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.

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

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

New strategies for the chemical characterization of multi-walled carbon nanotubes and their derivatives

Industrially relevant characterization of multi-walled carbon nanotubes (MWCNT) is still a challenging task. The aim of this work is to show novel and fast concepts for the chemical characterization of carbon nanotubes (CNT) by a combination of analytical techniques. Information obtained by individual tools like Fourier transform infrared spectroscopy (FTIR), attenuated total reflection infrared spectroscopy or Raman spectroscopy is not providing a full picture of the functionalization of MWCNTs. However, a combination of tools such as FTIR or mass spectrometry with thermogravimetric methods proved to be very useful. Sample preparation for FTIR and Raman spectroscopy is another focus of this contribution because of its strong effect on the results obtained. We also are suggesting methods for sample preparation that lead to highly reproducibility results. Measurements have been carried out on typical CNT samples such as commercially available pristine, carboxylated and amino-functionalized MWCNTs, and on polystyrenegrafted MWCNTs. The results may serve as a guidance for the qualitative and quantitative characterization of CNT.

Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A

A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target.

Quantum dots for positional registration in live cell-based arrays

For a better understanding of complex biological processes, it is desirable to simultaneously follow the dynamics of multiple components in living cells or organisms in real time. An encoding scheme was developed that enables the observation of multiple cell populations with single-cell resolution. Specifically, different yeast cell types were labeled with quantum dots and added to an array of microwells, where they randomly self-assemble into the complementary-sized cavities. Quantum dots conjugated to cells externally, internally, or in combination generated unique optical patterns to differentiate various cell types in the array. For the model system described herein, cells were monitored for their lacZ expression levels through the processing of a fluorescent precursor by ß-galactosidase. The encoding schemes employed were independent of the reporter emission and had no affect on the cellular activity. The live cell array platform allowed analysis of hundreds of individual cells simultaneously and continuously in real time. By coupling this platform with quantum dot cell labeling, the utility of this array format is extended to mixed cellular populations.

Direct observation of bulk and surface chemical morphologies of Ginkgo biloba leaves by Fourier transform mid- and near-infrared microspectroscopic imaging

Fourier transform infrared microspectroscopy is a powerful tool to obtain knowledge about the spatial and/or temporal distributions of the chemical compositions of plants for better understanding of their biological properties. However, the chemical morphologies of plant leaves in the plane of the blade are barely studied, because sections in this plane for mid-infrared transmission measurements are difficult to obtain. Besides, native compositions may be changed by chemical reagents used when plant samples are microtomed. To improve methods for direct infrared microspectroscopic imaging of plant leaves in the plane of the blade, the bulk and surface chemical morphologies of nonmicrotomed Ginkgo biloba leaves were characterized by near-infrared transmission and mid-infrared attenuated total reflection microspectroscopic imaging. A new self-modeling curve resolution procedure was proposed to extract the spectral and concentration information of pure compounds. Primary and secondary metabolites of secretory cavities, veins, and mesophylls of Ginkgo biloba leaf blades were analyzed, and the distributions of cuticle, protein, calcium oxalate, cellulose, and ginkgolic acids on the adaxial surface were determined. By the integration of multiple infrared microspectroscopic imaging and chemometrics methods, it is possible to analyze nonmicrotomed leaves and other plant samples directly to understand their native chemical morphologies in detail.

Metabonomic classification and detection of small molecule biomarkers of malignant pleural effusions

To date, most research has been focused on the benign molecules in pleural effusions, and diagnosis of malignant ones still remains challenging. In the present study, targeting the small molecules as potential biomarkers to predict the malignancy of the effusions, the metabolic profiles of 81 clinical pleural effusions (41 malignant effusions from lung cancer and 40 benign ones) were investigated through a NMR-based metabonomic approach. In ¹H NMR analysis, a total of ten small molecules in the effusions were simultaneously determined. Significantly higher mean values of valine, lactate, and alanine and markedly lower signal intensities of acetoacetate, trimethylamine-N-oxide, and α- and β-glucose were observed in malignant pleural effusions compared with those in benign ones. DFA modeling of NMR spectra subjected to a validation allowed the malignant effusions to be discriminated from benign ones in both training and validation groups. Currently, the conventional clinical analyses on chemical constituents in effusions could not provide a reliable prediction of malignancy of the effusions; the present results revealed that the small molecules might serve as useful biomarkers for diagnosis of the effusions, and the present NMR-based metabonomic approach provided a valuable potential to rapidly and sensitively predict the malignancy of the pleural effusions.

