Raman spectroscopy and imaging: applications in human breast cancer diagnosis

The applications of spectroscopic methods in cancer detection open new possibilities in early stage diagnostics. Raman spectroscopy and Raman imaging represent novel and rapidly developing tools in cancer diagnosis. In the study described in this paper Raman spectroscopy has been employed to examine noncancerous and cancerous human breast tissues of the same patient. The most significant differences between noncancerous and cancerous tissues were found in regions characteristic for the vibrations of carotenoids, lipids and proteins. Particular attention was paid to the role played by unsaturated fatty acids in the differentiation between the noncancerous and the cancerous tissues. Comparison of Raman spectra of the noncancerous and the cancerous tissues with the spectra of oleic, linoleic, α-linolenic, γ-linolenic, docosahexaenoic and eicosapentaenoic acids has been presented. The role of sample preparation in the determination of cancer markers is also discussed in this study.     

Differentiation Between Anti-Epidermal Growth Factor Receptors Antibody Conjugated and Unconjugated Gold Nanoparticles Using Attenuated Total Reflectance–Fourier Transform Infrared Spectroscopy

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) along with UV-Vis was used to distinguish between structural differences between a normal human breast cell line (MCF-12 F) and a cancerous breast cell line (MCF-7) after both being treated with conjugated gold nanoparticles and unconjugated gold nanoparticles. The MCF-7 cell line possessed a small amount of epidermal growth factor receptors (EGFR) on its cell surface whereas the MCF-12 F cell line did not. The anti-EGFR antibodies blocked the binding of epidermal growth factors to this receptor which plays an important role in the regulation of cell growth, proliferation, and differentiation. The infrared (IR) spectra for both cell lines yield several differences between normal biological samples and cancerous samples. As the malignancy of the sample increases, the peak intensity and wavenumber positions decrease. The same conclusion was drawn from thin-layers of conjugated nanoparticles and nonconjugated nanoparticles. The presence of conjugated nanoparticles increased the peak intensity of the MCF-7 cell line whereas it decreased the peak intensity for the MCF-12 F cell line. UV-Vis was used to show the presence of the anti-EGFR antibodies on the surface of the gold nanoparticles which was 5 nm red shifted compared to the gold nanoparticles solution.     

High-wavenumber FT-Raman spectroscopy for in vivo and ex vivo measurements of breast cancer

The identification of normal and cancer breast tissue of rats was investigated using high-frequency (HF) FT-Raman spectroscopy with a near-infrared excitation source on in vivo and ex vivo measurements. Significant differences in the Raman intensities of prominent Raman bands of lipids and proteins structures (2,800?C3,100?cm^?1) as well as in the broad band of water (3,100?C3,550?cm^?1) were observed in mean normal and cancer tissue spectra. The multivariate statistical analysis methods of principal components analysis (PCA) and linear discriminant analysis (LDA) were performed on all high-frequency Raman spectra of normal and cancer tissues. LDA results with the leave-one-out cross-validation option yielded a discrimination accuracy of 77.2, 83.3, and 100% for in vivo transcutaneous, in vivo skin-removed, and ex vivo biopsy HF Raman spectra. Despite the lower discrimination value for the in vivo transcutaneous measurements, which could be explained by the breathing movement and skin influences, our results showed good accuracy in discriminating between normal and cancer breast tissue samples. To support this, the calculated integration areas from the receiver-operating characteristic (ROC) curve yielded 0.86, 0.94, and 1.0 for in vivo transcutaneous, in vivo skin-removed, and ex vivo biopsy measurements, respectively. The feasibility of using HF Raman spectroscopy as a clinical diagnostic tool for breast cancer detection and monitoring is due to no interfering contribution from the optical fiber in the HF Raman region, the shorter acquisition time due to a more intense signal in the HF Raman region, and the ability to distinguish between normal and cancerous tissues.     

Raman Spectroscopy and Fluorescence Photon Migration for Breast Cancer Diagnosis and Imaging

We are developing optical methods based on near infra-red Raman spectroscopy and fluorescence photon migration for diagnosis and localization of breast cancer. We demonstrate the ability of Raman spectroscopy to classify accurately normal, benign and malignant breast tissues, an important step in developing Raman spectroscopic needle probes as a tool for improving the accuracy of needle biopsy. We also show that photon migration imaging can be used to localize accurately small fluorescent objects imbedded in a thick turbid medium with realistic optical properties, thus demonstrating the potential of this technique for optical imaging.     

