Raman spectroscopic identification of single bacterial cells under antibiotic influence

The identification of pathogenic bacteria is a frequently required task. Current identification procedures are usually either time-consuming due to necessary cultivation steps or expensive and demanding in their application. Furthermore, previous treatment of a patient with antibiotics often renders routine analysis by culturing difficult. Since Raman microspectroscopy allows for the identification of single bacterial cells, it can be used to identify such difficult to culture bacteria. Yet until now, there have been no investigations whether antibiotic treatment of the bacteria influences the Raman spectroscopic identification. This study aims to rapidly identify bacteria that have been subjected to antibiotic treatment on single cell level with Raman microspectroscopy. Two strains of Escherichia coli and two species of Pseudomonas have been treated with four antibiotics, all targeting different sites of the bacteria. With Raman spectra from untreated bacteria, a linear discriminant analysis (LDA) model is built, which successfully identifies the species of independent untreated bacteria. Upon treatment of the bacteria with subinhibitory concentrations of ampicillin, ciprofloxacin, gentamicin, and sulfamethoxazole, the LDA model achieves species identification accuracies of 85.4, 95.3, 89.9, and 97.3 %, respectively. Increasing the antibiotic concentrations has no effect on the identification performance. An ampicillin-resistant strain of E. coli and a sample of P. aeruginosa are successfully identified as well. General representation of antibiotic stress in the training data improves species identification performance, while representation of a specific antibiotic improves strain distinction capability. In conclusion, the identification of antibiotically treated bacteria is possible with Raman microspectroscopy for diverse antibiotics on single cell level.

Tumour cell identification by means of Raman spectroscopy in combination with optical traps and microfluidic environments

Raman spectroscopy has been recognized to be a powerful tool for label-free discrimination of cells. Sampling methods are under development to utilize the unique capabilities to identify cells in body fluids such as saliva, urine or blood. The current study applied optical traps in combination with Raman spectroscopy to acquire spectra of single cells in microfluidic glass channels. Optical traps were realized by two 1070 nm single mode fibre lasers. Microflows were controlled by a syringe pump system. A novel microfluidic glass chip was designed to inject single cells, modify the flow speed, accommodate the laser fibres and sort cells after Raman based identification. Whereas the integrated microchip setup used 514 nm for excitation of Raman spectra, a quartz capillary setup excited spectra with 785 nm laser wavelength. Classification models were trained using linear discriminant analysis to differentiate erythrocytes, leukocytes, acute myeloid leukaemia cells (OCI-AML3), and breast tumour cells BT-20 and MCF-7 with accuracies that are comparable with previous Raman experiments of dried cells and fixed cells in a Petri dish. Implementation into microfluidic environments enables a high degree of automation that is required to improve the throughput of the approach for Raman activated cell sorting.     

Quantitative analysis of serum and serum ultrafiltrate by means of Raman spectroscopy

The fast and reliable determination of concentrations of blood, plasma or serum constituents is a major requirement in clinical chemistry. We explored Raman spectroscopy as a reagent-free tool for predicting the concentrations of different parameters in serum and serum ultrafiltrate. In an investigation using samples from 247 blood donors (which we believe to be the largest study on Raman spectroscopy of serum so far) the concentrations of glucose, triglycerides, urea, total protein, cholesterol, high density lipoprotein, low density lipoprotein and uric acid were determined with an accuracy within the clinically interesting range. After training a multivariate algorithm for data analysis, using 148 samples, concentrations were predicted blindly for the remaining 99 serum samples based solely on the Raman spectra. Relative errors of prediction around 12% were obtained. Moreover, to the best of our knowledge, differentiation between HDL and LDL cholesterol as well as the quantification of uric acid was for the first time successfully accomplished for serum-based Raman spectroscopy. Finally, we showed that ultrafiltration can efficiently reduce fluorescent light background to improve prediction accuracy such that the relative coefficient of variation was reduced for glucose and urea in ultrafiltrate by more than a factor of 2 when compared to serum.     

