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.     

Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy

Using the shifted-excitation Raman difference spectroscopy technique and an optical fibre featuring a negative curvature excitation core and a coaxial ring of high numerical aperture collection cores, we have developed a portable, background and fluorescence free, endoscopic Raman probe. The probe consists of a single fibre with a diameter of less than 0.25 mm packaged in a sub-millimetre tubing, making it compatible with standard bronchoscopes. The Raman excitation light in the fibre is guided in air and therefore interacts little with silica, enabling an almost background free transmission of the excitation light. In addition, we used the shifted-excitation Raman difference spectroscopy technique and a tunable 785 nm laser to separate the fluorescence and the Raman spectrum from highly fluorescent samples, demonstrating the suitability of the probe for biomedical applications. Using this probe we also acquired fluorescence free human lung tissue data.     

Surface‐enhanced Raman spectroscopy for on‐site analysis: A review of recent developments

On‐site identification and quantification of chemicals is critical for promoting food safety, human health, homeland security risk assessment, and disease diagnosis. Surface‐enhanced Raman spectroscopy (SERS) has been widely considered as a promising method for on‐site analysis due to the advantages of nondestructive, abundant molecular information, and outstanding sensitivity. However, SERS for on‐site application has been restricted not only by the cost, performance, and portability of portable Raman instruments, but also by the sampling ability and signal enhancing performance of the SERS substrates. In recent years, the performance of SERS for on‐site analysis has been improved through portable Raman instruments, SERS substrates, and other combined technologies. In this review, popular commercial portable Raman spectrometers and the related technologies for on‐site analysis are compared. In addition, different types of SERS substrates for on‐site application are summarized. SERS combined with other technologies, such as electrochemical and microfluidics are also presented. The future perspective of SERS for on‐site analysis is also discussed.     

Raman spectroscopic monitoring of Lactarius latex

Lactarius is a genus of Basidiomycotina with mainly agaricoid representatives, which are characterised by the excretion of a typical milky fluid. In particular, the colour, changes and taste of this latex-like milk are often used as a taxonomically important character. When it is exuded several chemical reactions occur. To date, NMR spectroscopy is generally used for chemical investigation of this latex. However, as a vibrational spectroscopic technique Raman spectroscopy has several advantageous properties for this type of research. The aim of this study is to investigate whether Raman spectroscopy can be used as an alternative analytical technique to monitor the chemical reactions in Lactarius latex. Therefore, this paper presents the first Raman spectra of Lactarius latex and provides an interpretation of the Raman bands that are present. L. lacunarum latex spectra are thoroughly investigated by 2D correlation analysis and are compared with latex spectra of other species (L. chrysorrheus, L. deterrimus, L. fluens, L. glyciosmus and L. salmonicolor).     

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.     

Noninvasive assessments of skin glycated proteins by fluorescence and Raman techniques in diabetics and nondiabetics

Diabetes is a complex metabolic disease and has chronic complications. It has been considered a serious public health problem. The aim of the current study was to evaluate skin glycated proteins through fluorescence and Raman techniques. One hundred subjects were invited to participate in the study. Six volunteers did not attend due to exclusion criteria or a change of mind about participating. Therefore, 94 volunteers were grouped according to age range (20‐80 years), health condition (nondiabetic, with insulin resistance [IR] and/or diabetic) and Fitzpatrick skin type (I‐VI). The fluorescence spectrometer and the portable Raman spectroscopy system were used to measure glycated proteins from the skin. There was elevated skin autofluorescence in healthy middle‐aged and elderly subjects, as well as in patients with IR and/or diabetes. Regarding Raman spectroscopy, changes in the skin hydration state, degradation of type I collagen and greater glycation were related for diabetes and chronological aging. Weak and positive correlation between the skin autofluorescence and the Raman peaks ratio (855/876) related to the glycated proteins was also found. Raman spectroscopy shows several bands for spectral analyses, complementing the fluorescence data. Therefore, this study contributes to understanding of the optical of human skin for noninvasive diabetes screening.      

