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.

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.

Hyperspectral unmixing of Raman micro-images for assessment of morphological and chemical parameters in non-dried brain tumor specimens

Hyperspectral unmixing is an unsupervised algorithm to calculate a bilinear model of spectral endmembers and abundances of components from Raman images. Thirty-nine Raman images were collected from six glioma brain tumor specimens. The tumor grades ranged from astrocytoma WHO II to glioblastoma multiforme WHO IV. The abundance plots of the cell nuclei were processed by an image segmentation procedure to determine the average nuclei size, the number of nuclei, and the fraction of nuclei area. The latter two morphological parameters correlated with the malignancy. A combination of spectral unmixing and non-negativity constrained linear least squares fitting is introduced to assess chemical parameters. First, endmembers of the most abundant and most dissimilar components were defined that represent all data sets. Second, the content of the obtained components’ proteins, nucleic acids, lipids, and lipid to protein ratios were determined in all Raman images. Except for the protein content, all chemical parameters correlated with the malignancy. We conclude that the morphological and chemical information offer new ways to develop Raman-based classification approaches that can complement diagnosis of brain tumors. The role of non-linear Raman modalities to speed-up image acquisition is discussed.

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.

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.

The effect of optical substrates on micro-FTIR analysis of single mammalian cells

The study of individual cells with infrared (IR) microspectroscopy often requires living cells to be cultured directly onto a suitable substrate. The surface effect of the specific substrates on the cell growth—viability and associated biochemistry—as well as on the IR analysis—spectral interference and optical artifacts—is all too often ignored. Using the IR beamline, MIRIAM (Diamond Light Source, UK), we show the importance of the substrate used for IR absorption spectroscopy by analyzing two different cell lines cultured on a range of seven optical substrates in both transmission and reflection modes. First, cell viability measurements are made to determine the preferable substrates for normal cell growth. Successively, synchrotron radiation IR microspectroscopy is performed on the two cell lines to determine any genuine biochemically induced changes or optical effect in the spectra due to the different substrates. Multivariate analysis of spectral data is applied on each cell line to visualize the spectral changes. The results confirm the advantage of transmission measurements over reflection due to the absence of a strong optical standing wave artifact which amplifies the absorbance spectrum in the high wavenumber regions with respect to low wavenumbers in the mid-IR range. The transmission spectra reveal interference from a more subtle but significant optical artifact related to the reflection losses of the different substrate materials. This means that, for comparative studies of cell biochemistry by IR microspectroscopy, it is crucial that all samples are measured on the same substrate type.

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.

Rapid and sensitive detection of bacteria via platinum-labeled antibodies and on-particle ionization and enrichment prior to MALDI-TOF mass spectrometry

We introduce a rapid and sensitive approach to study the interactions of an affinity probe with the bacterial wall. Immunoglobulin was immobilized on platinum nanoparticles, and the resulting probe nanoparticles bind to bacterial walls as confirmed by transmission electron microscopy. A MALDI-MS assay was developed that can detect ~10⁵ cfu mL^?1 of S. marcescens and E. coli. This approach enables simple, rapid and straightforward detection of bacterial proteins, with high resolution and sensitivity, and without the requirement for tedious washing/separation steps.

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.

Electrode interfaces switchable by physical and chemical signals for biosensing, biofuel, and biocomputing applications

This review outlines advances in designing modified electrodes with switchable properties controlled by various physical and chemical signals. Irradiation of the modified electrode surfaces with various light signals, changing the temperature of the electrolyte solution, application of a magnetic field or electrical potentials, changing the pH of the solutions, and addition of chemical/biochemical substrates were used to change reversibly the electrode activity. The increasing complexity in the signal processing was achieved by integration of the switchable electrode interfaces with biomolecular information processing systems mimicking Boolean logic operations, thus allowing activation and inhibition of electrochemical processes on demand by complex combinations of biochemical signals. The systems reviewed range from simple chemical compositions to complex mixtures modeling biological fluids, where the signal substrates were added at normal physiological and elevated pathological concentrations. The switchable electrode interfaces are considered for future biomedical applications where the electrode properties will be modulated by the biomarker concentrations reflecting physiological conditions.

