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.     

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but new advanced techniques like coherent Raman scattering (CRS) and surface enhanced Raman scattering (SERS) gradually superseded these issues.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.     

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 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.     

Raman, fluorescence, and time-resolved light scattering as optical diagnostic techniques to separate diseased and normal biomedical media.

Studies of Raman scattering, fluorescence and time-resolved light scattering were conducted on cancer and normal biomedical media. Fourier transform Raman spectroscopic measurements were performed on human normal, benign and cancerous tissues from gynecological (GYN) tracts. A comparison of the intensity differences between various Raman modes as well as the number of Raman lines, enables one to distinguish normal GYN tissues from diseased tissues. Fluorescence spectroscopic measurements on human breast tissues show that the ratio of fluorescence intensity at 340 nm to that at 440 nm can be used to distinguish between cancerous and non-cancerous tissues. Separate studies on normal and cancerous breast cell lines show spectral differences. The measurements of back-scattered ultrafast laser pulses from human breast tissues show differences in the scattered pulse profiles for different tissues. These studies show that various optical techniques have the potential to be used in medical diagnostic applications.     

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.     

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.     

Imaging of epidermal growth factor receptor on single breast cancer cells using surface-enhanced Raman spectroscopy

Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells.     

质谱成像技术及其在乳腺癌研究中的应用

乳腺癌是女性最常见的恶性肿瘤,其发病率在世界范围内呈现上升趋势,是威胁女性健康的重要疾病之一。随着现代医学技术的快速发展,早期有效的诊断和筛查方法能够改善乳腺癌患者生存率和提高其生活质量。由于乳腺癌肿瘤具有非常显著的异质性,这对于诊断和筛查带来了较大困难,亟须在肿瘤演进时间信息中,继续引入生物分子的空间信息,从而对其异质性、肿瘤微环境等进行准确的追踪。质谱成像技术,可在免标记的前提下利用离子质荷比的特性发现生物组织中的各种分子,并研究这些分子的时间和空间信息,对其进行准确的定性、定量和空间定位。目前,通过质谱成像技术可直接获取药物及其代谢物、内源性代谢物、脂质、多肽和蛋白质等在组织中的空间分布信息,为肿瘤分子分型诊断和确认以及相关抗肿瘤药物的筛选提供了新的思路和研究方向。该综述以乳腺癌相关的生物样品制备和研究进展为主要内容,从小分子样本、大分子样本、石蜡包埋样本、基质喷涂方式、常用离子源等方面阐述质谱成像中样本制备的重要性以及样品制备过程中存在的难点问题。同时,以细胞模型、动物模型和临床肿瘤标本为研究对象,汇总了质谱成像技术在乳腺癌方面的应用进展,并进行了展望,为开展癌症精准分型研究和药物药效的快速筛查提供了重要依据。     

Selective sampling using confocal Raman spectroscopy provides enhanced specificity for urinary bladder cancer diagnosis

In recent years, Raman spectroscopy has shown substantive promise in diagnosing bladder cancer, especially due to its exquisite molecular specificity. The ability to reduce false detection rates in comparison to existing diagnostic tools such as photodynamic diagnosis makes Raman spectroscopy particularly attractive as a complementary diagnostic tool for real-time guidance of transurethral resection of bladder tumor (TURBT). Nevertheless, the state-of-the-art high-volume Raman spectroscopic probes have not reached the expected levels of specificity thereby impeding their clinical translation. To address this issue, we propose the use of a confocal Raman probe for bladder cancer diagnosis that can boost the specificity of the diagnostic algorithm based on its suppression of the out-of-focus non-analyte-specific signals emanating from the neighboring normal tissue. In this article, we engineer and apply such a probe, having depth of field of approximately 280?μm, for Raman spectral acquisition from ex vivo normal and cancerous TURBT samples. Using this clinical dataset, a diagnostic algorithm based on principal component analysis and logistic regression is developed. We demonstrate that this approach results in comparable sensitivity but significantly higher specificity in relation to high-volume Raman spectral data. The application of only two principal components is sufficient for the discrimination of the samples underlining the robustness of the algorithm. Further, no discordance between replicate spectra is observed emphasizing the reproducible nature of the current diagnostic assessment. The high levels of sensitivity and specificity achieved in this proof-of-concept study opens substantive avenues for application of a confocal Raman probe during endoscopic procedures related to diagnosis and treatment of bladder cancer.

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.     

Distinguishing breast cancer cells using surface-enhanced Raman scattering

The detection and identification of epidermal growth factor receptor 2 (HER2)-positive breast cancer cells is crucial for the clinic therapy of breast cancer. For the aim of the detection, a novel surface-enhanced Raman scattering (SERS) probe for distinguishing breast cancers at different HER2 statuses is reported in this paper. In such a probe, anti-HER2 antibody-conjugated silver nanoparticles have been synthesized for specific targeting of HER2-positive breast cancer cells. More importantly, different from the previously reported SERS probe for targeting cancer cells, p-mercaptobenzoic acid is utilized as both the Raman reporter and the conjugation agent for attaching antibody molecules, which leads to a much simplified structure. For investigating the ability of such a probe to distinguish breast cancer cells, SKBR3 and MCF7 cells were chosen as two model systems, which are HER2-positive- and HER2-negative-expressing cells, respectively. The experimental results reveal that SKBR3 cells exhibit much stronger SERS signals than MCF7 cells, indicating that the probe could be utilized to distinguish breast cancer cells at different HER2 statuses. This kind of SERS probe holds a potential for a direct detection of living breast cancer cells with the advantages of easy fabrication, high SERS sensitivity, and biocompatibility.     

