Imaging of Oral SCC Cells by Raman Micro-Spectroscopy Technique

We used Raman micro-spectroscopy technique to analyze the molecular changes associated with oral squamous cell carcinoma (SCC) cells in the form of frozen tissue. Previously, Raman micro-spectroscopy technique on human tissue was mainly based on spectral analysis, but we worked on imaging of molecular structure. In this study, we evaluated the distribution of four components at the cell level (about 10 μm) to describe the changes in protein and molecular structures of protein belonging to malignant tissue. We analyzed ten oral SCC samples of five patients without special pretreatments of the use of formaldehyde. We obtained cell level images of the oral SCC cells at various components (peak at 935 cm⁻¹: proline and valine, 1004 cm⁻¹: phenylalanine, 1223 cm⁻¹: nucleic acids, and 1650 cm⁻¹: amide I). These mapping images of SCC cells showed the distribution of nucleic acids in the nuclear areas; meanwhile, proline and valine, phenylalanine, and amide I were detected in the cytoplasm areas of the SCC cells. Furthermore, the peak of amide I in the cancer area shifts to the higher wavenumber side, which indicates the α-helix component may decrease in its relative amounts of protein in the β-sheet or random coil conformation. Imaging of SCC cells with Raman micro-spectroscopy technique indicated that such a new observation of cancer cells is useful for analyzing the detailed distribution of various molecular conformation within SCC cells.     

Raman spectroscopic imaging for in vivo detection of cerebral brain metastases

We report for the first time a proof-of-concept experiment employing Raman spectroscopy to detect intracerebral tumors in vivo by brain surface mapping. Raman spectroscopy is a non-destructive biophotonic method which probes molecular vibrations. It provides a specific fingerprint of the biochemical composition and structure of tissue without using any labels. Here, the Raman system was coupled to a fiber-optic probe. Metastatic brain tumors were induced by injection of murine melanoma cells into the carotid artery of mice, which led to subcortical and cortical tumor growth within 14 days. Before data acquisition, the cortex was exposed by creating a bony window covered by a calcium fluoride window. Spectral contributions were assigned to proteins, lipids, blood, water, bone, and melanin. Based on the spectral information, Raman images enabled the localization of cortical and subcortical tumor cell aggregates with accuracy of roughly 250 μm. This study demonstrates the prospects of Raman spectroscopy as an intravital tool to detect cerebral pathologies and opens the field for biophotonic imaging of the living brain. Future investigations aim to reduce the exposure time from minutes to seconds and improve the lateral resolution.     

Unsupervised unmixing of Raman microspectroscopic images for morphochemical analysis of non-dried brain tumor specimens

Raman microspectroscopic imaging provides molecular contrast in a label-free manner with subcellular spatial resolution. These properties might complement clinical tools for diagnosis of tissue and cells in the future. Eight Raman spectroscopic images were collected with 785 nm excitation from five non-dried brain specimens immersed in aqueous buffer. The specimens were assigned to molecular and granular layers of cerebellum, cerebrum with and without scattered tumor cells of astrocytoma WHO grade III, ependymoma WHO grade II, astrocytoma WHO grade III, and glioblastoma multiforme WHO grade IV with subnecrotic and necrotic regions. In contrast with dried tissue section, these samples were not affected by drying effects such as crystallization of lipids or denaturation of proteins and nucleic acids. The combined data sets were processed by use of the hyperspectral unmixing algorithms N-FINDR and VCA. Both unsupervised approaches calculated seven endmembers that reveal the abundance plots and spectral signatures of cholesterol, cholesterol ester, nucleic acids, carotene, proteins, lipids, and buffer. The endmembers were correlated with Raman spectra of reference materials. The focus of the single mode laser near 1 μm and the step size of 2 μm were sufficiently small to resolve morphological details, for example cholesterol ester islets and cell nuclei. The results are compared for both unmixing algorithms and with previously reported supervised spectral decomposition techniques.     