Glycosaminoglycan profiling in different cell types using infrared spectroscopy and imaging

We recently identified vibrational spectroscopic markers characteristic of standard glycosaminoglycan (GAG) molecules. The aims of the present work were to further this investigation to more complex biological systems and to characterize, via their spectral profiles, cell types with different capacities for GAG synthesis. After recording spectral information from individual GAG standards (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate) and GAG-GAG mixtures, GAG-defective mutant Chinese hamster ovary (CHO)-745 cells, wild-type CHO cells, and chondrocytes were analyzed as suspensions by high-throughput infrared spectroscopy and as single isolated cells by infrared imaging. Spectral data were processed and interpreted by exploratory unsupervised chemometric methods based on hierarchical cluster analysis and principal component analysis. Our results showed that the spectral information obtained was discriminant enough to clearly delineate between the different cell types both at the cell suspension and single-cell levels. The abilities of the technique are to perform spectral profiling and to identify single cells with different potentials to synthesize GAGs. Infrared microspectroscopy/imaging could therefore be developed for cell screening purposes and further for identifying GAG molecules in normal tissues during physiological conditions (aging, healing process) and numerous pathological states (arthritis, cancer). Figure

FTIR imaging for profiling GAG-synthesizing cells     

Perceiving the chemical language of Gram-negative bacteria: listening by high-resolution mass spectrometry

Gram-negative bacteria use N-acylhomoserine lactones (AHLs) as their command language to coordinate population behavior during invasion and colonization of higher organisms. Although many different bacterial bioreporters are available for AHLs monitoring, in which a phenotypic response, e.g. bioluminescence, violacin production, and β-galactosidase activity, is exploited, mass spectrometry (MS) is the most versatile detector for rapid analysis of AHLs in complex microbial samples, with or without prior separation steps. In this paper we critically review recent advances in the application of high-resolution MS to analysis of the quorum sensing (QS) signaling molecules used by Gram-negative bacteria, with much emphasis on AHLs. A critical review of the use of bioreporters in the study of AHLs is followed by a short methodological survey of the capabilities of high-resolution mass spectrometry (HRMS), including Fourier-transform ion cyclotron resonance (FTICR) MS and quadrupole time-of-flight (qTOF) MS. Use of infusion electrospray ultrahigh-resolution FTICR MS (12 Tesla) enables accurate mass measurements for determination of the elemental formulas of AHLs in Acidovorax sp. N35 and Burkholderia ubonensis AB030584. Results obtained by coupling liquid chromatography with a hybrid quadrupole linear ion trap-FTICR mass spectrometer (LC–LTQ-FTICRMS, 7-T) for characterization of acylated homoserine lactones in the human pathogen Pseudomonas aeruginosa are presented. UPLC–ESI-qTOF MS has also proved to be suitable for identification of 3O-C₁₀HSL in Pseudomonas putida IsoF cell culture supernatant. Aspects of sample preparation and the avoidance of analytical pitfalls are also emphasized.

Atomic force microscopy of model lipid membranes

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Overcoming the adverse effects of crosslinking in biosensors via addition of PEG: Improved sensing of hydrogen peroxide using immobilized peroxidase

Glutaraldehyde (GA) is widely used as a crosslinker to immobilize enzymes, for examples in biosensors, but often causes partial denaturation. We find that the proper use of poly(ethylene glycol) (PEG) during the crosslinking process can fully preserve the native state and activity of horseradish peroxidase (HRP). An amperometric biosensor was developed based on these findings for the direct determination of hydrogen peroxide. UV-Vis and FTIR spectroscopy reveal that the HRP entrapped in a polypyrrole matrix retains its native structure. The addition of PEG increases the sensitivity and stability of the biosensor and prevents many of effects caused by intra-crosslinking via GA. The biosensor was operated at a potential of ?350?mV (vs Ag/AgCl) without any mediator and gave a linear response to H₂O₂ in the 5 to 190???M concentration range. The apparent Michaelis-Menten constant is 3.37?mM, and maximal current is as high as 3.43???A. The surface of the biosensor was characterized by atomic force microscopy operated in the tapping mode.

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.