傅里叶变换红外光谱法用于体表无创性检测乳腺肿瘤的研究

乳腺肿瘤是女性常见的疾病之一,其中乳腺癌的发病率在女性恶性肿瘤中占首位,并且发病率呈不断增加的趋势,严重危害妇女的身体健康．乳腺癌的早期诊断和治疗是提高乳腺癌患者整体生存率的关键．目前大多采用传统的X线、超声、CT和核磁共振等技术进行影像诊断,这些诊断方法需要等到肿块形成具有占位效应时才能检测出来．而振动光谱法是分子结构变化的灵敏探针,它在癌症形成的初级阶段即可观察到分子结构的改变,因此傅里叶变换中红外光谱技术有可能发展成为一种无损伤、快速和方便的肿瘤早期诊断方法．     

The auxiliary determination of the binding site of berberine binding to human serum albumin by surface-enhanced Raman scattering

We report on the joint application of fluorescence, ultraviolet-visible (UV-Vis) and Raman spectroscopy to the study of berberine with human serum albumin (HSA). We propose the surface-enhanced Raman scattering (SERS) technique to improve the understanding of the quenching interaction caused by berberine which could be applied in recognition process of fluorescent drugs with large biomolecules. The fluorescence and UV-Vis spectroscopic results show that the fluorescence intensity of HSA is significantly decreased in the presence of berberine, and the quenching mechanism is static. The SERS technique demonstrates clear advantages over direct measurements in physiological conditions. By means of this method, we are able to deduce important information concerning the binding property of berberine when interacting with HSA. We show the nitrogen atom is free but the dioxolane is involved in the spontaneously electrostatic inducement and subsequently hydrophobic binding.     

Raman Imaging: An Impending Approach Towards Cancer Diagnosis

In accordance with the recent studies, Raman spectroscopy is well experimented as a highly sensitive analytical and imaging technique in biomedical research, mainly for various disease diagnosis including cancer. In comparison with other imaging modalities, Raman spectroscopy facilitate numerous assistances owing to its low background signal, immense spatial resolution, high chemical specificity, multiplexing capability, excellent photo stability and non-invasive detection capability. In cancer diagnosis Raman imaging intervened as a promising investigative tool to provide molecular level information to differentiate the cancerous vs non-cancerous cells, tissues and even in body fluids. Anciently, spontaneous Raman scattering is very feeble due to its low signal intensity and long acquisition time but new advanced techniques like coherent Raman scattering (CRS) and surface enhanced Raman scattering (SERS) gradually superseded these issues. So, the present review focuses on the recent developments and applications of Raman spectroscopy-based imaging techniques for cancer diagnosis.     

Depth profiling of calcifications in breast tissue using picosecond Kerr-gated Raman spectroscopy

Breast calcifications are found in both benign and malignant lesions and their composition can indicate the disease state. Calcium oxalate (dihydrate) (COD) is associated with benign lesions, however calcium hydroxyapatite (HAP) is found mainly in proliferative lesions including carcinoma. The diagnostic practices of mammography and histopathology examine the morphology of the specimen. They can not reliably distinguish between the two types of calcification, which may indicate the presence of a cancerous lesion during mammography. We demonstrate for the first time that Kerr-gated Raman spectroscopy is capable of non-destructive probing of sufficient biochemical information from calcifications buried within tissue, and this information can potentially be used as a first step in identifying the type of lesion. The method uses a picosecond pulsed laser combined with fast temporal gating of Raman scattered light to enable spectra to be collected from a specific depth within scattering media by collecting signals emerging from the sample at a given time delay following the laser pulse. Spectra characteristic of both HAP and COD were obtained at depths of up to 0.96 mm, in both chicken breast and fatty tissue; and normal and cancerous human breast by utilising different time delays. This presents great potential for the use of Raman spectroscopy as an adjunct to mammography in the early diagnosis of breast cancer.     

