Sample illumination configurations for spatially resolved Raman spectrometry using a charge-coupled device detector

In this paper two simple configurations for sample illumination using a charge-coupled device (CCD) detector have been shown. The choice of an appropriate sample illumination can be crucial to obtain spatial and spectral information of complex samples. It is demonstrated that simultaneous Raman spectra of a heterogeneous sample of three compounds can be obtained using a vertical sample illumination. Spatially resolved resonant Raman and surface-enhanced resonant Raman spectra of a complex (Ni-PAPH) have been observed with a low integration time. Dividing the CCD in two region; and with horizontal multiline sample illumination (argon-ion laser at 488 nm and HeNe laser at 632.8 nm) spatially resolved fluorescence spectra of a homogeneous mixture of dyes have been obtained. The total image was acquired in only 1 s.     

Transmission Raman measurement directly through packed corn kernels to improve sample representation and accuracy of compositional analysis

The potential of transmission Raman spectroscopy for direct analysis of packed granular samples, one of the most frequently encountered sample types in the field of non-destructive spectroscopic analysis, has been evaluated. For this purpose, transmission Raman spectra were collected by laser illumination through packed corn kernels to determine their protein concentration. Back-scattering Raman spectra of the same samples were also collected for comparison. Raman spectral features of the major kernel constituents were initially characterized, and Raman mapping over the whole kernel face was performed to investigate the inhomogeneous distribution of constituents in a kernel. Possible variations of transmission spectral features depending on the laser illumination on different locations of a kernel were investigated, since the orientation of kernels in the packing was essentially random. Rotation of kernel packing during spectral collection was helpful in improving the compositional representation of packed kernels. With partial least squares (PLS) regression, the protein concentrations were determined using both spectral collection methods and the resulting accuracies were compared. As a result, the transmission measurement provided a more accurate determination of protein concentration since it enabled deeper sampling across the packed kernels, leading to a better compositional representation of them. By contrast, in the back-scattering measurement, kernels on the top of the packing were mainly sampled for the spectral acquisition. Moreover, the back-scattering spectral feature, more weighted to constituents localized at the outer portion of a kernel, was short of representing the overall composition of a kernel.     

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.     

Raman spectroscopy in combination with background near-infrared autofluorescence enhances the in vivo assessment of malignant tissues

The diagnostic ability of optical spectroscopy techniques, including near-infrared (NIR) Raman spectroscopy, NIR autofluorescence spectroscopy and the composite Raman and NIR autofluorescence spectroscopy, for in vivo detection of malignant tumors was evaluated in this study. A murine tumor model, in which BALB/c mice were implanted with Meth-A fibrosarcoma cells into the subcutaneous region of the lower back, was used for this purpose. A rapid-acquisition dispersive-type NIR Raman system was employed for tissue Raman and NIR autofluorescence spectroscopic measurements at 785-nm laser excitation. High-quality in vivo NIR Raman spectra associated with an autofluorescence background from mouse skin and tumor tissue were acquired in 5 s. Multivariate statistical techniques, including principal component analysis (PCA) and linear discriminant analysis (LDA), were used to develop diagnostic algorithms for differentiating tumors from normal tissue based on their spectral features. Spectral classification of tumor tissue was tested using a leave-one-out, cross-validation method, and the receiver operating characteristic (ROC) curves were used to further evaluate the performance of diagnostic algorithms derived. Thirty-two in vivo Raman, NIR fluorescence and composite Raman and NIR fluorescence spectra were analyzed (16 normal, 16 tumors). Classification results obtained from cross-validation of the LDA model based on the three spectral data sets showed diagnostic sensitivities of 81.3%, 93.8% and 93.8%; specificities of 100%, 87.5% and 100%; and overall diagnostic accuracies of 90.6%, 90.6% and 96.9% respectively, for tumor identification. ROC curves showed that the most effective diagnostic algorithms were from the composite Raman and NIR autofluorescence techniques.     

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.