Spectroscopic analysis of bacterial biological warfare simulants and the effects of environmental conditioning on a bacterial spectrum

The ability to distinguish bacteria from mixed samples is of great interest, especially in the medical and defence arenas. This paper reports a step towards the aim of differentiating pathogenic endospores in situ, to aid any required response for hazard management using infrared spectroscopy combined with multivariate analysis. We describe a proof-of-principle study aimed at discriminating biological warfare simulants from common environmental bacteria. We also report an evaluation of multiple pre-processing techniques and subsequent differences in cross-validation of two pattern recognition models (Support Vector Machines and Principal Component–Linear Discriminant Analysis) for a six-class classification (bacterial classification). These classifications were possible with an average sensitivity of 88.0 and 86.9?%, and an average specificity of 97.6 and 97.5?% for the SVM and the PC-LDA models, respectively. Most spectroscopic models are built upon spectra from bacteria that have been specifically prepared for analysis by a particular method; this paper will comment upon the differences in the bacterial spectrum that occur between specific preparations when the bacteria have spent 30?days in the simulated weather conditions of a hot dry climate.

Spectral signatures for the classification of microbial species using Raman spectra

In general, classification-based methods based on confocal Raman microscopy are focused on targeted studies under which the spectral libraries are collected under controlled instrument parameters, which facilitate analyses via standard multivariate data analysis methods and cross-validation. We develop and compare approaches to transform spectra collected at different spectral ranges and varying levels of resolution into a single lower-dimension spectral signature library. This will result in a more robust analysis method able to accommodate spectra accumulated at different times and conditions. We demonstrate these approaches on a relevant test case; the identification of microbial species from a natural environment. The training data were based on samples prepared for three unique species collected at two time points and the test data consisted of blinded unknowns prepared and analyzed at a later date with different instrument parameters. The results indicate that using reduced dimension representations of the spectral signatures improves classification accuracy over basic alignment protocols. In particular, utilizing the microbial species partial least squares discriminant analysis classifier on the blinded samples based on alignment achieved ~78 % accuracy, while both binning and peak selection approaches yielded 100 % accuracy.

Comprehensive and simultaneous coverage of lipid and polar metabolites for endogenous cellular metabolomics using HILIC-TOF-MS

The comprehensive metabolomic analyses using eukaryotic and prokaryotic cells are an effective way to identify biomarkers or biochemical pathways which can then be used to characterize disease states, differences between cell lines or inducers of cellular stress responses. One of the most commonly used extraction methods for comprehensive metabolomics is the Bligh and Dyer method (BD) which separates the metabolome into polar and nonpolar fractions. These fractions are then typically analysed separately using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) liquid chromatography (LC), respectively. However, this method has low sample throughput and can also be biased to either polar or nonpolar metabolites. Here, we introduce a MeOH/EtOH/H₂O extraction paired with HILIC-time-of-flight (TOF)-mass spectrometry (MS) for comprehensive and simultaneous detection of both polar and nonpolar metabolites that is compatible for a wide array of cellular species cultured in different growth media. This method has been shown to be capable of separating polar metabolites by a HILIC mechanism and classes of lipids by an adsorption-like mechanism. Furthermore, this method is scalable and offers a substantial increase in sample throughput compared to BD with comparable extraction efficiency. This method was able to cover 92.2 % of the detectable metabolome of Gram-negative bacterium Sinorhizobium meliloti, as compared to 91.6 % of the metabolome by a combination of BD polar (59.4 %) and BD nonpolar (53.9 %) fractions. This single-extraction HILIC approach was successfully used to characterize the endometabolism of Gram-negative and Gram-positive bacteria as well as mammalian macrophages.

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.

High-resolution MALDI-TOF mass spectrometry of bacterial proteins using a Tris-EDTA buffer approach

The performance of matrix assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF) of bacterial proteins strongly depends on sample preparation. It is found that the mass spectral profiles obtained from direct MALDI-TOF MS of the protein extracts are much weaker for individual bacterial cells than compared to those prepared by the Tris-EDTA buffer approach (TEBA). Characteristic mass spectral peaks were observed in the mass range from 3,000 to 15,000?Da. The mass peaks reported earlier and claimed to serve as species-specific biomarkers are consistently found here as well. Mass peaks at m/z of 3636, 5466, 5750, 6315, 6547, 7274, 9192, and 9742 are found for Escherichia coli studied and assigned as specific biomarkers. Similarly, specific mass peaks have been identified at m/z 5443, 7270, 7724, and 9888 for Bacillus subtilis, and at 3603, 5496, 6800, 8858 and 9531 for Serratia marcescens. The detection limits for the three target bacteria range from 2.4?×?10⁵ to 3.3?×?10⁵?cfu·mL^-1. We conclude that the TE buffer approach can produce reliable data for rapid classification, high-resolution and highly sensitive detection of bacteria.