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.

Isolating stem cells in the inter-follicular epidermis employing synchrotron radiation-based Fourier-transform infrared microspectroscopy and focal plane array imaging

Normal function and physiology of the epidermis is maintained by the regenerative capacity of this tissue via adult stem cells (SCs). However, definitive identifying markers for SCs remain elusive. Infrared (IR) spectroscopy exploits the ability of cellular biomolecules to absorb in the mid-IR region (λ?=?2.5–25?μm), detecting vibrational transitions of chemical bonds. In this study, we exploited the cell’s inherent biochemical composition to discriminate SCs of the inter-follicular skin epidermis based on IR-derived markers. Paraffin-embedded samples of human scalp skin (n?=?4) were obtained, and 10-μm thick sections were mounted for IR spectroscopy. Samples were interrogated in transmission mode using synchrotron radiation-based Fourier-transform IR (FTIR) microspectroscopy (15?×?15?μm) and also imaged employing globar-source FTIR focal plane array (FPA) imaging (5.4?×?5.4?μm). Dependent on the location of derived spectra, wavenumber–absorbance/intensity relationships were examined using unsupervised principal component analysis. This approach showed clear separation and spectral differences dependent on cell type. Spectral biomarkers concurrently associated with segregation of SCs, transit-amplifying cells and terminally-differentiated cells of epidermis were primarily PO ₂ ^? vibrational modes (1,225 and 1,080?cm^?1), related to DNA conformational alterations. FPA imaging coupled with hierarchical cluster analysis also indicated the presence of specific basal layer cells potentially originating from the follicular bulge, suggested by co-clustering of spectra. This study highlights PO ₂ ^? vibrational modes as potential putative SC markers.

Local surface plasmon resonance of single silver nanorice particles in the near-infrared

We report on the synthesis and optical spectra of silver nanorice particles. Two strong absorption bands are resolved in the near UV and near-IR region, and the dark field scattering spectra are consistent with the absorption spectra. Finite-difference time-domain simulations reveal that the peak in the IR region can be attributed to the E field that is parallel to the long axis, while the peak in the UV can be attributed to the E field perpendicular to the short axis of the silver nanorice particles.

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.

Novel μ-membrane module for online determination of the free fatty acid content in the dispersed phase of oil-in-water emulsions

Monitoring the dispersed phase of an oil-in-water (O–W) emulsion by means of Fourier transform infrared (FTIR) spectroscopy is a challenging task, restricted to the continuous phase that is in contact with the FTIR probe. Nonetheless, real-time measurement and kinetic analysis by FTIR, including analysis of the dispersed, often non-polar phase containing substrates and/or products, is desirable. Enzymatic hydrolysis of sunflower oil was performed in an O–W emulsion. After separation of the oil phase by use of a newly developed μ-membrane module, infrared spectra were collected using an attenuated total reflectance (ATR) cell. Different chemometric models were calibrated using the partial least squares (PLS) algorithm. Online application of a chemometric model based on the FTIR spectra enabled real-time monitoring of free fatty acid concentrations in the oil phase.

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.

Chemometric tool for identification of iron–gall inks by use of visible–near infrared fibre optic reflection spectroscopy

A method has been developed for identification of corrosive iron–gall inks in historical drawings and documents. The method is based on target-factor analysis of visible–near infrared fibre optic reflection spectra (VIS–NIR FORS). A set of reference spectra was obtained from model samples of laboratory-prepared inks covering a wide range of mixing ratios of basic ink components deposited on substrates and artificially aged. As criteria for correspondence of a studied spectrum with a reference spectrum, the apparent error in target (AET) and the empirical function SPOIL according to Malinowski were used. The capability of the proposed tool to distinguish corrosive iron–gall inks from bistre and sepia inks was evaluated by use of a set of control samples of bistre, sepia, and iron–gall inks. Examples are presented of analysis of historical drawings from the 15th and 16th centuries and written documents from the 19th century. The results of analysis based on the tool were confirmed by XRF analysis and colorimetric spot analysis.

