The puzzling hyper-fine structure and an accurate equilibrium geometry of succinic anhydride.

An accurate semiexperimental equilibrium structure of succinic anhydride has been determined from a combination of experiment and theory. The cm-wave and mm-wave rotational spectra of succinic anhydride, 3,4-dihydrofuran-2,5-dione, were recorded in a pulsed supersonic jet using Fourier-transform microwave spectroscopy and in a free-jet using mm-wave absorption spectroscopy. Many lines in the cm-wave spectrum show fine structure and after eliminating all other possibilities the origin of this fine structure is determined to be from spin-spin interaction. Accurate rotational and quartic centrifugal distortion constants are determined. Assignments of 13C and 18O singly substituted isotopologues in natural abundance were used to obtain a substitution geometry for the heavy atoms of succinic anhydride. Theoretical approaches permitted the calculation of a Born-Oppenheimer ab initio structure and the determination of a semiexperimental equilibrium structure in which computed rovibrational corrections were utilized to convert vibrational ground state rotational constants into equilibrium constants. The agreement between the semiexperimental structure and the Born-Oppenheimer ab initio structure is excellent. Succinic anhydride has been shown to have a planar heavy atom equilibrium structure with the effects of a large amplitude vibration apparent in the resultant rotational constants.

Parasites under the Spotlight: Applications of Vibrational Spectroscopy to Malaria Research.

New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.

In vivo atomic force microscopy-infrared spectroscopy of bacteria.

A new experimental platform for probing nanoscale molecular changes in living bacteria using atomic force microscopy-infrared (AFM-IR) spectroscopy is demonstrated. This near-field technique is eminently suited to the study of single bacterial cells. Here, we report its application to monitor dynamical changes occurring in the cell wall during cell division in Staphylococcus aureus using AFM to demonstrate the division of the cell and AFM-IR to record spectra showing the thickening of the septum. This work was followed by an investigation into single cells, with particular emphasis on cell-wall signatures, in several bacterial species. Specifically, mainly cell wall components from S. aureus and Escherichia coli containing complex carbohydrate and phosphodiester groups, including peptidoglycans and teichoic acid, could be identified and mapped at nanometre spatial resolution. Principal component analysis of AFM-IR spectra of six living bacterial species enabled the discrimination of Gram-positive from Gram-negative bacteria based on spectral bands originating mainly from the cell wall components. The ability to monitor in vivo molecular changes during cellular processes in bacteria at the nanoscale opens a new platform to study environmental influences and other factors that affect bacterial chemistry.

Multispectral Atomic Force Microscopy-Infrared Nano-Imaging of Malaria Infected Red Blood Cells.

Atomic force microscopy-infrared (AFM-IR) spectroscopy is a powerful new technique that can be applied to study molecular composition of cells and tissues at the nanoscale. AFM-IR maps are acquired using a single wavenumber value: they show either the absorbance plotted against a single wavenumber value or a ratio of two absorbance values. Here, we implement multivariate image analysis to generate multivariate AFM-IR maps and use this approach to resolve subcellular structural information in red blood cells infected with Plasmodium falciparum at different stages of development. This was achieved by converting the discrete spectral points into a multispectral line spectrum prior to multivariate image reconstruction. The approach was used to generate compositional maps of subcellular structures in the parasites, including the food vacuole, lipid inclusions, and the nucleus, on the basis of the intensity of hemozoin, hemoglobin, lipid, and DNA IR marker bands, respectively. Confocal Raman spectroscopy was used to validate the presence of hemozoin in the regions identified by the AFM-IR technique. The high spatial resolution of AFM-IR combined with hyperspectral modeling enables the direct detection of subcellular components, without the need for cell sectioning or immunological/biochemical staining. Multispectral-AFM-IR thus has the capacity to probe the phenotype of the malaria parasite during its intraerythrocytic development. This enables novel approaches to studying the mode of action of antimalarial drugs and the phenotypes of drug-resistant parasites, thus contributing to the development of diagnostic and control measures.

Vibrational spectroscopy combined with transcriptomic analysis for investigation of bacterial responses towards acid stress.