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor,Neratinib, in Cancer Cells

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label‐free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC‐MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.     

Plasmonics nanoprobes: detection of single-nucleotide polymorphisms in the breast cancer BRCA1 gene

This paper describes the application of plasmonics-based nanoprobes that combine the modulation of the plasmonics effect to change the surface-enhanced Raman scattering (SERS) of a Raman label and the specificity of a DNA hairpin loop sequence to recognize and discriminate a variety of molecular target sequences. Hybridization with target DNA opens the hairpin and physically separates the Raman label from the metal nanoparticle thus reducing the plasmonics effect and quenching the SERS signal of the label. We have successfully demonstrated the specificity and selectivity of the nanoprobes in the detection of a single-nucleotide polymorphism (SNP) in the breast cancer BRCA1 gene in a homogenous solution at room temperature. In addition, the potential application of plasmonics nanoprobes for quantitative DNA diagnostic testing is discussed.     

Subsurface probing of calcifications with spatially offset Raman spectroscopy (SORS): future possibilities for the diagnosis of breast cancer

Breast calcifications are often the only mammographic features indicating the presence of a cancerous lesion. Calcium oxalate (type I) may be found in and around benign lesions, however calcium hydroxyapatite (type II) is usually found within proliferative lesions, which can include both benign and malignant pathologies. However, the composition of type II calcifications has been demonstrated to vary between benign and malignant proliferative lesions, and could be an indicator for the possible disease state. Raman spectroscopy has previously been demonstrated as a powerful tool for non-destructive analysis of tissues, utilising laser light to probe chemical composition. Raman spectroscopy is traditionally a surface technique. However, we have recently developed methods that permit its application for obtaining sample composition to clinically relevant depths of many mm. We report the first demonstration of spatially offset Raman spectroscopy (SORS) for potential in vivo breast analysis. This study evaluates the possibility of utilising SORS for measuring calcification composition through varying thicknesses of tissues (2 to 10 mm), which is about one to two orders of magnitude deeper than has been possible with conventional Raman approaches. SORS can be used to distinguish non-invasively between calcification types I and II (and carbonate substitution of phosphate in calcium hydroxyapatite) within tissue of up to 10 mm deep. This result secures the first step in taking this technique forward for clinical applications seeking to use Raman spectroscopy as an adjunct to mammography for early diagnosis of breast cancer, by utilising both soft tissue and calcification signals. Non-invasive elucidation of calcification composition, and hence type, associated with benign or malignant lesions, could eliminate the requirement for biopsy in many patients.     

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.     

Chemical Applications of Raman Spectroscopy

Raman spectorscopy is—like infrared spectroscopy—a method for the study of vibrations of molecules and crystals. The two methods are complementary: if a vibration results in a change of the polarizability of a molecule, it is Raman active; if a change in the molecular dipole moment results, it is infrared active Vibrations of nonpolar groups and totally symmetrical vibrations of molecules are often only Raman active. IR and Raman spectra together give information about the symmetries and structures of molecules and crystals and about the properties of chemical bonds and intermolecular interactions. Until about 10 years ago Raman spectra could only be recorded on relatively large amounts of essentially colorless substances. After the advent of laser light sources the situation changed completely. The amount of sample substance required is now in the region of milli- and micrograms. Gases, liquids and solid samples, especially air-sensitive and reactive substances, single crystals, crystal needles and filaments as well as aqueous solutions can be readily investigated. The identification of molecules and the elucidation of molecular structures, biochemical analysis, and control of evnivornmental pollution are important aplications of Raman spectroscopy. Raman spectroscopy now constitutes an additional powerful tool in instrumental analysis     

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.     

Raman imaging analysis of pharmaceutical tablets by two-dimensional (2D) correlation spectroscopy

Chemical properties of active substances and insoluble excipient within tablets such as crystalline structures can be seen as an important index for solubility of ingredients. Spectroscopic imaging can potentially be a solid solution to understanding mechanisms at the molecular level and it may bring useful insight in terms of process analytical technique. In the present study, generalized two-dimensional (2D) correlation spectroscopy is utilized for the Raman image analysis of pharmaceutical tablets to reveal molecular interactions between chemical components. By using a spatial distance as a perturbation variable in 2D correlation scheme, synchronous and asynchronous correlation analysis becomes possible. Two kinds of pharmaceutical tablets, pentoxifylline (PTX) as an active substance and palmitic acid (PA) as an insoluble excipient, are prepared with different grinding times, 0.5 and 45 min. The 2D correlation analysis of Raman images of the tablets clearly reveals both physical and chemical effects of grinding process on the properties of the tablets. Asynchronous correlations indicate that a specific molecular structural change of PTX related to the crystallinity is induced by the grinding process. Namely, the crystallinity of PTX based on CH₂ structure is a key factor to control the solubility of the tablets. Some properties of pharmaceutical tablets, i.e. solubility or distribution of components in turn may become possible by the simple grinding process. Detailed analysis of Raman images becomes possible by the 2D correlation spectroscopy.     

Coherent anti-Stokes Raman scattering microscopy for polymers

Coherent anti-stokes Raman scattering (CARS) microscopy is a label-free chemical imaging modality capable of interrogating local molecular composition, concentration, and even orientation. In comparison to traditional Raman spectroscopy/imaging, CARS generates signals that are typically orders-of-magnitude stronger, enabling high-throughput and large-area imaging with superior spectroscopic fidelity. In this review, we present an overview of CARS microscopy as applied to polymer science, covering such timely and important topics as drug release and reaction kinetics to 3D molecular structures and orientation. We also discuss outstanding opportunities and challenges to using CARS microscopy as a quantitative measurement method.