Versatile synthesis of probes for high-throughput enzyme activity screening

Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination.

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.

Vacuum Ultraviolet Action Spectroscopy of Polysaccharides

We studied the optical properties of gas-phase polysaccharides (maltose, maltotetraose, and maltohexaose) ions by action spectroscopy using the coupling between a quadrupole ion trap and a vacuum ultraviolet (VUV) beamline at the SOLEIL synchrotron radiation facility (France) in the 7 to 18 eV range. The spectra provide unique benchmarks for evaluation of theoretical data on electronic transitions of model carbohydrates in the VUV range. The effects of the nature of the charge held by polysaccharide ions on the relaxation processes were also explored. Finally the effect of isomerization of polysaccharides (with melezitose and raffinose) on their photofragmentation with VUV photons is presented.

Synchrotron FTIR microspectroscopy of Escherichia coli at single-cell scale under silver-induced stress conditions

The present work was focused on elucidating biochemical changes in the model bacterium Escherichia coli exposed to ionic silver mediated stress, at a single-cell scale. In order to achieve this, in situ synchrotron Fourier-transform infrared (sFTIR) microspectroscopy was performed, for the first time, on individual cells by attenuated total reflectance (ATR) combined with the use of zinc-selenide hemisphere for high spatial resolution. In a first part, the potential of the method was evaluated on bacteria subjected to a lethal 100 μM AgNO₃ concentration for 2 h compared to untreated 100 % viable cells. Differences in cell composition were assessed for the C–H stretching and protein spectral regions, indicating that the inhibitory action was targeted against both fatty acids and proteins. Transmission electron microscopy (TEM) confirmed morphological damages of the cell ultrastructure. The relevance of ATR-sFTIR microspectroscopy for highlighting the heterogeneity in Ag⁺-mediated effects within a given bacterial population was also pointed out. In a second part, cells were exposed to sub-lethal Ag⁺ concentrations (<10 μM AgNO₃) tested under “dynamic” growth mode: early addition vs. pulse in the mid-exponential phase, and compared to simultaneously batch-grown untreated bacteria or cells sampled just before the pulse, respectively. sFTIR microspectroscopy and TEM imaging were performed in close relation with growth kinetics characterization. No significant effect of the Ag⁺ pulses was detected, in accordance with macrokinetics data. For early-treated cells, effects on fatty acid composition were shown, although no major alteration of protein secondary structure was noticed. These partial effects were consistent with TEM observations and growth kinetics.

Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common ‘capping’ unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in ‘beam-type’ CID (HCD) and ETD experiments, at the same time—since the acidic carboxyl group was ‘neutralized’—it will display a different retention time than its unmodified counterpart. We also suggest solutions that—when incorporated into existing search engines—may significantly improve the reliability of glycopeptide assignments.

Oral cancer diagnostics based on infrared spectral markers and wax physisorption kinetics

Infrared microspectroscopy is an emerging approach for disease analysis owing to its capability for in situ chemical characterization of pathological processes. Synchrotron-based infrared microspectroscopy (SR-IMS) provides ultra-high spatial resolution for profiling biochemical events associated with disease progression. Spectral alterations were observed in cultured oral cells derived from healthy, precancerous, primary, and metastatic cancers. An innovative wax-physisorption-based kinetic FTIR imaging method for the detection of oral precancer and cancer was demonstrated successfully. The approach is based on determining the residual amount of paraffin wax (C₂₅H₅₂) or beeswax (C₄₆H₉₂O₂) on a sample surface after xylene washing. This amount is used as a signpost of the degree of physisorption that altered during malignant transformation. The results of linear discriminant analysis (LDA) of oral cell lines indicated that the methylene (CH₂) and methyl group (CH₃) stretching vibrations in the range of 3,000–2,800 cm^?1 have the highest accuracy rate (89.6 %) to discriminate the healthy keratinocytes (NHOK) from cancer cells. The results of wax-physisorption-based FTIR imaging showed a stronger physisorption with beeswax in oral precancerous and cancer cells as compared with that of NHOK, which showed a strong capability with paraffin wax. The infrared kinetic study of oral cavity tissue showed a consistency in the wax physisorption of the cell lines. On the basis of our findings, these results show the potential use of wax-physisorption-based kinetic FTIR imaging for the early screening of oral cancer lesions and the chemical changes during oral carcinogenesis.