Comparison of subcellular responses for the evaluation and prediction of the chemotherapeutic response to cisplatin in lung adenocarcinoma using Raman spectroscopy

Confocal Raman Micro-spectroscopy (CRM) is employed to examine the chemical and physiological effects of anticancer agents, using cisplatin and A549 adenocarcinoma cells as a model compound and test system respectively. Spectral responses of the membrane and cytoplasm of the cell are analysed independently and the results are compared to previously reported spectroscopic studies of the nucleus. Moreover, Raman spectra from the proteins extracted from the control and exposed samples are acquired and analysed to confirm the origin of the molecular changes of the cell membrane and cytoplasm of the A549 cells. Multivariate data analysis techniques including Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR) along with PLS-Jackknifing are used to analyse the data measured from the cell membrane and cytoplasm of the A549 cells and results are correlated with parallel measurements from the cytotoxicity assay MTT. A PLSR model is used to differentiate between the chemical effect of the chemotherapeutic agent and the physiological response of the A549 cells and to identify regions of the spectrum that are associated with these processes respectively. The PLSR model is also employed to predict, on the basis of the Raman spectra, the effective dose as well as the level of physiological response, using spectra data from the cytoplasmic and cell membrane regions. The effectiveness of the models based on spectral datasets from the cell membrane and cytoplasm is compared to similar models constructed using spectral data from the nuclear region as well as one combining spectral data from all regions. In all cases, higher prediction accuracy is found for regression against the cisplatin dose, and for both regression against the dose and the physiological response, nuclear data yield higher precision.     

Expanding the Range of Bioorthogonal Tags for Multiplex Stimulated Raman Scattering Microscopy

Multiplex optical detection in live cells is challenging due to overlapping signals and poor signal-to-noise associated with some chemical reporters. To address this, the application of spectral phasor analysis to stimulated Raman scattering (SRS) microscopy for unmixing three bioorthogonal Raman probes within cells is reported. Triplex detection of a metallacarborane using the B−H stretch at 2480–2650 cm⁻¹, together with a bis-alkyne and deuterated fatty acid can be achieved within the cell-silent region of the Raman spectrum. When coupled to imaging in the high-wavenumber region of the cellular Raman spectrum, nine discrete regions of interest can be spectrally unmixed from the hyperspectral SRS dataset, demonstrating a new capability in the toolkit of multiplexed Raman imaging of live cells.     

Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy

The molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures.

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

Three dimensional collagen gels have been used as matrices for the imaging of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. The lateral resolution of the system has been estimated to be <1.5 μm making it possible to access the subcellular organization. Using K-means clustering analysis, it is shown that the different subcellular compartments of individual cells can be identified and differentiated. The biochemical specificity of the information contained in the Raman spectra allows the visualization of differences in the molecular signature of the different sub-cellular structures. Furthermore, to enhance the chemical information obtained from the spectra, principal component analysis has been employed, allowing the identification of spectral windows with a high variability. The comparison between the loadings calculated and spectra from pure biochemical compounds enables the correlation of the variations observed with the molecular content of the different cellular compartments.     

Stimulated Raman scattering microscopy with spectral phasor analysis: applications in assessing drug–cell interactions

Statins have displayed significant, although heterogeneous, anti-tumour activity in breast cancer disease progression and recurrence. They offer promise as a class of drugs, normally used for cardiovascular disease control, that could have a significant impact on the treatment of cancer. Understanding their mode of action and accurately assessing their efficacy on live cancer cells is an important and significant challenge. Stimulated Raman scattering (SRS) microscopy is a powerful, label-free imaging technique that can rapidly characterise the biochemical responses of live cell populations following drug treatment. Here, we demonstrate multi-wavelength SRS imaging together with spectral phasor analysis to characterise a panel of breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231 cells) treated with two clinically relevant statins, atorvastatin and rosuvastatin. Label-free SRS imaging within the high wavenumber region of the Raman spectrum (2800–3050 cm⁻¹) revealed the lipid droplet distribution throughout populations of live breast cancer cells using biocompatible imaging conditions. A spectral phasor analysis of the hyperspectral dataset enables rapid differentiation of discrete cellular compartments based on their intrinsic SRS characteristics. Applying the spectral phasor method to studying statin treated cells identified a lipid accumulating phenotype in cell populations which displayed the lowest sensitivity to statin treatment, whilst a weaker lipid accumulating phenotype was associated with a potent reduction in cell viability. This study provides an insight into potential resistance mechanisms of specific cancer cells towards treatment with statins. Label-free SRS imaging provides a novel and innovative technique for phenotypic assessment of drug-induced effects across different cellular populations and enables effective analysis of drug–cell interactions at the subcellular scale.

Stimulated Raman scattering microscopy with spectral phasor analysis provides a label-free approach for phenotypic evaluation of drug-induced effects.     