Autofluorescence spectroscopy of normal and malignant human breast cell lines

The fluorescence of tryptophan, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized in normal human breast cells as well as in malignant human breast cells of similar and dissimilar genetic origins. Fluorescence measurements of each cell line were made over a wide range of cell concentrations, and the fluorescence per cell was determined from the slope in the linear range of the fluorescence intensity vs cell concentration plot. All of the malignant cells showed a statistically significant decrease in the tryptophan fluorescence per cell relative to that of the normal cells. No statistically significant differences were observed in the NAD(P)H or FAD fluorescence per cell between the normal and any of the malignant cell types. NAD(P)H fluorescence was also imaged from monolayers of the normal and malignant cells (of similar genetic origin) using two-photon fluorescence microscopy. A statistically significant decrease in the NAD(P)H fluorescence with malignancy was observed, suggesting that fluorescence imaging of single cells or the cell monolayer preparation may provide more contrast than volume-averaged fluorescence measurements of cells in suspension. In conclusion, the differences in normal and malignant human breast tissue fluorescence spectra may be attributed in part to differences in the intrinsic cellular fluorescence of normal and malignant breast epithelial cells.     

SERS in Salt Wells

We report herein a simple, inexpensive fabrication methodology of salt microwells, and define the utility of the latter as nanoparticle containers for highly sensitive surface‐enhanced Raman scattering (SERS) studies. AFM characterization of Ag and Au loaded salt microwells reveal the ability to contain favorable nanostructures such as nanoparticle dimers, which can significantly enhance the Raman intensity of molecules. By performing diffraction‐limited confocal Raman microscopy on salt microwells, we show high sensitivity and fidelity in the detection of dyes, peptides, and proteins, as a proof of our concept. The SERS limit of detection (accumulation time of 1 s) for rhodamine B and TAT contained in salt mircowells is 10 pM and 1 nM , respectively. The Raman characterization measurements of salt microwells with three different laser lines (532 nm, 632.81 nm, 785 nm) reveal low background intensity and high signal‐to‐noise ratio upon nanoparticle loading, which makes them suitable for enhanced Raman detection. SERS mapping of these sub‐femtoliter containers show spatial confinement of the relevant analyte to a few microns, which make them potential candidates for microscale bioreactors.     

Growth substrate induced functional changes elucidated by FTIR and Raman spectroscopy in in–vitro cultured human keratinocytes

Non-invasive measurements of cellular function in in vitro cultured cell lines using vibrational spectroscopy require the use of spectroscopic substrates such as quartz, ZnSe and MirrIR etc. These substrates are generally dissimilar to the original in vivo extracellular environment of a given cell line and are often tolerated poorly by cultured cell lines resulting in morphological and functional changes in the cell. The present study demonstrates various correlations between vibrational spectroscopic analyses and biochemical analyses in the evaluation of the interaction of a normal human epithelial keratinocyte cell line (HaCaT) with MirrIR and quartz substrates coated with fibronectin, laminin and gelatin. The findings of this study suggest that there is a correlation between quantitative measurements of cellular proliferative capacity and viability and peak area ratios in FTIR spectra, with replicated differences in similar areas of the observed Raman spectra. Differences in the physiology of cells were observed between the two spectroscopic substrates coated in fibronectin and laminin, but little differences were observed when the cells were attached to gelatin-coated quartz and MirrIR slides. The correlations demonstrate the sensitivity of the spectroscopic techniques to evaluate the physiology of the system. Furthermore the study suggests that gelatin is a suitable coating for use in spectroscopic measurements of cellular function in human keratinocytes, as it provides a material that normalises the effect of substrate attachment on cellular physiology. This effect is likely to be cell-line dependent, and it is recommended that similar evaluations of this effect are performed for those combinations of spectroscopic substrate and cell lines that are to be used in individual experiments.     

In and ex vivo breast disease study by Raman spectroscopy

In this work, Raman spectra in the 900?C1,800?cm^?1 wavenumber region of in vivo and ex vivo breast tissues of both healthy mice (normal) and mice with induced mammary gland tumors (abnormal) were measured. In the case of the in vivo tissues, the Raman spectra were collected for both transcutaneous (with skin) and skin-removed tissues. To identify the spectral differences between normal and cancer breast tissue, the paired t-test was carried out for each wavenumber using the whole spectral range from both groups. Quadratic discriminate analysis based on principal component analysis (PCA) was also used to determine and evaluate differences in the Raman spectra for the various samples as a basis for diagnostic purposes. The differences in the Raman spectra of the samples were due to biochemical changes at the molecular, cellular and tissue levels. The sensitivity and specificity of the classification scheme based on the differences in the Raman spectra obtained by PCA were evaluated using the receiver operating characteristic (ROC) curve. The in vivo transcutaneous normal and abnormal tissues were correctly classified based on their measured Raman spectra with a discriminant proportion of 73%, while the in vivo skin-removed normal and abnormal tissues were correctly classified again based on their measured Raman spectra with a discriminant proportion of 86%. This result reveals a strong influence due to the skin of the breast, which decreased the specificity by 11%. Finally, the results from ex vivo measurements gave the highest specificity and sensitivity: 96 and 97%, respectively, as well as a largest percentage for correct discrimination: 94%. Now that the important bands have been experimentally determined in this and other works, what remains is for first principles molecular-level simulations to determine whether the changes are simply due to conformational changes, due to aggregation, due to changes in the environment, or complex interactions of all of the above.     