Identifying and localizing intracellular nanoparticles using Raman spectroscopy

Raman microscopy is employed to spectroscopically image biological cells previously exposed to fluorescently labelled polystyrene nanoparticles and, in combination with K-means clustering and principal component analysis (PCA), is demonstrated to be capable of localising the nanoparticles and identifying the subcellular environment based on the molecular spectroscopic signatures. The neutral nanoparticles of 50 nm or 100 nm, as characterised by dynamic light scattering, are shown to be non-toxic to a human lung adenocarcinoma cell-line (A549), according to a range of cytotoxicity assays including Neutral Red, Alamar Blue, Coomassie Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confocal fluorescence microscopy identifies intracellular fluorescence due to the nanoparticle exposure, but the fluorescence distribution is spatially diffuse, potentially due to detachment of the dye from the nanoparticles, and the technique fails to unambiguously identify the distribution of the nanoparticles within the cells. Raman spectroscopic mapping of the cells in combination with K-means cluster analysis is used to clearly identify and localise the polystyrene nanoparticles in exposed cells, based on their characteristic spectroscopic signatures. PCA identifies the local environment as rich in lipidic signatures which are associated with localisation of the nanoparticles in the endoplasmic reticulum. The importance of optimised cell growth conditions and fixation processes is highlighted. The preliminary study demonstrates the potential of the technique to unambiguously identify and locate nonfluorescent nanoparticles in cells and to probe not only the local environment but also changes in the cell metabolism which may be associated with cytotoxic responses.     

Characterization of metal ions in coordinating solvent mixtures by means of Raman spectroscopy.

Titration Raman spectroscopy has been developed for studying the solvation structure of metal ions in solution. The method affords us the solvation number, and the value thus obtained in neat solvents is in good agreement with that determined by EXAFS. The method is then applied to solvent mixtures, and the individual solvation number for each solvent is extracted. In a solvent mixture of N,N-dimethylformamide (DMF) and N,N,N',N'-tetramethylurea (TMU), the metal ion prefers DMF to TMU, which is ascribed to the solvation steric effect. The same applies also for the solvent mixture of N,N-dimethylpropionamide (DMPA) and DMF. However, unlike TMU, DMPA changes its conformation from the planar cis to non-planar staggered upon solvation to the metal ion. The enthalpy for the conformational change of DMPA is positive in the bulk, while it is significantly negative in the coordination sphere of the manganese(II) ion. Here, we briefly describe the procedure of measurements and analyses for the titration Raman spectroscopy, and review the solvation structure of the alkaline earth, first transition metal(II) and lanthanide(III) ions in some solvent mixtures in view of solvation steric effect.     

Label-free Raman spectroscopy for accessing intracellular anticancer drug release on gold nanoparticles

We investigated glutathione (GSH)-induced purine or pyrimidine anticancer drug release on gold nanoparticle (AuNP) surfaces by means of label-free Raman spectroscopy. GSH-triggered releases of 6-thioguanine (6TG), gemcitabine (GEM), acycloguanosine (ACY), and fadrozole (FAD) were examined in a comparative way by means of surface-enhanced Raman scattering (SERS). The GSH-induced dissociation constant of GEM (or ACY/FAD) from AuNPs was estimated to be larger by more than 38 times than that of 6TG from the kinetic relationship. Tripeptide control experiments were presented to check the turn-off Raman signalling mechanism. Dark-field microscopy (DFM) and transmission electron microscopy (TEM) indicated the intracellular AuNP loads. After their cellular uptake, GEM, ACY, and FAD would not show SERS intensities as strong as 6TG. This may be due to easier release of GEM, ACY, and FAD than 6TG by intracellular reducing species including GSH. We observed fairly strong SERS signals of GEM and 6TG in cell culture media solution. Our CCK-8 cytotoxicity assay data support that 6TG-AuNPs did not exhibit a substantial decrease in cell viability presumably due to strong binding. Label-free confocal Raman spectroscopy can be utilized as an effective tool to access intracellular anticancer drug release.     