Molecular imaging of paper cross sections by FT-IR spectroscopy and principal component analysis

The molecular imaging of paper cross sections containing the wet-strength additive poly(amidoamine)–epichlorohydrin (PAE) was effected by Fourier transform infrared (FT-IR) spectroscopic imaging. Thin cross sections of laboratory sheet samples were prepared and transferred onto CaF₂ substrates. A laboratory sheet sample without PAE acted as a reference. Principal component analysis (PCA) was applied to identify and to reveal the distribution of PAE across the section. Differences in the loading plots of the fourth and fifth principal components for the sheets with and without PAE were found in the region of the amide I, amide II, and amine bands within a variance of 0.4–0.8 %. The score images of the PCA reveal inhomogeneous distribution of PAE. Small areas of higher concentration of PAE occur across the cross section. The aim of this study was to demonstrate that FT-IR spectroscopic imaging provides spatially resolved quantitative information about the chemical composition of paper, which was successfully achieved.

Profiling of secondary metabolites in tissues from Rheum palmatum L. using laser microdissection and liquid chromatography mass spectrometry

Evaluating the quality of herbal medicines by morphological features is a convenient, quick, and practical method compared with other methods that mostly depend on modern instruments. Here, laser microdissection and ultra-performance liquid chromatography are combined with mass spectrometry to map the distribution of secondary metabolites in cells or tissues of a herb itself for correlating its bioactive components and morphological features. The root and rhizome of Rheum palmatum L. were taken as research target, which is the Chinese medicine, Radix et Rhizoma Rhei. According to fluorescent microscopic characteristics, 12 herbal cells or tissues of Radix et Rhizoma Rhei were separated by laser microdissection. Thirty-eight compounds were identified or tentatively characterized in the microdissected tissues. (+)-Catechin, 1-O-galloyl-2-O-cinnamoyl-β-d-glucose, and emodin were found to be the major components in most of the tissues. The brown ergastic substances found in rays of normal and anomalous vascular bundles as well as the parenchymatous cells of rhizome pith and the parenchymatous cells of root xylem contained higher than average amounts of these three components and more kinds of secondary metabolites. Overall, results suggest that Radix et Rhizoma Rhei of larger size and with conspicuous “brocaded patterns” and star spots are of higher quality as they tend to have greater contents of bioactive components. The study provides quantitative and specific criteria by which the quality of Radix et Rhizoma Rhei can be judged. This research also established a new, reliable, and practical method for direct profiling and imaging of secondary metabolites in any herbal tissue.

Raman microspectroscopic chemical mapping and chemometric classification for the identification of gunshot residue on adhesive tape

A novel approach utilizing automated Raman microspectroscopic mapping for gunshot residue (GSR) detection was investigated. A well-established technique for GSR recovery (tape lifting) was utilized for GSR particle collection. Uncontaminated samples of the substrate (tape), organic GSR (OGSR), and inorganic GSR (IGSR) particles were characterized to generate three respective Raman spectroscopic training sets. Automated Raman mapping was used to rapidly collect spectra over areas of the tape substrate populated with GSR particles. Raman spectra collected from the maps were classified against the training sets via partial least squares discriminant analysis (PLS-DA) to determine if GSR was present. We report the application of Raman chemical mapping as a proof of concept for the positive detection of GSR particles of varying morphologies. The estimated size of GSR particles, which could be readily detected by this method, is about 3.4 μm. The efficiency of the classification was quantitated with rates of true positives and negatives. Validation studies scrutinizing the practicality of this approach as a viable tool for potential forensics investigations are currently in progress.