Nonphotochemical hole-burning study of selectively stained normal and cancerous human ovarian tissues

Results are presented of nonphotochemical hole-burning (HB) experiments on cancerous ovarian and analogous normal peritoneal in vitro tissues stained with the mitochondrial-selective dye rhodamine 800. A comparison of fluorescence excitation spectra, hole-growth kinetics data, and external electric field (Stark) effects on the shape of spectral holes burned in cancerous and normal tissues stained with rhodamine 800 revealed significant differences only in the dipole moment change (fDeltamu) measured by a combination of HB and Stark spectroscopies. It is shown that the permanent dipole moment change for the S0--> S1 transition of the rhodamine 800 molecules in cancerous tissue is higher than that of normal tissue by a factor of about 1.4. The finding is similar to the HB results obtained earlier for human ovarian surface epithelial cell lines, i.e., OV167 carcinoma and OSE(tsT)-14 normal cells stained with the same mitochondria-specific dye (Walsh et al. Biophys. J. 2003, 84, 1299). We propose that the observed difference in the permanent dipole moment change in cancerous ovarian tissue is related to a modification in mitochondrial membrane potential.     

中红外光纤技术用于口腔肿瘤在体原位检测的研究

肿瘤是严重威胁人类健康和生命的疾病,早期诊断和及时治疗是提高肿瘤病人存活率的重要因素,肿瘤的发生和发展一般可分为3个阶段：(1)基因突变;(2)生物分子组成和结构发生改变;(3)细胞和组织形态发生变化,目前常用的影像学方法只能检测较大的肿块,组织标本的病理诊断法需在     

The orientation of protoberberine alkaloids and their binding activities to human serum albumin by surface-enhanced Raman scattering

Raman and surface-enhanced Raman scattering (SERS) technique are reliably used to compare relative intensity shifts and to investigate the adsorption geometry of protoberberine alkaloids on Ag nanoparticles. We report joint application of fluorescence and SERS spectroscopy to study the interaction between protoberberine alkaloids and human serum albumin (HSA). We propose SERS technique to improve the quenching interaction caused by protoberberine alkaloids which are used to be applied in recognition process of fluorescent drugs with large biomolecules. The fluorescence results show that the fluorescence intensity of HSA is significantly decreased in presence of protoberberine alkaloids. The SERS technique demonstrates obvious advantages over direct measurements in discriminating and identifying pharmaceutical molecules. By means of this method, we are able to detect important information concerning the orientation of protoberberine alkaloids when interacting with HSA. We also show that the nitrogen atom is free, but a benzene ring and two adjacent methoxy groups are involved in the spontaneously electrostatic inducement and subsequently binding with HSA.     

水溶液中碳纳米管的表面增强拉曼光谱研究

合成了碳纳米管和金纳米颗粒的复合物, 测量了水溶液相中复合物的表面增强拉曼光谱, 结果表明, 碳纳米管的巯基化修饰可以提高碳纳米管与金纳米颗粒复合的效率, 随着金纳米颗粒负载量的增加, 碳纳米管的拉曼信号逐渐增强. 加入己二胺分子可以减小金纳米颗粒之间的距离使表面增强效应更显著, 碳纳米管的拉曼光谱得到进一步的增强. 还可进一步在复合体系中加入对巯基苯胺和罗丹明B等小分子拉曼探针, 利用金纳米颗粒的表面增强效应, 这种多元复合体系有望作为多通道拉曼成像探针材料.     