The ability of bacteria to tolerate acid stress plays an important role in their growth and survival. In particular, aciduric bacteria have several survival systems that prevent cell damage from acid stress. In this study, the effect of the bacterial stress induced by pre-adaptation at different pH values on the cellular macromolecules of Lactobacillus plantarum was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy. The expression of key genes was also quantified to provide understanding of the transcriptional response of the cells to lethal acid stress conditions. Principal component analysis of the spectra exhibited marked differences in the spectral regions associated with carbohydrates, lipids, proteins, and nucleic acids for all acid-stressed cells compared to those of untreated control cells. The changes in spectroscopic and transcriptomic profiles that were observed revealed alterations in bacterial cell wall composition after acid treatment. The results suggest the existence of a complex bacterial stress response in which modifications of cellular compounds from pre-adaption at low pH are involved. This study demonstrates the potential application of vibrational spectroscopy techniques to discriminate between intact and injured bacterial cells as well as to study their stress responses after exposure to acid environments during food processing.

In situ quantification of ß-carotene partitioning in oil-in-water emulsions by confocal Raman microscopy.

Confocal Raman microscopy (CRM) was able to quantify the ß-carotene concentration in oil droplets and determine the partitioning characteristics of ß-carotene within the emulsion system in situ. The results were validated by a conventional method involving solvent extraction of ß-carotene separately from the total emulsion as well as the aqueous phase separated by centrifugation, and quantification by absorption spectrophotometry. CRM also enabled the localization of ß-carotene in an emulsion. From the Raman image, the ß-carotene partitioning between the aqueous and oil phases of palm olein-in-water emulsions stabilized by whey protein isolate (WPI) was observed. Increasing the concentration of ß-carotene in an emulsion (from 0.1 to 0.3g/kg emulsion) with a fixed gross composition (10% palm olein:2% WPI) decreased the concentration of ß-carotene in the oil droplet. CRM is a powerful tool for in situ analyses of components in heterogeneous systems such as emulsions.

Simultaneous ATR-FTIR Based Determination of Malaria Parasitemia, Glucose and Urea in Whole Blood Dried onto a Glass Slide.

New diagnostic tools that can detect malaria parasites in conjunction with other diagnostic parameters are urgently required. In this study, Attenuated Total Reflection Fourier transform infrared (ATR-FTIR) spectroscopy in combination with Partial Least Square Discriminant Analysis (PLS-DA) and Partial Least Square Regression (PLS-R) have been applied as a point-of-care test for identifying malaria parasites, blood glucose, and urea levels in whole blood samples from thick blood films on glass slides. The specificity for the PLS-DA was found to be 98% for parasitemia levels >0.5%, but a rather low sensitivity of 70% was achieved because of the small number of negative samples in the model. In PLS-R the Root Mean Square Error of Cross Validation (RMSECV) for parasite concentration (0-5%) was 0.58%. Similarly, for glucose (0-400 mg/dL) and urea (0-250 mg/dL) spiked samples, relative RMSECVs were 16% and 17%, respectively. The method reported here is the first example of multianalyte/disease diagnosis using ATR-FTIR spectroscopy, which in this case, enabled the simultaneous quantification of glucose and urea analytes along with malaria parasitemia quantification using one spectrum obtained from a single drop of blood on a glass microscope slide.

The radio spectra of planar aromatic heterocycles: how to quantify and predict the negative inertial defects.

The simplest tricyclic aromatic nitrogen heterocyclic molecules 5,6-benzoquinoline and 7,8-benzoquinoline are possible candidates for detection of aromatic systems in the interstellar medium. Therefore the pure rotational spectra have been recorded using frequency-scanned Stark modulated, jet-cooled millimetre wave absorption spectroscopy (48-87 GHz) and Fourier Transform Microwave (FT-MW) spectroscopy (2-26 GHz) of a supersonic rotationally cold molecular jet. Guided by theoretical molecular orbital predictions, spectral analysis of mm-wave spectra, and higher resolution FT-MW spectroscopy provided accurate rotational and centrifugal distortion constants together with 14N nuclear quadrupole coupling constants for both species. The tricyclic frames of these species undergo low energy out-of-plane zero-point vibrations resulting in deviations from the moments of inertia that the rigid structure would exhibit. The determined inertial defects, along with those of similar species are used to develop an empirical formula for calculation of inertial defects of aromatic ring systems. The predictive ability of the formula is shown to be excellent in general for planar species with a number of pronounced out-of-plane vibrations. The resultant constants for the benzoquinolines are of sufficient accuracy to be used in astrophysical searches for planar aromatic heterocycles.