Micro‐Raman spectroscopy of algae: Composition analysis and fluorescence background behavior

Preliminary feasibility studies were performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella sorokiniana (UTEX #1230) and Neochloris oleoabundans (UTEX #1185), were chosen for this study. Both species were considered to be candidates for biofuel production. Raman signals due to storage lipids (specifically triglycerides) were clearly identified in the nitrogen‐starved C. sorokiniana and N. oleoabundans, but not in their healthy counterparts. On the other hand, signals resulting from the carotenoids were found to be present in all of the samples. Composition mapping was conducted in which Raman spectra were acquired from a dense sequence of locations over a small region of interest. The spectra obtained for the mapping images were filtered for the wavelengths of characteristic peaks that correspond to components of interest (i.e., triglyceride or carotenoid). The locations of the components of interest could be identified by the high intensity areas in the composition maps. Finally, the time evolution of fluorescence background was observed while acquiring Raman signals from the algae. The time dependence of fluorescence background is characterized by a general power law decay interrupted by sudden high intensity fluorescence events. The decreasing trend is likely a result of photo‐bleaching of cell pigments due to prolonged intense laser exposure, while the sudden high intensity fluorescence events are not understood. Biotechnol. Bioeng. 2010;105: 889–898. © 2009 Wiley Periodicals, Inc.     

A hybrid least squares and principal component analysis algorithm for Raman spectroscopy

Raman spectroscopy is a powerful technique for detecting and quantifying analytes in chemical mixtures. A critical part of Raman spectroscopy is the use of a computer algorithm to analyze the measured Raman spectra. The most commonly used algorithm is the classical least squares method, which is popular due to its speed and ease of implementation. However, it is sensitive to inaccuracies or variations in the reference spectra of the analytes (compounds of interest) and the background. Many algorithms, primarily multivariate calibration methods, have been proposed that increase robustness to such variations. In this study, we propose a novel method that improves robustness even further by explicitly modeling variations in both the background and analyte signals. More specifically, it extends the classical least squares model by allowing the declared reference spectra to vary in accordance with the principal components obtained from training sets of spectra measured in prior characterization experiments. The amount of variation allowed is constrained by the eigenvalues of this principal component analysis. We compare the novel algorithm to the least squares method with a low-order polynomial residual model, as well as a state-of-the-art hybrid linear analysis method. The latter is a multivariate calibration method designed specifically to improve robustness to background variability in cases where training spectra of the background, as well as the mean spectrum of the analyte, are available. We demonstrate the novel algorithm's superior performance by comparing quantitative error metrics generated by each method. The experiments consider both simulated data and experimental data acquired from in vitro solutions of Raman-enhanced gold-silica nanoparticles.     

Use of near-infrared raman spectroscopy to detect IgG and IgM antibodies against Toxoplasma gondii in serum samples of domestic cats.

Near-infrared Raman spectroscopy (NIRS) is a particularly promising technique that is being used in recent years formany biomedical applications. Optical spectroscopy has gained increasing prominence as a tool for quantitative analysis of biological samples, clinical diagnostic, concentration measurements of blood metabolites and therapeutic drugs, and analysis of the chemical composition of human tissues. Toxoplasmosis is an important zoonosis in public health, and domestic cats are the most important transmitters of the disease. This disease can be detected by several serological tests, which usually have a high cost and require a long time. The goal of this work was to investigate a new method to diagnosis Toxoplasma gondii infections using NIRS. In order to confirm antibody detection, 24 cat blood serum samples were analyzed by the Raman spectra, from which 23 presented positive serology to toxoplasmosis and one was a reference negative serum. Characteristic Raman peaks allowed differentiation between negative and positive sera, confirming the possibility of antibody detection by Raman spectroscopy. These results give the first evidence that this technique can be useful to quantify antibodies in cat sera.     

Parallelized shifted‐excitation Raman difference spectroscopy for fluorescence rejection in a temporary varying system

A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted‐excitation Raman difference spectroscopy (SERDS), in which a slow (typically 1 Hz) modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted‐excitation spectra, has been used to separate Raman scattering from excitation‐shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested.      

Rapid epidemiological analysis of Acinetobacter strains by Raman spectroscopy

From earlier publications, we noticed that Raman spectra could potentially be used for subspecies identification of microorganisms. Here we evaluated the technique for its use as a typing tool of Acinetobacter species, using a collection of well-characterised strains from five hospital outbreaks. The strains were previously analysed using molecular techniques as cell envelope protein profiling and ribotyping. In this study, we have typed the strains by AFLP analysis and Raman spectroscopy. We compared the results using hierarchical cluster analysis, which showed highly similar groupings by both techniques. There seemed to be some misclassification between two sets of outbreak strains in the Raman analysis. We ascribe this to the clonal relationship between the strains of both outbreaks, described earlier. This results from a highly similar biochemical composition of the strains involved, and hence a highly similar Raman spectrum. We conclude that Raman spectroscopy could be an easy-to-use alternative in epidemiological studies of Acinetobacter strains and a promising starting point for the development of epidemiological studies in general.     