Differences in the hippocampal frequency of creatine inclusions between the acute and latent phases of pilocarpine model defined using synchrotron radiation-based FTIR microspectroscopy

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. Of the animal models developed to investigate the pathogenesis of TLE, the one with pilocarpine-induced seizures is most often used. After pilocarpine administration in animals, three distinct periods—acute, latent, and chronic—can be distinguished according to their behavior. The present paper is the continuation of our previous study which has shown an increased occurrence of creatine inclusions in rat hippocampal formations from the acute phase of pilocarpine-induced status epilepticus (SE) and positive correlation between their quantity and the total time of seizure activity within the observation period. In this paper, we tried to verify if anomalies in hippocampal creatine accumulation were the temporary or permanent effect of pilocarpine-evoked seizures. To realize this purpose, male Wistar rats in the latent phase (3 days after pilocarpine administration) were examined. The results obtained for the period when stabilization of animal behavior and EEG occurs were afterwards compared with ones obtained for the acute phase of pilocarpine-induced SE and for naive controls. To investigate the frequency of creatine inclusions within the hippocampal formation as well as in its selected areas (sectors 1–3 of Ammon’s horn (CA1–CA3), dentate gyrus (DG), and hilus of DG) and cellular layers (pyramidal, molecular, multiform, and granular cell layers), synchrotron radiation-based Fourier-transform infrared microspectroscopy was used. The applied technique, being a combination of light microscopy and infrared spectroscopy, allowed us to localize microscopic details in the analyzed samples and provided information concerning their chemical composition. Moreover, the use of a synchrotron source of IR radiation allowed us to carry out the research at the diffraction-limited spatial resolution which, because of the typical size of creatine inclusions (from a few to dozens of micrometers), was necessary for our study. The comparison of epileptic animals in the latent phase with controls showed statistically significant increase in the number of creatine inclusions for most of the analyzed hippocampal regions, all examined cellular layers, as well as the whole hippocampal formation. Moreover, for the hilus of the DG and CA3 area, the number of creatine deposits was higher in the latent than in the acute phase after pilocarpine injection. In light of the obtained results, an anomaly in the hippocampal accumulation of creatine is the long-term effect of pilocarpine-evoked seizures, and the intensity of this phenomenon may increase with time passing from the primary injury.

Synthesis of multi-branched gold nanoparticles by reduction of tetrachloroauric acid with Tris base, and their application to SERS and cellular imaging

A biosensor for hydrogen peroxide was constructed by immobilizing horseradish peroxidase on chitosan-wrapped NiFe₂O₄ nanoparticles on a glassy carbon electrode (GCE). The electron mediator carboxyferrocene was also immobilized on the surface of the GCE. UV?Cvis spectra, Fourier transform IR spectra, scanning electron microscopy, and electrochemical impedance spectra were acquired to characterize the biosensor. The experimental conditions were studied and optimized. The biosensor responds linearly to H₂O₂ in the range from 1.0?×?10^?5 to 2.0?×?10^?3?M and with a detection limit of 2.0?×?10^?6?M (at S/N?=?3).