Comparison of near-infrared and Raman spectroscopy for the determination of the density of polyethylene pellets

In this study, we compare near-infrared (NIR) and Raman spectroscopy for the determination of the density of linear low density polyethylene (PE) (in a pellet form). As generally known, Raman spectral features are more selective than those of NIR for most chemical samples. NIR spectroscopy has been more extensively used for the quantitative analysis of polymers, but Raman spectroscopy is the better choice as long as the problem of reproducibility of Raman measurements (especially for solid samples), mostly resulting from insufficient sample representation due to probing only localized chemical information and the sensitivity of sample placement with regard to the focal plane, can be overcome. To improve sample representation and reproducibility of Raman measurements, we have employed the wide area illumination (WAI) Raman scheme, capable of illuminating a laser onto a large sample area (28.3 mm²) for Raman spectral collection (a 6-mm laser spot with a focal length of 248 mm). Diffuse reflectance NIR spectra of PE pellets were collected using a sample moving system which allowed for the scanning of large areas. The prediction error was 0.0008 g cm⁻³ for Raman spectroscopy and 0.0011 g cm⁻³ for NIR spectroscopy. The harmonization of inherently selective Raman features and a reproducible spectral collection with correct sample representations using the WAI scheme led to an accurate determination of the density of the PE pellets.     

活性碳纤维阴极电芬顿反应降解微囊藻毒素研究

以具有高比表面积的活性碳纤维作为阴极,通过电芬顿反应降解水中微囊藻毒素(MCRR,MCLR)的电化学方法系统考察了电流密度、pH值和Fe2+浓度等因素对微囊藻毒素降解效果的影响.实验结果表明,在Fe2+浓度为1.0mmol/L和电流密度为6.6mA/cm2条件下,电化学处理60min,MCRR(8.81mg/L)去除率为75%,MCLR(6.36mg/L)去除率为94%.证明过氧化氢可以通过电化学还原在活性碳纤维阴极表面高效产生,微囊藻毒素可被高效降解去除.     

Direct on-line Raman measurement of flying solid samples: determination of polyethylene pellet density

We demonstrated an on-line Raman measurement of polyethylene (PE) pellet density when it is flying in a sample line. While in flight, pellets are sparsely populated at spectral collection, a spectral collection strategy covering a large spatial volume (larger number of pellets simultaneously) is necessary to acquire reasonable Raman intensity. In addition, the Raman measurement must be less sensitive to pellet position, because position and distribution are uncontrollable in a flying condition. To fulfill these requirements, a wide area illumination (WAI) scheme capable of covering a large sample volume (illumination volume: 0.7 cm³) was used when the pellets were flying in a 2.5-cm-diameter sample line. In addition, a long focal length (250 mm) was used so that minor changes in pellet position would not significantly affect the resulting Raman spectral feature. Although Raman intensity substantially decreased due to the large void space among flying pellets, a correct spectral feature representing PE was successfully obtained without any significant spectral distortion. Using partial least squares (PLS) regression, the prediction error under flying conditions was 0.0009 g cm⁻³, which was comparable to that acquired when the pellets were packed (0.0008 g cm⁻³). When a conventional Raman scheme covering a smaller sample volume with a short focal length was used, the PE intensity decreased dramatically, and the resulting signal-to-noise ratio was not proper for quantitative analysis.     

Direct, non-destructive quantitative measurement of an active pharmaceutical ingredient in an intact capsule formulation using Raman spectroscopy

The active pharmaceutical ingredient (ambroxol) in an intact capsule formulation has been non-destructively quantified using Raman spectroscopy. To improve the problem of insufficient representive sampling inherent in Raman measurements, we have employed a wide area illumination (WAI) scheme that enables much improved sample coverage through a circular excitation laser spot with a 6 mm diameter. One of the anticipated sources of variation for this measurement was variation in the capsule shells. However, the WAI scheme significantly decreased the spectral variation among empty capsules compared to a measurement with a traditional small-spot excitation. Therefore, measurement variations emanating from the capsule shell did not significantly influence the accuracy of the determination of ambroxol concentrations. The resulting standard error of prediction (SEP) using the WAI scheme was comparable to that from previous Raman measurements which used a conventional small-spot excitation and employed a sampling scheme that involved rotation of an ambroxol pellet. It is further noteworthy that the SEP was also similar to that obtained from the use of transmission NIR spectroscopy, which was achieved by collection of spectra of the powdered capsule contents removed from the shell. The proposed Raman measurement using the WAI scheme in this case was sufficient to achieve the quantitative measurement of the active pharmaceutical ingredient (API) content of capsules non-destructively.     