Prétraitements et méthodes de séparation de sources pour l’analyse des spectres Raman issus d’échantillons biologiques

Raman spectroscopy is a useful tool to investigate the molecular composition of biological samples. Source separation methods can be used to efficiently separate dense information recorded by Raman spectra. Distorting effects such as fluorescence background, peak misalignment or peak width heterogeneity break the linear generative model required by the source separation methods. Preprocessing steps are needed to compensate these deforming effects and make recorded Raman spectra fit the linear generative model of source separation methods. We show in this paper how efficiency of source separation methods is deeply dependent on preprocessing steps applied to raw dataset. Resulting improvements are illustrated through the study of the numerical dewaxing of Raman signal of a human skin biopsy. The applied source separation methods are a classical independent component analysis (ICA) algorithm named joint approximate diagonalization of eigenmatrices (JADE), and two positive source separation methods called non-negative matrix factorization (NMF) and maximum likelihood positive source separation (MLPSS).     

Direct observation of spectral differences between normal and basal cell carcinoma (BCC) tissues using confocal Raman microscopy

Raman spectroscopy has strong potential for providing noninvasive dermatological diagnosis of skin cancer. In this study, confocal Raman microscopy was applied to the dermatological diagnosis for one of the most common skin cancers, basal cell carcinoma (BCC). BCC tissues were obtained from 10 BCC patients using a routine biopsy and used for confocal Raman measurements. Autofluorescence signals from tissues, which interfere with the Raman signals, were greatly reduced using a confocal slit adjustment. Distinct Raman band differences between normal and BCC tissues for the amide I mode and the PO2- symmetric stretching mode showed that this technique has strong potential for use as a dermatological diagnostic tool without the need for statistical treatment of spectral data. It was also possible to precisely differentiate BCC tissue from surrounding noncancerous tissue using the confocal Raman depth profiling technique. We propose that confocal Raman microscopy provides a novel method for dermatological diagnosis since direct observations of spectral differences between normal and BCC tissues are possible.     

功能红细胞研究的拉曼光谱技术与进展

红细胞在人体新陈代谢和物质运输方面起着非常重要的作用。作为一种无损、快速而灵敏的光谱学检测方法,拉曼光谱技术在生物医学领域的应用日渐广泛。本文综述了近年来拉曼光谱技术在红细胞及血红蛋白研究中的应用进展,详尽评述当前研究功能红细胞中较有优势的拉曼技术—显微拉曼光谱技术和光镊拉曼光谱技术,并对其技术的改进做出总结,以期为利用激光拉曼光谱研究红细胞提供较系统的参考。     

Fast and robust two-dimensional inverse Laplace transformation of single-molecule fluorescence lifetime data

Fluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule two-dimensional (2D) fluorescence lifetime correlation spectroscopy is an emerging technique that holds promise for the study of protein and nucleic acid dynamics, as the technique is 1) capable of resolving conformational dynamics using a single chromophore, 2) resolves forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transform (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. Current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform one-dimensional (1D) and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data. We compare this approach to the widely used maximal entropy method.     

激光拉曼光谱技术在生物分子DNA研究中的应用和进展

激光技术的兴起使拉曼光谱成为激光分析中最活跃的研究领域之一,已被广泛地用于物质成分的分析和分子结构的鉴定。本文综述了拉曼技术在DNA研究中近年来的最新进展,包括：DNA的常规拉曼光谱分析;DNA的激光共振拉曼光谱分析;DNA在金属表面或电极上吸附行为的表面增强拉曼光谱研究;DNA的傅立叶变换拉曼光谱研究等。并对拉曼光谱技术在DNA等生物大分子领域中的研究前景做了进一步的展望。     

Near-infrared Fourier transform Raman and conventional Raman studies of calf gamma-crystallins in the lyophilized state and in solution.