Evaporation of material and influence of auxiliary spark gap on the spectral excitation by means of laser emission spectroscopy for local analysis of graphite

For the determination of impurities in graphite, laser-micro-emission spectroscopy allows the analysis of much smaller sample areas than possible by spectroscopic analysis of arc or spark discharges.Because the maximum quantity of material which can be evaporated by a laser beam is only approximately 8μg, it was found necessary to introduce a spark gap above the evaporation pit to provide additional excitation of the plasma, thereby increasing the sensitivity of the analysis. In this way, the radiation intensity of the plasma was increased by two orders of magnitude.The effects of the spark gap parameters, voltage, capacitance and inductance, on the spectral excitation were investigated. Voltage and capacitance determine the energy supplied to the spark gap, whereas capacitance, together with inductance, controls the duration and characteristic of the discharge. To obtain the optimum additional excitation, the duration of the spark discharge had to be matched with the time taken for the material to evaporate.The catalytic effect of some impurity elements on the corrosion of graphite was identified by using the technique to analyse material in the corrosion pits of irradiated graphite fuel elements.     

Study of ozone interaction with microfibrous filter material by IR Fourier and Raman spectroscopy

Interaction of ozone with microfibrous materials based on polystyrene, acrylonitrile, diacetylcellulose, vinyl chloride and carbon are studied by the methods of IR Fourier and Raman spectroscopy.     

Chemotaxonomical identification of spores of macrofungi: possibilities of Raman spectroscopy

Confocal Raman spectroscopy is a non-destructive analytical method which is useful to obtain detailed information about the molecular composition of biological samples. Its high spatial resolution was used to collect spectra of single basidiospores of macrofungi of the genera Collybia, Gymnopus, Laccaria, Lactarius, Mycena and Russula. These spectra can be divided into three major taxon-related groups, with general compositional differences, such as the relative amount of lipids compared to proteins. In this study, collapsing of thin-walled spores during storage was often observed, a phenomenon which has been given little attention in the literature. The Raman spectra are treated with different chemometric preprocessing techniques, including Savitsky–Golay, standard normal variate (SNV) preprocessing and extended multiplicative scatter correction (EMSC). By using linear discriminant analysis, approximately 90% of the spectra can be assigned to the correct genus, but identification on the species level was not possible.     

Automatic identification of novel bacteria using Raman spectroscopy and Gaussian processes

Raman spectroscopy is successfully used for the reliable classification of complex biological samples. Much effort concentrates on the accurate prediction of known categories for highly relevant tasks in a wide area of applications such as cancer detection and bacteria recognition. However, the resulting recognition systems cannot always be directly used in practice since unseen samples might not belong to classes present in the training set. Our work aims to tackle this problem of novelty detection using a recently proposed approach based on Gaussian processes. By learning novelty scores for a large bacteria Raman dataset comprising 50 different strains, we analyze the behavior of this method on an independent dataset which includes known as well as unknown categories. Our experiment reveals that non-parametric methods such as Gaussian processes can be successfully applied to the task of finding unknown bacterial strains, leading to encouraging results motivating their further utilization in this area.     

The identification of fibers by infrared and Raman microspectroscopy

The application of Raman and infrared microspectroscopy to fiber identification has been investigated. Natural and synthetic fibers, both organic and inorganic in nature, can be rapidly characterized by these techniques. In general, it has been found that infrared microspectroscopy offers a nonsubjective method of fiber identification that is quicker, easier, and occasionally, more selective than classical methods. Raman microspectroscopy has also been proven useful for these analyses. It provides low-frequency information, requires virtually no sample preparation, and supplies data complementary to that furnished by infrared microspectroscopy. In many cases one may use these methods for a quick differentiation of fibers of the same type which have undergone different chemical treatments.     

