The effect of cell fixation on the discrimination of normal and leukemia cells with laser tweezers Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the effect of different chemical fixation procedures on the Raman spectra of normal and leukemia cells. Individual unfixed, paraformaldehyde-fixed, and methanol-fixed normal and transformed lymphocytes from three different cell lines were analyzed with LTRS. When compared to the spectra of unfixed cells, the fixed cell spectra show clear, reproducible changes in the intensity of specific Raman markers commonly assigned to DNA, RNA, protein, and lipid vibrations (e.g. 785, 1230, 1305, 1660 cm(-1)) in mammalian cells, many of which are important markers that have been used to discriminate between normal and cancer lymphocytes. Statistical analyses of the Raman data and classification using principal component analysis and linear discriminant analysis indicate that methanol fixation induces a greater change in the Raman spectra than paraformaldehyde. In addition, we demonstrate that the spectral changes as a result of the fixation process have an adverse effect on the accurate Raman discrimination of the normal and cancer cells. The spectral artifacts created by the use of fixatives indicate that the method of cell preparation is an important parameter to consider when applying Raman spectroscopy to characterize, image, or differentiate between different fixed cell samples to avoid potential misinterpretation of the data.     

Fluorescence spectroscopy for detection of malignancy: H-ras overexpressing fibroblasts as a model.

Autofluorescence from intracellular chromophores upon illumination of cells by monochromatic light has been studied towards the development of novel noninvasive and sensitive technology for the early detection of cancer. To investigate the relationship between biochemical and morphological changes underlying malignant disease and resulting fluorescence spectra, an in vitro model system of a paired normal and malignant murine fibroblasts cell lines, differing in cancer-associated H-ras expression was employed. A comparison of fluorescence excitation and emission spectra of proliferative cells revealed that fluorescence intensity of malignant cells was significantly less than that of normal cells upon excitation at 290 nm. Fluorescence of both cell lines decreased with decreasing cell concentration, but at each concentration, normal cells had higher fluorescence intensity than malignant cells. Similar differences between the cell lines were observed when brought to quiescence or at stationary phase. Results suggested that the chromophore contributing most significantly to these spectra is tryptophan and its moieties in proteins. This model system demonstrates the specific contribution of H-ras to subcellular chromophores, resulting in a significant difference in their autofluorescence intensity, and implies the potential use of the technique for cancer detection. This model system is potent for analysis of the contribution of other oncogenes and their combinations towards spectral detection of cancer.     

Raman Spectroscopy Analysis of the Biochemical Characteristics of Molecules Associated with the Malignant Transformation of Gastric Mucosa

Objective

The purpose of this study was to comparatively analyze the signature Raman spectra of genomic DNA, nuclei, and tissue of normal gastric mucosa and gastric cancer and to investigate the biochemical transformation of molecules associated with gastric mucosa malignancy.

Method

Genomic DNA, nuclei, and tissue from normal gastric mucosa and gastric cancer were analyzed by Raman spectroscopy.

Results

1) The Raman spectrum of gastric cancer genomic DNA showed that two peaks appeared, one at approximately 1090 cm^-1 with a higher intensity than the peak at 1050 cm^-1 in the spectrum. Characteristic peaks appeared at 950 cm^-1, 1010 cm^-1, and 1100-1600 cm^-1. 2) Using a hematoxylin and eosin (H&E)-stained section, the intensity of the characteristic peak of nucleic acids at 1085 cm^-1 was increased and shifted to 1088 cm^-1 in cancer cells. The relative intensity of the characteristic peaks of nucleoproteins at 755 cm^-1 and 1607 cm^-1 was significantly increased in cancer cells compared with normal cells. 3) Compared with normal tissues, the peak representing PO^2- symmetric stretching vibration shifted from 1088 cm^-1 to 1083 cm^-1 in cancer tissue, and the characteristic peak for collagen at 938 cm^-1 shifted to 944 cm^-1. In addition, an extra characteristic peak indicating C = C stretching vibration appeared at 1379 cm^-1 in the lipid spectrum in cancer tissue.

Conclusions

The position, intensity, and shape of peaks in the Raman spectra of DNA, nuclei, and tissue from gastric cancer were significantly different compared with those of normal cells. These results indicate that the DNA phosphate backbone becomes unstable in cancer cells and might be broken; the relative content of histones is increased and stable; the relative collagen content is reduced, facilitating cancer cell metastasis; and the relative content of unsaturated fatty acids is increased, increasing the mobility of the plasma membrane of cancer cells.     