We present in this report a detailed structural study of calf gamma-crystallins both in the solid state and in solution by the newly developed technique of near-infrared (IR) Fourier transform (FT)-Raman spectroscopy as well as by the conventional Raman method. In comparison with conventional laser Raman spectroscopy, the near-IR FT-Raman approach exhibits several attractive features such as fluorescence rejection capability, frequency accuracy, and the FT's multiplex and throughput advantages. These distinct characteristics combined form the basis for the particular suitability of FT-Raman in crystallin structural analysis and elucidation. We have thus obtained evidence in support of the view that native calf gamma-II crystallin does not contain a disulfide bond either in the lyophilized state or in solution. In addition, conventional Raman spectra are examined for all four gamma-crystallin fractions. gamma-S, gamma-II, gamma-III, and gamma-IV, and the results indicate a high degree of structural similarities among them. It is also found that the sulfhydryl groups in all four gamma-crystallins are highly resistant to air oxidation and are capable of maintaining their reduced state during isolation in the absence of added reductants or such chelating agents as EDTA.     

激光喇曼光谱技术在食品科学中的应用

激光喇曼光谱技术是一种非侵入、非弹性的光散射技术,它能够无损地提供丰富的分子结构和物质成分的信息。近来它在食品工业领域表现出很大的应用潜力。本文综述了激光喇曼光谱技术在食品科学中的应用及其新进展。主要包括果蔬农药残留的检测、肉类产品质量检测、伪劣食品鉴定、食物蛋白的研究以及食品加工监控等方面的应用。并对喇曼光谱技术在这些方面的应用前景作了进一步的展望。     

Time-gated interferometric detection increases Raman scattering to fluorescence signal ratio in biological samples

Attainable levels of signal-to-background ratio (SBR) in Raman spectroscopy of biological samples is limited by the presence of endogenous fluorophores. It is customary to remove the ubiquitous fluorescence background using postacquisition data processing. However, new approaches are needed to reduce background contributions and maximize the fraction of the sensor dynamical range occupied by Raman photons. Time-resolved detection using pulsed lasers and time-gated measurements can be used to address the signal-to-background problem in biological samples by limiting light detection to nonresonant interaction phenomena with relaxation time scales occurring on sub-nanosecond time scales, thereby excluding contributions from resonant phenomena such as fluorescence. A time-gated Fourier-transform spectrometer was assembled using a commercially available interferometer, a single channel single-photon avalanche diode and time tagging electronics. A time gate of 300 ps increased the signal-to-background-ratio of the 1440 cm^?1 Raman band from 36% to 69% in an olive oil sample hereby demonstrating the potential of this approach for autofluorescence suppression.      

The application of spectroscopy techniques for diagnosis of malaria parasites and arboviruses and surveillance of mosquito vectors: A systematic review and critical appraisal of evidence

Spectroscopy-based techniques are emerging diagnostic and surveillance tools for mosquito-borne diseases. This review has consolidated and summarised recent research in the application of Raman and infrared spectroscopy techniques including near- and mid-infrared spectroscopy for malaria and arboviruses, identified knowledge gaps, and recommended future research directions. Full-length peer-reviewed journal articles related to the application of Raman and infrared (near- and mid-infrared) spectroscopy for malaria and arboviruses were systematically searched in PUBMED, MEDILINE, and Web of Science databases using the PRISMA guidelines. In text review of identified studies included the methodology of spectroscopy technique used, data analysis applied, wavelengths used, and key findings for diagnosis of malaria and arboviruses and surveillance of mosquito vectors. A total of 58 studies met the inclusion criteria for our systematic literature search. Although there was an increased application of Raman and infrared spectroscopy-based techniques in the last 10 years, our review indicates that Raman spectroscopy (RS) technique has been applied exclusively for the diagnosis of malaria and arboviruses. The mid-infrared spectroscopy (MIRS) technique has been assessed for the diagnosis of malaria parasites in human blood and as a surveillance tool for malaria vectors, whereas the near-infrared spectroscopy (NIRS) technique has almost exclusively been applied as a surveillance tool for malaria and arbovirus vectors.Conclusions/SignificanceThe potential of RS as a surveillance tool for malaria and arbovirus vectors and MIRS for the diagnosis and surveillance of arboviruses is yet to be assessed. NIRS capacity as a surveillance tool for malaria and arbovirus vectors should be validated under field conditions, and its potential as a diagnostic tool for malaria and arboviruses needs to be evaluated. It is recommended that all 3 techniques evaluated simultaneously using multiple machine learning techniques in multiple epidemiological settings to determine the most accurate technique for each application. Prior to their field application, a standardised protocol for spectra collection and data analysis should be developed. This will harmonise their application in multiple field settings allowing easy and faster integration into existing disease control platforms. Ultimately, development of rapid and cost-effective point-of-care diagnostic tools for malaria and arboviruses based on spectroscopy techniques may help combat current and future outbreaks of these infectious diseases.