅱ (PS Ⅱ )reaction center complex was characterized by four main bands, peaking at 1532 (νl), 1156 (ν2), 1010 (ν3) and 970 (ν4) cm -1, respectively, with several additional small Raman bands in the region between 1100 cm-1 and 1500 cm-1 It was suggested that β-carotene molecules of the reaction center complex were in all-trans configuration. The resonance Raman spectrum of an acetone extract from the reaction center complex also showed four main bands. The peak position of νl, ν3 and ν4 band shifted 5 cm-1 to the shorter wave number. The most dramatic changes were the reduction of the intensity of ν4. From the above results it was demon- strated that the conformation of β-carotene molecules in the PS Ⅰ reaction center was not the same as that of free β-carotene molecules in solution, but similar to that of carotenoid molecules in the photosynthetic bacterial reaction center, in other words, they are likely to be in a twisted conformation.     

Photoreductive titration of the resonance Raman spectra of cytochrome oxidase in whole mitochondria.

A photoreductive titration of the resonance Raman (RR) spectra of cytochrome c oxidase in whole mitochondria was recorded by exploiting the preferential enhancement of the Raman signals of reduced cytochrome oxidase excited at 441.6 nm. When the sample was cooled to about--10 degrees C, it was possible to slow down the photoreductive effect of the laser and to record RR spectra at various states of reduction. Compared to the earliest recorded scan (most oxidized), the dithionite-reduced sample shows the appearance of new bands at 216, 363, 560, and 1665 cm-1. At intermediate stages of photoreduction, the 216- and 560-cm-1 bands appear before the 363- and 1665-cm-1 bands; photoreduction induces full intensity in the former bands, whereas the latter bands are photoreduced to 50% of the dithionite-reduced intensity. The relative intensities of a doublet at 1609--1623 cm-1 are affected by reduction: the band at 1609 cm-1 is weaker in the earlier scans; in later scans this band has grown to equal intensity with the 1623-cm-1 band. We conclude that this reductive titration of the RR spectrum of cytochrome c oxidase reflects three states in its reduction. The behavior of the doublet at 1609--1623 cm-1 suggests that the two hemes are nonequivalent but interacting. The band at 216 cm-1 may be indicative of an iron-copper interaction that is affected by the presence of external ligands.     

Inter- and intramolecular disulfide bond formation and related structural changes in the lens proteins. A Raman spectroscopic study in vivo of lens aging

Raman spectra have been measured for intact rat lens nuclei at various stages of aging in an attempt to gain further insight into age-related structural changes in the lens proteins, especially changes concerning protein sulfhydryl groups. Two Raman bands at 2579 and 2561 cm-1 were observed to be assignable to SH stretching modes of the cysteine residues. These bands have been attributed to "exposed" and "buried" sulfhydryl groups of the lens proteins, respectively, on the basis of a model compound study. The relative intensities of both SH stretching modes decreased with lens aging, and concurrently the intensity of a S-S stretching mode at 509 cm-1 due to disulfide bridges increased, suggesting that not only exposed but also buried protein sulfhydryl groups are converted to disulfide groups as a result of aging. The rate of the intensity decrease in the 2561 cm-1 band was similar to that in the 2579 cm-1 band. Therefore, it seems likely that the sulfhydryl groups in the two distinct environments are nearly equally subjected to the oxidation. Cysteine and cystine residues of the lens proteins gave their C-S stretching modes at 708 cm-1, indicating that they predominantly assume PC and/or PN conformers. The intensity ratio of a tyrosine doublet near 840 cm-1 (I832/I855) changed from approximately 0.86 to approximately 0.81 with the aging of the rat lens. This result implies that some tyrosine residues undergo a change in their hydrogen bonding environments during the course of aging. Of particular importance is that the relative intensity change of the tyrosine doublet with normal aging and that with cataract formation are in opposite directions.