Towards detection and identification of circulating tumour cells using Raman spectroscopy

Body fluids are easily accessible and contain valuable indices for medical diagnosis. Fascinating tools are tumour cells circulating in the peripheral blood of cancer patients. As these cells are extremely rare, they constitute a challenge for clinical diagnostics. In this contribution we present the Raman spectroscopic-based identification of different single cells in suspension that are found in peripheral blood of cancer patients including healthy cells like leukocytes and erythrocytes, and tumour cells like leukaemic cells and cells originating from solid tumours. Leukocytes and erythrocytes were isolated from the peripheral blood of healthy donors while myeloid leukaemia cells (OCI-AML3) and breast carcinoma derived cells (MCF-7, BT-20) were obtained from cell cultures. A laser emitting 785 nm light was used for optical trapping the single cells in the laser focus and to excite the Raman spectrum. Support vector machines were applied to develop a supervised classification model with spectra of 1210 cells originating from three different donors and three independent cultivation batches. Distinguishing tumour cells from healthy cells was achieved with a sensitivity of >99.7% and a specificity of >99.5%. In addition, the correct cell types were predicted with an accuracy of approximately 92%.     

The hydrogen-bond network in propylene-glycol studied by Raman spectroscopy

In the present work, we report a polarized Raman study versus temperature of the complex O–H stretching vibrational band (3800–3000 cm⁻¹) performed in a glass forming liquid, namely propylene-glycol (PG), with chemical formula given by H[OCH(CH₃)CH₂]OH. The spectra were collected in bulk and confined state within a sol–gel controlled porous glass having highly interconnected 25 Å diameter pores and characterized by a huge number of silanol groups (Si–OH), able to interact with PG molecules via hydrogen bond. The goal was to investigate how the hydrophilic nature of the surface influenced the molecular mobility of this hydrogen-bonded system, by monitoring intra- and inter-molecular host–host and host–guest interactions. The analysed O–H spectral region was decomposed into Gaussian symmetrical profiles, each of them associated to a well-defined aggregate, triggered by the presence of H-bond. Passing from the bulk state to the confined one, a clear change of the dynamical properties has been revealed and related to the interactions with the surface. The observed results were discussed on the basis of current models for associated liquids.     

Molecular-level investigation on electrochemical interfaces by Raman spectroscopy

The structure and dynamics of electrode/liquid interfaces play an increasingly important role in electrochemistry. Raman spectroscopy is capable of providing detailed structural information at molecular level and new insight into the interfacial structure, adsorption, reaction, electrocatalysis and corro-sion. In this account we will summarize some progresses of surface Raman spectroscopy in the study of electrochemical interfaces, mainly based on our group's work, laying emphasis on the detection sensitivity, spectral resolution, time resolution and spatial resolution as well as the hyphenated technique.     

Nondestructive classification and identification of ballpoint pen inks by Raman spectroscopy for forensic document examinations

As a non-destructive analytical method, Raman spectroscopy often provides insufficient information to identify or differentiate the ink used for the preparation of a questioned document. In this study, blue and black ballpoint pen inks deposited on paper substrate were examined in situ by conventional Raman spectroscopy. Inks were successfully classified based on the total number of prominent bands in Raman spectra. It was found that more than 90% of the samples of the same type and color could be differentiated visually using only Raman spectra, i.e. 94 and 95% for blue and black inks, respectively. As a result of this study, a flow chart has been constructed for blue and black ballpoint pen inks allowing their systematic identification. Raman spectroscopy proved to be a fast and precise technique for forensic ink analysis.     