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.     

Surface-enhanced vibrational spectra of 2-nitrofluorene

The infrared and Raman spectra of the isolated adsorbate 2-nitrofluorene (2NF) have been registered and the spectral assignment was performed on the basis of both previous data concerning related molecules and density functional theory DFT calculations. The theoretical calculations were compared to the experimental results, obtaining a good agreement with the IR and Raman data. The surface-enhanced infrared and Raman spectra, SEIRA and SERS, of 2NF on different metal surfaces were registered; the best spectra were obtained by using the 633nm laser line. The most probable orientation and organization of the adsorbate on the surface were inferred from the reflection-absorption infrared spectrum RAIRS and SERS and SEIRA data.     

Single vibronic level resonant Raman scattering of molecular ions in solid glass: PMDA anion and naphthalene cation

Resonant Raman spectra were obtained for the radical anion of pyromellitic dianhydride (PMDA) in MTHF glass and the radical cation of naphthalene in alkyl halide glass at 77 K by using a pulsed tunable dye laser. The intensity pattern of the single vibronic level resonant Raman resembles that of hot fluorescence when the homogeneous line width of the intermediate level is narrow. Overtones and combinations of the excited vibrational mode prevail in the spectra.     

A new non-invasive, quantitative Raman technique for the determination of an active ingredient in pharmaceutical liquids by direct measurement through a plastic bottle

The concentration of an active pharmaceutical ingredient (povidone) in a commercial eyewash solution has been measured directly through a plastic (low-density polyethylene: LDPE) container using a wide area illumination (WAI) Raman scheme. The WAI scheme allows excitation using a 6 mm laser spot (focal length: 248 mm) that is designed to cover a wide sample area. As a result, it has the potential to improve the reliability Raman measurements by significantly enhancing representative sample interrogation, thus improving the reproducibility of sampling. It also decreases the sensitivity of sample placement with regard to the excitation focal plane. Simultaneously, isobutyric anhydride was placed in front of the bottles to use for a synchronous external standard configuration. This helps to correct the problematic variation of Raman intensity from the inherent fluctuation in laser power. Using the WAI Raman scheme combined with the synchronous standard method, the povidone concentration was successfully measured with spectral collection that was performed through a plastic barrier. The conventional Raman scheme was difficult to employ for the same purpose because of the degraded spectral reproducibility resulting from the smaller laser illumination area and the sensitivity of such an approach to the position of the sample bottle. The result from this study suggests that the WAI scheme exhibits a strong potential for the non-destructive quantitative analysis of pharmaceuticals measured directly in plastic containers. Preliminary work also shows that similar measurements can also be made in glass bottles. If implemented, this technique could be utilized as a simple and rugged method for quality assurance of final products in a manner consistent with Process analytical technology (PAT) requirements.     

Evaluating the Conservation State of Naturally Aged Paper with Raman and Luminescence Spectral Mapping: Toward a Non-Destructive Diagnostic Protocol

Micro-Raman and luminescence spectroscopy were combined with morphological analysis to study the conservation state of differently degraded paper samples, dated from 1873 to 2021. The aim of the work reported in this paper was to obtain ageing markers based on variations of Raman and fluorescence spectral features. Raman and luminescence spectra were acquired by scanning non-printed areas of books, and Raman and fluorescence maps were built by contrasting spectral parameters point by point, obtaining a micron-scale space resolved imaging of the degradation pattern. Complementary information on paper morphology and surface compactness were obtained by high-resolution scanning electron and atomic force microscopy. The proposed non-destructive procedure is particularly interesting for precious and ancient samples to analyze their degradation processes and to evaluate the performance and effectiveness of restoration treatments.     