A simple strategy for detecting synthetic polymers on solid surfaces using liquid crystal

In this study, we developed a liquid crystal (LC)-based detection method for polymer films synthesized on solid surfaces. A dark to bright transition in the optical appearance of nematic 4-cyano-4′-pentylbiphenyl (5CB) was observed after transferring a poly(methyl methacrylate) (PMMA) film onto a glass substrate functionalized with n-octyltrichlorosilane (OTS). This phenomenon indicates an orientational transition of 5CB from a homeotropic to a planar-random state. The optical response of 5CB was then evaluated directly through polymerization reactions on the OTS-functionalized glass substrate. Polymer films of PMMA, poly(glycidyl methacrylate) (PGMA), and poly(dimethylsiloxane) (PDMS) were synthesized on OTS surfaces covered with their reaction mixtures. All polymer films displayed bright signals of 5CB, which corresponded to the planar-random orientation of LCs. However, no change in orientation was observed for the control experiments. We confirmed the formation of polymer films on the OTS surface using atomic force microscopy. Overall, our results suggest that LCs can be used to construct optical monitoring systems for the product of polymerization reactions.

Label-free microcantilever-based immunosensors for highly sensitive determination of avian influenza virus H9

We report on label-free immunosensors for the highly sensitive detection of avian influenza virus. The method makes use of the microcantilevers of an atomic force microscope onto which monoclonal antibodies against avian influenza virus were covalently immobilized. The factors influencing the performance of the resulting immunosensors were optimized by measuring the deflections of the cantilever via optical reflection, and this resulted in low detection limits and a wide analytical range. The differential deflection signals revealed specific antigen binding and their intensity is proportional to the logarithm of the concentrations of the virus in solution. Under optimal conditions, the immunosensors exhibit a linear response in the 7.6 ng mL^?1 to 76 μg mL^?1 concentration range of avian influenza virus, and the detection limit is 1.9 ng mL^?1.

Particle Production in Reflection and Transmission Mode Laser Ablation: Implications for Laserspray Ionization

Particles were ablated from laser desorption and inlet ionization matrix thin films with a UV laser in reflection and transmission geometries. Particle size distributions were measured with a combined scanning mobility particle sizer (SMPS) and aerodynamic particle sizer (APS) system that measured particles in the size range from 10 nm to 20 μm. The matrixes investigated were 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 2,5-dihydroxy-acetophenone (DHAP), and 2-nitrophloroglucinol (NPG). Nanoparticles with average diameters between 20 and 120 nm were observed in both transmission and reflection geometry. The particle mass distribution was significantly different in reflection and transmission geometry. In reflection geometry, approximately equal mass was distributed between particles in the 20 to 450 nm range of diameters and particles in the 450 nm to 1.5 μm diameter range. In transmission mode, the particle mass distribution was dominated by large particles in the 2 to 20 μm diameter range. Ablation of inlet ionization matrices DHAP and NPG produced particles that were 3 to 4 times smaller compared with the other matrices. The results are consistent with ion formation by nanoparticle melting and breakup or melting and breakup of the large particles through contact with heated inlet surfaces.

Matrix Segregation as the Major Cause for Sample Inhomogeneity in MALDI Dried Droplet Spots

The segregation in dried droplet MALDI sample spots was analyzed with regard to the matrix-to-sample ratio using optical microscopy, MALDI imaging mass spectrometry (MALDI MSI) and IR imaging spectroscopy. In this context, different polymer/matrix/solvent systems usually applied in the analysis of synthetic polymers were investigated. The use of typical matrix concentrations (10 mg mL^?1) in almost every case resulted in ring patterns, whereas higher concentrated matrix solutions always led to homogeneous sample spot layers. The data revealed that segregation is predominantly caused by matrix transport in the drying droplet, whereas polymer segregation seems to be only secondary.

A surface plasmon resonance study on the optical properties of gold nanoparticles on thin gold films

We report on an investigation of the optical properties of gold nanoparticles assembled as thin films of different thickness. The nanoparticles were linked to the surface of a gold chip by dithiol reagents and studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. There is good correlation between the experimental findings and theoretical simulation, and the respective data reveal the presence of ordered nanostructures in the assemblies. The shift in the SPR angle is linearly dependent on the particle size and the ratio of the different particles. SPR spectroscopy also reveals important information in terms of the optical constants of such films. This shall be further applied to in-situ quality control in the fabrication of optoelectronic, solar cell and semiconductor devices.