Raman spectral imaging of single cancer cells: probing the impact of sample fixation methods

Raman spectroscopy has proven its potential for the analysis of cell constituents and processes. However, sample preparation methods compatible with clinical practice must be implemented for collection of accurate spectral information. This study aims at assessing, using micro-Raman imaging, the effects of some routinely used fixation methods such as formalin-fixation, formalin-fixation/air drying, cytocentrifugation, and air drying on intracellular spectral information. Data were compared with those acquired from single living cells. In parallel to these spectral information, cell morphological modifications that accompany sample preparation were compared. Spectral images of isolated cells were first analyzed in an unsupervised way using hierarchical cluster analysis (HCA), which allowed delimitation of the cellular compartments. The resulting nuclei cluster centers were compared and revealed at the molecular level that fixation induced changes in spectral information assigned to nucleic acids and proteins. In a second approach, a supervised fitting procedure using model spectra of DNA, RNA, and proteins, chemically extracted from living cells, revealed very small modifications at the level of the localization and quantification of these macromolecules. Finally, HCA and principal components analysis (PCA) performed on individual spectra randomly selected from the nuclear regions showed that formalin-fixation and cytocentrifugation are sample preparation methods that have little impact on the biochemical information as compared to living conditions. Any step involving cell air drying seems to accentuate the spectral deviations from the other preparation methods. It is therefore important in a future context of spectral cytology to take into account these variations.     

Live‐Cell Stimulated Raman Scattering Imaging of Alkyne‐Tagged Biomolecules

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman‐silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label‐free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state‐of‐the‐art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non‐invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live‐cell imaging.     

Mechanical Trap Surface‐Enhanced Raman Spectroscopy for Three‐Dimensional Surface Molecular Imaging of Single Live Cells

Reported is a new shell‐based spectroscopic platform, named mechanical trap surface‐enhanced Raman spectroscopy (MTSERS), for simultaneous capture, profiling, and 3D microscopic mapping of the intrinsic molecular signatures on the membrane of single live cells. By leveraging the functionalization of the inner surfaces of the MTs with plasmonic gold nanostars, and conformal contact of the cell membrane, MTSERS permits excellent signal enhancement, reliably detects molecular signatures, and allows non‐perturbative, multiplex 3D surface imaging of analytes, such as lipids and proteins on the surface of single cells. The demonstrated ability underscores the potential of MTSERS to perform 3D spectroscopic microimaging and to furnish biologically interpretable, quantitative, and dynamic molecular maps in live cell populations.     

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

The in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy’s fixative and methanol–acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level.     

Crisp and soft multivariate methods visualize individual cell nuclei in Raman images of liver tissue sections

Raman and infrared spectroscopy have been recognized to be promising tools in clinical diagnostics because they provide molecular contrast without external stains. Here, vertex component analysis (VCA) was applied to Raman and Fourier transform infrared (FTIR) images of liver tissue sections and the results were compared with K-means cluster analysis, fuzzy C-means cluster analysis and principal component analysis. The main components of VCA from three Raman images were assigned to the central vein, periportal vein, cell nuclei, liver parenchyma and bile duct. After resonant Mie scattering correction, VCA of FTIR images identified veins, liver parenchyma, cracks, but no cell nuclei. The advantages of VCA in the context of tissue characterization by vibrational spectroscopic imaging are that the tissue architecture is visualized and the spectral information is reconstructed. Composite images were constructed that revealed a high molecular contrast and that can be interpreted in a similar way like hematoxylin and eosin stained tissue sections.     

Methodology for fiber-optic Raman mapping and FTIR imaging of metastases in mouse brains

The objectives of this study were to optimize the preparation of pristine brain tissue to obtain reference information, to optimize the conditions for introducing a fiber-optic probe to acquire Raman maps, and to transfer previous results obtained from human brain tumors to an animal model. Brain metastases of malignant melanomas were induced by injecting tumor cells into the carotid artery of mice. The procedure mimicked hematogenous tumor spread in one brain hemisphere while the other hemisphere remained tumor free. Three series of sections were prepared consecutively from whole mouse brains: dried, thin sections for FTIR imaging, hematoxylin and eosin-stained thin sections for histopathological assessment, and pristine, 2-mm thick sections for Raman mapping. FTIR images were recorded using a spectrometer with a multi-channel detector. Raman maps were collected serially using a spectrometer coupled to a fiber-optic probe. The FTIR images and the Raman maps were segmented by cluster analysis. The color-coded cluster memberships coincided well with the morphology of mouse brains in stained tissue sections. More details in less time were resolved in FTIR images with a nominal resolution of 25 microm than in Raman maps collected with a laser focus 60 microm in diameter. The spectral contributions of melanin in tumor cells were resonance enhanced in Raman spectra on excitation at 785 nm which enabled their sensitive detection in Raman maps. Possible reasons why metastatic cells of malignant melanomas were not identified in FTIR images are discussed.     