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。     

Optimisation of Wavelength Modulated Raman Spectroscopy: Towards High Throughput Cell Screening

In the field of biomedicine, Raman spectroscopy is a powerful technique to discriminate between normal and cancerous cells. However the strong background signal from the sample and the instrumentation affects the efficiency of this discrimination technique. Wavelength Modulated Raman spectroscopy (WMRS) may suppress the background from the Raman spectra. In this study we demonstrate a systematic approach for optimizing the various parameters of WMRS to achieve a reduction in the acquisition time for potential applications such as higher throughput cell screening. The Signal to Noise Ratio (SNR) of the Raman bands depends on the modulation amplitude, time constant and total acquisition time. It was observed that the sampling rate does not influence the signal to noise ratio of the Raman bands if three or more wavelengths are sampled. With these optimised WMRS parameters, we increased the throughput in the binary classification of normal human urothelial cells and bladder cancer cells by reducing the total acquisition time to 6 s which is significantly lower in comparison to previous acquisition times required for the discrimination between similar cell types.     

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。     

Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells

Healthy human males produce sperm cells of which about 25–40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro‐Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA‐packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in‐vitro fertilization. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Micro-Raman spectroscopy detects individual neoplastic and normal hematopoietic cells

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific, slow, biologically perturbing, or a combination thereof. Here, we show that single-cell micro-Raman spectroscopy averts these shortcomings and can be used to discriminate between unfixed normal human lymphocytes and transformed Jurkat and Raji lymphocyte cell lines based on their biomolecular Raman signatures. We demonstrate that single-cell Raman spectra provide a highly reproducible biomolecular fingerprint of each cell type. Characteristic peaks, mostly due to different DNA and protein concentrations, allow for discerning normal lymphocytes from transformed lymphocytes with high confidence (p < 0.05). Spectra are also compared and analyzed by principal component analysis to demonstrate that normal and transformed cells form distinct clusters that can be defined using just two principal components. The method is shown to have a sensitivity of 98.3% for cancer detection, with 97.2% of the cells being correctly classified as belonging to the normal or transformed type. These results demonstrate the potential application of confocal micro-Raman spectroscopy as a clinical tool for single cancer cell detection based on intrinsic biomolecular signatures, therefore eliminating the need for exogenous fluorescent labeling.     

Combining near-infrared-excited autofluorescence and Raman spectroscopy improves in vivo diagnosis of gastric cancer

This study aims to evaluate the diagnostic utility of the combined near-infrared (NIR) autofluorescence (AF) and Raman spectroscopy for improving in vivo detection of gastric cancer at clinical gastroscopy. A rapid Raman endoscopic technique was employed for in vivo spectroscopic measurements of normal (n=1098) and cancer (n=140) gastric tissues from 81 gastric patients. The composite NIR AF and Raman spectra in the range of 800-1800 cm(-1) were analyzed using principal component analysis (PCA) and linear discriminant (LDA) to extract diagnostic information associated with distinctive spectroscopic processes of gastric malignancies. High quality in vivo composite NIR AF and Raman spectra can routinely be acquired from the gastric within 0.5s. The integrated intensity over the range of 800-1800 cm(-1) established the diagnostic implications (p=1.6E-14) of the change of NIR AF intensity associated with neoplastic transformation. PCA-LDA diagnostic modeling on the in vivo tissue NIR AF and Raman spectra acquired yielded a diagnostic accuracy of 92.2% (sensitivity of 97.9% and specificity of 91.5%) for identifying gastric cancer from normal tissue. The integration area under the receiver operating characteristic (ROC) curve using the combined NIR AF and Raman spectroscopy was 0.985, which is superior to either the Raman spectroscopy or NIR AF spectroscopy alone. This work demonstrates that the complementary Raman and NIR AF spectroscopy techniques can be integrated together for improving the in vivo diagnosis and detection of gastric cancer at endoscopy.     

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.     

Quantitative fluorescence spectrophotometry of acridine orange-stained unfixed cells. Potential for automated detection of human uterine cancer.

After staining with acridine orange (AO), the nuclei of unfixed cells from the human female genital tract exhibited the same fluorescence behavior previously observed for human and murine leukocytes and mouse ascites tumor cells. With staining conditions chosen to assure saturation of the green-fluorescing AO-nucleic acid complex in normal cells, corrected fluorescence emission spectra were recorded from the entire nucleus of 341 cells taken from 32 normal and 28 abnormal patients. Intensity of the recorded spectra was expressed in phosphor particle units, a fixed arbitrary unit of fluorescence intensity, to display intensity differences among the spectra from the various cell types. In all abnormal samples, one or more cells were found with 530-nm nuclear fluorescence intensity considerably greater than the maximum intensity recorded from normal cells. Determination of the adequacy of 530-nm nuclear fluorescence intensity as a criterion for cancer detection requires additional investigation. Additional criteria, if needed, may be supplied by the metachromasy of AO-stained unfixed cells.     