Automated quantitative analysis of lipid accumulation and hydrolysis in living macrophages with label-free imaging

The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R ²?>?0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K _i. Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.

Spectroscopic detection and quantification of heme and heme degradation products

Heme and heme degradation products play critical roles in numerous biological phenomena which until now have only been partially understood. One reason for this is the very low concentrations at which free heme, its complexes and the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution, the potential of nondestructive Raman spectroscopy for the detection, quantification and discrimination of heme and heme degradation products is investigated. Resonance Raman spectroscopy using different excitation wavelengths (413, 476, 532, and 752?nm) is employed to estimate the limit of detection for hemin, myoglobin, biliverdin, and bilirubin. Concentrations in the low micromolar range (down to 3?μmol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the surface-enhanced Raman spectroscopy (SERS) detection of hemin in the presence of other cellular components, such as the highly similar cytochrome c, DNA, and the important antioxidant glutathione, is presented. A microfluidic device was used to reproducibly create a segmented flow of aqueous droplets and oil compartments. Those aqueous droplets acted as model chambers where the analytes have to compete for the colloid. With the help of statistical analysis, it was possible to detect and differentiate the pure substances as well as the binary mixtures and gain insights into their interaction.

Temperature-assisted ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for the determination of PCBs and PBDEs in water and urine samples

We report on silver–gold core-shell nanostructures that contain Methylene Blue (MB) at the gold–silver interface. They can be used as reporter molecules in surface-enhanced Raman scattering (SERS) labels. The labels are stable and have strong SERS activity. TEM imaging revealed that these nanoparticles display bright and dark stripe structures. In addition, these labels can act as probes that can be detected and imaged through the specific Raman signatures of the reporters. We show that such SERS probes can identify cellular structures due to enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the core-shell nanostructures. They also provide structural information on the cellular environment as demonstrated for these nanoparticles as new SERS-active and biocompatible substrates for imaging of live cells.

Mirion—A Software Package for Automatic Processing of Mass Spectrometric Images

Mass spectrometric imaging (MSI) techniques are of growing interest for the Life Sciences. In recent years, the development of new instruments employing ion sources that are tailored for spatial scanning allowed the acquisition of large data sets. A subsequent data processing, however, is still a bottleneck in the analytical process, as a manual data interpretation is impossible within a reasonable time frame. The transformation of mass spectrometric data into spatial distribution images of detected compounds turned out to be the most appropriate method to visualize the results of such scans, as humans are able to interpret images faster and easier than plain numbers. Image generation, thus, is a time-consuming and complex yet very efficient task. The free software package “Mirion,” presented in this paper, allows the handling and analysis of data sets acquired by mass spectrometry imaging. Mirion can be used for image processing of MSI data obtained from many different sources, as it uses the HUPO-PSI-based standard data format imzML, which is implemented in the proprietary software of most of the mass spectrometer companies. Different graphical representations of the recorded data are available. Furthermore, automatic calculation and overlay of mass spectrometric images promotes direct comparison of different analytes for data evaluation. The program also includes tools for image processing and image analysis.

Analysis of human blood plasma and hen egg white by chiroptical spectroscopic methods (ECD, VCD, ROA)

Chiroptical methods are widely used in structural and conformational analyses of biopolymers. The application of these methods to investigations of biofluids would provide new avenues for the molecular diagnosis of protein-misfolding diseases. In this work, samples of human blood plasma and hen egg white were analyzed using a combination of conventional and chiroptical methods: ultraviolet absorption/electronic circular dichroism (UV/ECD), Fourier transform infrared absorption/vibrational circular dichroism (FTIR/VCD), and Raman scattering/Raman optical activity (Raman/ROA). For comparison, the main components of these substances—human serum albumin (HSA) and ovalbumin (Ova)—were also analyzed by these methods. The ultraviolet region of the ECD spectrum was analyzed using the CDNN CD software package to evaluate the secondary structures of the proteins. The UV/ECD, FTIR/VCD, and Raman/ROA spectra of the substances were quite similar to those of the corresponding major proteins, while some differences were also detected and explained. The conclusions drawn from the FTIR/VCD and Raman/ROA data were in good agreement with the secondary structures calculated from ECD. The results obtained in this work demonstrate that the chiroptical methods used here can be applied to analyze not only pure protein solutions but also more complex systems, such as biological fluids.