Raman Spectra of Laser Crystals NAB and NYAB and the Ligand Vibrations of Their Active Ions

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Spatial tissue distribution of polyacetylenes in carrot root

The presented results show the usefulness of Raman spectroscopy in the investigation of polyacetylenes in carrot root. The components are measured directly in the plant tissue without any preliminary sample preparation. Compared with the strong polyacetylene signals the spectral impact of the surrounding biological matrix is weak, except for carotenoids, and therefore it does not contribute significantly to the obtained results. Three different Raman mapping techniques applied here have revealed essential information about the investigated compounds. Using point acquisition several spectra have been measured to demonstrate the complex composition of the polyacetylene fraction in carrot root. The molecular structures of falcarinol, falcarindiol and falcarindiol 3-acetate are similar but their Raman spectra exhibit differences demonstrated by the shift of their -C triple bond C- mode. Line mapping performed along the diameter of transversely cut carrot roots has been used to investigate the relative concentration of polyacetylenes and carotenoids. An area map provides detailed information regarding the distribution of both components. It has been found that high accumulation of polyacetylenes is located in the outer section of the root, namely the pericyclic parenchyma, and in the phloem part close to the secondary cambium. The highest concentration of carotenes is seen in the immediate vicinity to polyacetylene conglomerates.     

Investigation of skin and skin lesions by NIR-FT-Raman spectroscopy

There is a vast demand for in vivo methods for the detection of skin cancer, one of the most dangerous skin lesions. Use of near infrared Fourier transform (NIR-FT)-Raman spectroscopy virtually eliminates the fluorescence of the normal cell constituents and provides a signal to noise ratio, r _SN, large enough to successfully evaluate the spectra using chemometric methods. A novel fiber optic probe for NIR-FT-Raman spectroscopy was used, which allows sterilization and the prevention of hazards due to laser radiation and makes in vivo measurements possible. The Raman spectra of normal skin are dominated by the connective tissue, mainly collagen type I. The Raman spectra of skin with inflammatory diseases show an increased lipid and water content. Kaposi sarcomas show typical features of tumors mainly in the amide III and the protein backbone range. A clear separation of Raman spectra of normal skin from those of benign and malignant neoplasms can be achieved by cluster analysis. However, the unequivocal diagnosis of skin cancer needs investigation of a larger number of more defined skin samples, taking into consideration the concurrent appearance of different skin symptoms like coloring and inflammation. Received: 25 July 1997 / Revised: 16 September 1997 / Accepted: 20 September 1997     

Resonance Raman spectra of cytochrome C and oxyhemoglobin using pulsed laser excitation and optical multichannel detection

The resonance Raman spectra of dilute cytochrome c and oxyhemoglobin solutions obtained with high peak power (>1 MW) laser excitation at 532.0 nm and optical multichannel analysis (OMA) are presented. The frequencies and intensities of bands in resonance Raman spectra obtained under these conditions are directly comparable to spectra derived from 0.15 W peak power excitation using cw lasers. No anomalous spectral features are observed. These pulsed-laser/OMA spectra are used to comment on the applicability of such techniques for time-resolved resonance Raman spectroscopy of biopolymers.     

Raman microspectroscopic mapping: a tool for the characterisation of polymer surfaces

Raman mapping by point illumination of polymer surfaces is discussed with examples taken from the plasma treatment of polypropylene (PP) and subsequent grafting of polystyrene (PS). Maps can be constructed for surface properties such as crystallinity, blend components and distribution of grafted PS. The Raman sampling volume was estimated for confocal operation using a 50x objective lens.     

Use of the Gerchberg–Saxton algorithm in optimal coherent anti-Stokes Raman spectroscopy

We are utilizing recent advances in ultrafast laser technology and recent discoveries in optimal shaping of laser pulses to significantly enhance the stand-off detection of explosives via control of molecular processes at the quantum level. Optimal dynamic detection of explosives is a method whereby the selectivity and sensitivity of any of a number of nonlinear spectroscopic methods are enhanced using optimal shaping of ultrafast laser pulses. We have recently investigated the Gerchberg–Saxton algorithm as a method to very quickly estimate the optimal spectral phase for a given analyte from its spontaneous Raman spectrum and the ultrafast laser pulse spectrum. Results for obtaining selective coherent anti-Stokes Raman spectra (CARS) for an analyte in a mixture, while suppressing the CARS signals from the other mixture components, are compared for the Gerchberg–Saxton method versus previously obtained results from closed-loop machine-learning optimization using evolutionary strategies.