Novel dual-reporter preclinical screen for antiastrocytoma agents identifies cytostatic and cytotoxic compounds

Astrocytoma/glioblastoma is the most common malignant form of brain cancer and is often unresponsive to current pharmacological therapies and surgical interventions. Despite several potential therapeutic agents against astrocytoma and glioblastoma, there are currently no effective therapies for astrocytoma, creating a great need for the identification of effective antitumor agents. The authors have developed a novel dual-reporter system in Trp53/Nf1-null astrocytoma cells to simultaneously and rapidly assay cell viability and cell cycle progression as evidenced by activity of the human E2F1 promoter in vitro. The dual-reporter high-throughput assay was used to screen experimental therapeutics for activity in Trp53/Nf1-null astrocytoma. Several compounds were identified demonstrating selectivity for astrocytoma over primary astrocytes. The dual-reporter system described here may be a valuable tool for identifying potential antitumor treatments that specifically target astrocytoma.     

Keratin orientation in wool and feathers by polarized raman spectroscopy

Good quality polarized Raman spectra of a single wool fiber and an intact feather barbule are presented. The intensity ratio of the alpha-helix component of the amide I band measured parallel and perpendicular to the wool fiber axis was 0.39 +/- 0.05. This is consistent with theoretical predictions based on orientational calculations using the normal Raman polarizability tensor for an alpha-helical amide I band where the protein strands are aligned roughly parallel with the fiber axis. However, the depolarized spectral intensity of the alpha-helix mode was greater than expected. For the feather barbule, despite high quality spectra, a unique orientation of the beta-sheet structure could not be determined using the Raman intensity ratios of the amide I band alone. Using previously developed methods, the protein chains were found to be oriented between 60 and 90 degrees from the long axis of the barbule compared to an angle of 51 degrees calculated from polarized IR spectra of the same barbule. The Raman tensor methods for the determination of protein orientation in these fibers was found to be constrained by the complexity of the materials and the limitations of the band fitting methods used to apportion the intensity among the various vibrational modes of their spectra. Other advantages and limitations of polarized Raman microscopic methods of structural determination are discussed.     

Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes.

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.     

对硝基苯硫酚分子内嵌的星形表面增强拉曼散射金 "套娃 "纳米颗粒用于肿瘤拉曼影像的初步研究

目的:制备对硝基苯硫酚(4-Nitrobenzenethiol,4-NBT)分子内嵌的星形表面增强拉曼散射(Surface enhanced Raman Scattering,SERS)金"套娃"纳米颗粒,测定其拉曼增强效果和应用于细胞以及活体肿瘤拉曼影像的可行性。方法:以种子介导法先后制备金纳米星及星形SERS金"套娃"纳米颗粒,采用透射电镜观察其形貌,激光粒度分析仪测定其粒径及Zeta电位,拉曼光谱仪测定其拉曼光谱,考察其对A549细胞的拉曼成像效果,建立A549皮下瘤模型,考察其对活体皮下瘤的成像效果。结果:制备并优化的金纳米星粒径较小,为60.5 nm,其针尖密度较高,以此为核心制备的星形SERS金"套娃"纳米颗粒形态规整,粒径约为66.7nm,Zeta电位约为-16.6 m V,拉曼增强效果提升至其前驱体金纳米星的5.3倍,能够实现对A549细胞及A549皮下瘤的拉曼成像。结论:所制备的星形SERS金"套娃"纳米颗粒形态规整均一,拉曼增强效果较好,能实现对细胞及活体肿瘤的拉曼影像。     

Lipid droplets in prostate cancer cells and effect of irradiation studied by Raman microspectroscopy

Lipid droplets (LDs) are key organelles in cancer cells proliferation, growth, and response to stress. These nanometric structures can aggregate to reach the size of microns becoming important cell components. Although it is known that LDs contain various lipids, their chemical composition is still under investigation. Moreover, their function in cell's response to exogenous factors is also not fully understood. Raman spectroscopy, together with chemometrics, has been shown to be a powerful tool for analytical analyses of cancer cell components on the subcellular level. It provides the opportunity to analyse LDs in a label-free manner in live cells. In the current study, this method was applied to investigate LDs composition in untreated and irradiated with X-ray beams prostate cancer cells. Raman mapping technique proved lipids accumulation in PC-3 cells and allowed visualization of LDs spatial distribution in cytoplasm. A heterogeneous composition of LDs was revealed by detailed analysis of Raman spectra. Interestingly, PC-3 cells were found to accumulate either triacylglycerols or cholesteryl esters. Finally, effect of X-ray radiation on the cells was investigated using Raman spectroscopy and fluorescence staining. Significant influence of LDs in the process of cell response was confirmed and time dependence of this phenomenon was determined.     

Effects of organic solutes on the Raman spectra of barnacle muscle fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.