The structure of different cellulosic fibres characterized by Raman spectroscopy

Different samples of cellulosic materials were analyzed by Raman spectroscopy and wood chips from Pinus elliottii, treated with acidic and alkaline aqueous solutions, were used to evaluate diagnostic signatures of the chemical structure of the cellulosic fibres. Cotton and whiskers synthesized from cotton, ancient Egyptian linen from a mummy wrapping, and five different paper sheets used in museum handling were compared. The complementarity of the Raman spectroscopic and scanning electron microscopic data facilitated the evaluation of the crystallinity, the level of organization and chain sizes of the fibres and the identification of different oxidation products. Intensity ratios measured from pairs of key bands were used to characterize the crystallinity, chain lengths and presence of oxidative decomposition in the range of the studied samples. Finally, the Raman spectra of the ancient Egyptian linen specimen indicated a potential future application of the proposed analysis for the characterization of archaeological pieces composed of linen.     

Characterization of alkali treated flax fibres by means of FT Raman spectroscopy and environmental scanning electron microscopy

Flax fibres grown under well managed conditions were submitted to NaOH chemical treatments, so called Mercerization. The extent of the polymorphic transformation of cellulose I into cellulose II taking place within the crystalline domains of the fibre cellulose was dependent on the alkali concentration. FT Raman spectroscopy turned out to represent an ideal tool for detecting the polymorphic transformation of the cellulosic fine structure of the flax fibres in vivo. In addition to the differences of the FT Raman spectra in the frequency range below 1500 cm(-1), second derivatives of the spectra in the range of the CH stretching vibrations could also be used to distinguish the two polymorphic modifications. The intensity ratio R of the stretching modes v(s)COC and v(as)COC represents a spectral parameter characterising the molecular structure of the flax fibres. As a supplementary tool, Environmental scanning electron microscopy (ESEM) was used to visualize the microstructural fibre properties dependent on the alkali concentrations during the Mercerization.     

Raman spectroscopy as a means for the identification of plattnerite (PbO2), of lead pigments and of their degradation products

The Raman spectra of plattnerite [lead(IV) oxide, PbO2] and of the lead pigments red lead (Pb3O4), lead monoxide [PbO, litharge (tetragonal) and massicot (orthorhombic)], lead white [basic lead carbonate, 2PbCO3.Pb(OH)2] and of their laser-induced degradation products were recorded using a range of different excitation lines, spectrometer systems and experimental conditions. The degradation of PbO2 is more extensive along the pathway PbO2-->Pb3O4-->PbO (litharge)-->PbO (massicot) the shorter the wavelength of the excitation line and the higher its power. The Raman spectrum of PbO2, which is black and of the rutile structure, is particularly difficult to obtain but three bands, at 653, 515 and 424 cm-1, were identified as arising from the b2g, a1g and e(g) modes respectively, by analogy with the corresponding modes of isostructural SnO2 (776, 634 and 475 cm-1). A further oxide was identified, PbO1.55, the Raman spectrum of which does not correspond to that of any of the laser-induced degradation products of PbO2 at any of the wavelengths used. The Raman results are critical to the future use of Raman microscopy for the identification of lead pigments on artworks.     

Experimental problems in Raman spectroscopy applied to pigment identification in mixtures

Raman spectroscopy provides useful information to detect and identify pictorial materials in artworks, although some problems are involved when the identification of individual pigments in mixtures is treated. With the hypothesis of the Principle of superposition, the mixture spectrum should be the direct sum of each pondered individual spectrum. In this work, we will show several mixtures where it can be noticed that the mixture spectrum is not qualitatively proportional to the sum of pondered individual spectra. Also there were some cases where the bands of one of the pigments could not be detected in the mixture spectrum. This non-linear behaviour could be attributed to specific proprieties of each material that are revealed when they interact with each other. We conjecture that, for instance, the different reflectances or the wavelength of the laser source could be determinant factors of the obtained results. In this paper an experimental method has been designed in order to characterize the quantitative behaviour of the Raman bands corresponding to each pigment in a mixture. Adequate coefficients are defined and calculated to facilitate the study of the spectral contribution of the different components of a mixture.