Classification of normal and malignant human gastric mucosa tissue with confocal Raman microspectroscopy and wavelet analysis

Thirty-two samples from the human gastric mucosa tissue, including 13 normal and 19 malignant tissue samples were measured by confocal Raman microspectroscopy. The low signal-to-background ratio spectra from human gastric mucosa tissues were obtained by this technique without any sample preparation. Raman spectral interferences include a broad featureless sloping background due to fluorescence and noise. They mask most Raman spectral feature and lead to problems with precision and quantitation of the original spectral information. A preprocessed algorithm based on wavelet analysis was used to reduce noise and eliminate background/baseline of Raman spectra. Comparing preprocessed spectra of malignant gastric mucosa tissues with those of counterpart normal ones, there were obvious spectral changes, including intensity increase at approximately 1156cm(-1) and intensity decrease at approximately 1587cm(-1). The quantitative criterion based upon the intensity ratio of the approximately 1156 and approximately 1587cm(-1) was extracted for classification of the normal and malignant gastric mucosa tissue samples. This could result in a new diagnostic method, which would assist the early diagnosis of gastric cancer.     

Near infrared Raman spectroscopic mapping of native brain tissue and intracranial tumors

This study assessed the diagnostic potential of Raman spectroscopic mapping by evaluating its ability to distinguish between normal brain tissue and the human intracranial tumors gliomas and meningeomas. Seven Raman maps of native specimens were collected ex vivo by a Raman spectrometer with 785 nm excitation coupled to a microscope with a motorized stage. Variations within each Raman map were analyzed by cluster analysis. The dependence of tissue composition on the tissue type in cluster averaged Raman spectra was shown by linear combinations of reference spectra. Normal brain tissue was found to contain higher levels of lipids, intracranial tumors have more hemoglobin and lower lipid to protein ratios, meningeomas contain more collagen with maximum collagen content in normal meninges. One sample was studied without freezing. Whereas tumor regions did not change significantly, spectral changes were observed in the hemoglobin component after snap freezing and thawing to room temperature. The results constitute a basis for subsequent Raman studies to develop classification models for diagnosis of brain tissue.     

Multistep energy transfer in single molecular photonic wires

We demonstrate the synthesis and spectroscopic characterization of an unidirectional photonic wire based on four highly efficient fluorescence energy-transfer steps (FRET) between five spectrally different chromophores covalently attached to double-stranded DNA. The DNA-based modular conception enables the introduction of various chromophores at well-defined positions and arbitrary interchromophore distances. While ensemble fluorescence measurements show overall FRET efficiencies between 15 and 30%, single-molecule spectroscopy performed on four spectrally separated detectors easily uncovers subpopulations that exhibit overall FRET efficiencies of up to approximately 90% across a distance of 13.6 nm and a spectral range of approximately 200 nm. Fluorescence trajectories of individual photonic wires show five different fluorescence intensity patterns which can be ascribed to successive photobleaching events.     

Non-destructive Phenotyping of Chili Pepper Ripening Using Spectroscopic Probes: A Potential Approach for Shelf-Life Measurement

This study explores the application of spectroscopic techniques (laser induced fluorescence, Raman and attenuated total reflectance Fourier transform infrared) coupled with principal component analysis for the nondestructive, extraction free and rapid evaluation of biochemical changes associated with ripening of chili peppers at four stages (mature, pre-ripe, ripe and post-ripe). The analysis of the fluorescence spectra of the exocarp of chili pepper shows a decrease in the intensity of chlorophyll bands at 685 and 735?nm and an increase in the intensity of carotenoid fluorescence bands at 490–500?nm and 565–580?nm with progress in the ripening stages. These changes are regarded to be significant phenotypic markers for the ripening of chili peppers. The observed changes in the position of carotenoid bands in Raman spectra at 1004, 1156, 1188, and 1524?cm^?1 with increase in their intensity indicating the accumulation of carotenoids and change in the carotenoid composition from β-carotene in the mature chilis to capsanthin in the ripe chilis. In addition, the infrared spectra show changes in the carbohydrates, amide II, amide I and cutin at various stages of ripening. Also, the variation in the position of pectin bands indicates change in its molecular mass with decreasing content. The determined spectral signatures can be used as biomolecular index for effective monitoring of the ripening of chili peppers. The commercial application of noninvasive spectroscopic probes will be advantageous for the phenotyping of economically important plant parts, screening, grading, shelf life estimation and quality standardization.