Nanoscale Chemical Imaging of Interfacial Monolayers by Tip‐Enhanced Raman Spectroscopy

We report an investigation of interfacial fluorinated hydrocarbon (carboxylic‐fantrip) monolayers by nanoscale imaging using tip‐enhanced Raman spectroscopy (TERS) and density functional theory (DFT) calculations. By comparing TERS images of a sub‐monolayer prepared by spin‐coating and a π–π‐stacked monolayer on Au(111) in which the molecular orientation is confined, specific Raman peaks shift and line widths narrow in the transferred LB monolayer. Based on DFT calculations that take into account dispersion corrections and surface selection rules, these specific effects are proposed to originate from π–π stacking and molecular orientation restriction. TERS shows the possibility to distinguish between a random and locked orientation with a spatial resolution of less than 10 nm. This work combines experimental TERS imaging with theoretical DFT calculations and opens up the possibility of studying molecular orientations and intermolecular interaction at the nanoscale and molecular level.     

Seeing what was unseen: new analytical methods for molecular imaging

For nondestructive analysis of chemical processes in living cells, we developed novel intracellular fluorescent indicators for second messengers, protein phosphorylation, and protein/protein interactions that work in single living cells. Key molecules and steps of cellular signaling pathways were visualized under a confocal laser microscope in target live cells using developed fluorescent indicators. A second new approach to molecular imaging is also described. When chemically modified tips were used for STM measurements, contrast enhancements at specific regions in the STM images occurred on the basis of hydrogen bond and metal-coordination interactions. This enabled us to detect not only the distribution of specific chemical species and functional groups but also the orientation of functional groups. The contrast enhancements reflect the increase in a tunneling current due to the overlap of electronic wave functions induced by the chemical interactions between tip and sample.     

Distinctive Molecular Abnormalities in Benign and Malignant Skin Lesions: Studies by Raman Spectroscopy

Abstract— Near-infrared Fourier transform Raman spectroscopy is an analytical, nondestructive technique that provides information about the molecular structure of the investigated sample. The molecular structure of proteins and lipids differs between neoplastic and normal tissues and therefore Raman spectroscopy has been considered promising for the diagnosis of cancer. We aimed to compare the molecular structure of normal skin, benign and malignant skin lesions by the near-infrared Fourier transform Raman spectroscopy. Biopsies were obtained from the following skin lesions: skin tag, dermatofibroma, seborrhoeic keratosis, actinic keratosis, keratoacan-thoma, basal cell carcinoma, squamous cell carcinoma, nevus intradermal, nevus compositus, dysplastic nevus and lentigo maligna. Control skin was harvested from the vicinity of these lesions. In the Raman spectra, the secondary structure of the proteins was reflected by the amide vibrations of peptide bonds. The principal lipid vibrations were twisting and wagging (CH₂) and CH stretching vibrations. Histologically distinguishable lesions showed specific combinations of band changes indicating alterations in the protein conformation and in the molecular structure of the lipids. Histogenetically related lesions (actinic keratosis and sqamous cell carcinoma) produced similar but not identical patterns of spectral changes. Because the examined skin lesions produced reproducible and unique spectra, we suggest that Raman spectroscopy will be useful for diagnosis of skin lesions.     

Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using Orthogonal Fluorescent Sensors

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid‐binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems‐level investigation of lipid‐mediated cell signaling and regulation.     

基于拉曼光谱成像的食品中化学添加剂的无损检测

基于拉曼光谱成像技术对小麦粉中过氧化苯甲酰和L-抗坏血酸进行快速、 无损、 原位检测, 并对2种添加剂的空间分布进行了可视化研究. 采用实验室自行搭建的线扫描式拉曼光谱成像系统, 激发光源波长为785 nm, 有效光谱范围为0~2885.7 cm^-1. 分别在小麦粉中添加含量为0.1%~30%的过氧化苯甲酰和L-抗坏血酸, 对制备的样品进行拉曼光谱扫描, 选取感兴趣区域的光谱信号进行平均, 得到平均光谱代表该样品的拉曼信息. 分别选取过氧化苯甲酰和L-抗坏血酸的2个特征峰, 与该物质在小麦粉中的含量建立线性关系, 其决定系数R²分别为0.9828 和0.9912. 采集的特征波段拉曼图像经过自适应迭代重加权惩罚最小二乘(airPLS)方法扣除荧光背景后, 选取合适的特征峰强度作为阈值, 对校正拉曼图像进行二值化分析, 得到添加物的空间分布可视化图像. 该方法与点检测拉曼技术相比, 具有检测结果准确且检测时间较短的优势, 且可以实现不均匀样品中多种物质的同时检测与分布可视化.     

Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins.

Detection of F?rster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater F?rster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.