Patterned growth of vertically aligned silicon nanowire arrays for label-free DNA detection using surface-enhanced Raman spectroscopy

Patterning is of paramount importance in many areas of modern science and technology. As a good candidate for novel nanoscale optoelectronics and miniaturized molecule sensors, vertically aligned silicon nanowire (SiNW) with controllable location and orientation is highly desirable. In this study, we developed an effective procedure for the fabrication of vertically aligned SiNW arrays with micro-sized features by using single-step photolithography and silver nanoparticle-induced chemical etching at room temperature. We demonstrated that the vertically aligned SiNW arrays can be used as a platform for label-free DNA detection using surface-enhanced Raman spectroscopy (SERS), where the inherent “fingerprint” SERS spectra allows for the differentiation of closely related biospecies. Since the SiNW array patterns could be modified by simply varying the mask used in the photolithographic processing, it is expected that the methodology can be used to fabricate label-free DNA microarrays and may be applicable to tissue engineering, which aims to create living tissue substitutes from cells seeded onto 3D scaffolds.

Chloroaluminum phthalocyanine thin films: chemical reaction and molecular orientation

The chemical transformation of the polar chloroaluminum phthalocyanine, AlClPc, to μ-(oxo)bis(phthalocyaninato)aluminum(III), (PcAl)₂O, in thin films on indium tin oxide is studied and its influence on the molecular orientation is discussed. The studies were conducted using complementary spectroscopic techniques: Raman spectroscopy, X-ray photoelectron spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. In addition, density functional theory calculations were performed in order to identify specific vibrations and to monitor the product formation. The thin films of AlClPc were annealed in controlled environmental conditions to obtain (PcAl)₂O. It is shown that the chemical transformation in the thin films can proceed only in the presence of water. The influence of the reaction and the annealing on the molecular orientation was studied with Raman spectroscopy and NEXAFS spectroscopy in total electron yield and partial electron yield modes. The comparison of the results obtained from these techniques allows the determination of the molecular orientation of the film as a function of the probing depth.

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.

Pharmaceutical Analysis by Solid-Substrate Electrospray Ionization Mass Spectrometry with Wooden Tips

Electrospray ionization (ESI) using wooden tips as solid substrates allows direct ionization of various samples and their simple and efficient analyses by mass spectrometry (MS). In this study, wooden-tip ESI-MS was used for pharmaceutical analysis. A wide variety of active components present in pharmaceuticals with forms of tablets, capsules, granules, dry suspensions, suspensions, drops, and oral liquids, etc., were all successfully ionized directly for mass spectrometric analysis. Trace degradation products were also sensitively detected using wooden-tip ESI-MS. This strategy was extended to construct chemical fingerprints of herbal products containing complex and unknown components, and the fingerprints provided valuable information for their quality assessment and origin tracing. Our experimental data demonstrated that wooden-tip ESI-MS is a useful tool for rapid pharmaceutical analysis, with high sensitivity and wide applicability, showing promising perspectives for quality assessment and control, authentication, and origin tracing of pharmaceuticals.

Implementation of a Gaussian Beam Laser and Aspheric Optics for High Spatial Resolution MALDI Imaging MS

We have investigated the use of a Gaussian beam laser for MALDI Imaging Mass Spectrometry to provide a precisely defined laser spot of 5 μm diameter on target using a commercial MALDI TOF instrument originally designed to produce a 20 μm diameter laser beam spot at its smallest setting. A Gaussian beam laser was installed in the instrument in combination with an aspheric focusing lens. This ion source produced sharp ion images at 5 μm spatial resolution with signals of high intensity as shown for images from thin tissue sections of mouse brain.