Screening of Wolbachia Endosymbiont Infection in Aedes aegypti Mosquitoes Using Attenuated Total Reflection Mid-Infrared Spectroscopy.

Dengue fever is the most common mosquito transmitted viral infection afflicting humans, estimated to generate around 390 million infections each year in over 100 countries. The introduction of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquitoes has the potential to greatly reduce the public health burden of the disease. This approach requires extensive polymerase chain reaction (PCR) testing of the Wolbachia-infection status of mosquitoes in areas where Wolbachia-A. aegypti are released. Here, we report the first example of small organism mid-infrared spectroscopy where we have applied attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy and multivariate modeling methods to determine sex, age, and the presence of Wolbachia (wMel strain) in laboratory mosquitoes and sex and age in field mosquitoes. The prediction errors using partial least squares discriminant analysis (PLS-DA) discrimination models for laboratory studies on independent test sets ranged from 0 to 3% for age and sex grading and 3% to 5% for Wolbachia infection diagnosis using dry mosquito abdomens while field study results using an artificial neural network yielded a 10% error. The application of FT-IR analysis is inexpensive, easy to use, and portable and shows significant potential to replace the reliance on more expensive and laborious PCR assays.

Effect of Drying Methods on Protein and DNA Conformation Changes in Lactobacillus rhamnosus GG Cells by Fourier Transform Infrared Spectroscopy.

Microencapsulation protects cells against environmental stress encountered during the production of probiotics, which are used as live microbial food ingredients. Freeze-drying and spray-drying are used in the preparation of powdered microencapsulated probiotics. This study examines the ability of Fourier transform infrared (FTIR) spectroscopy to detect differences in cells exposed to freeze-drying and spray-drying of encapsulated Lactobacillus rhamnosus GG cells. The FTIR analysis clearly demonstrated there were more significant molecular changes in lipid, fatty acid content, protein, and DNA conformation of nonencapsulated compared to encapsulated bacterial cells. The technique was also able to differentiate between spray-dried and freeze-dried cells. The results also revealed the extent of protection from a protein-carbohydrate-based encapsulant matrix on the cells depending on the type drying process. The extent of this protection to the dehydration stress was shown to be less in spray-dried cells than in freeze-dried cells. This suggests that FTIR could be used as a rapid, noninvasive, and real-time measurement technique to detect detrimental drying effects on cells.

Raman spectroscopic analysis of Lactobacillus rhamnosus GG in response to dehydration reveals DNA conformation changes.

Dehydration of bacterial cells elicits cellular stress responses in bacteria. Microencapsulation has been used to protect cells against the environmental stress. In this study, Confocal Raman Spectroscopy was used to examine DNA changes in the chemical composition of non-encapsulated and microencapsulated Lactobacillus rhamnosus GG and the reversibility of these changes upon freeze drying and rehydration. The viability of cells upon freeze drying was also enumerated using culture methods and membrane integrity was measured using BacLight Live/Dead staining. Raman analyses show changes in the spectral features associated with various biochemical compounds, which are interpreted as the result of detrimental freeze drying effects on the bacterial cells. Specifically, analyses based on Principal Components Analysis (PCA) of Raman spectra, confirm that microencapsulation protects cells from environmental stress. The results also reveal a B- to A-like DNA conformation change in dormant cells that provided insights into the extent of reversibility of this transition upon rehydration. The extent of this reversibility is less in non-encapsulated than in microencapsulated cells. These findings indicate the potential application of Raman spectroscopy in rapid sensing of microbial dehydration stress responses.

Heavy snow: IR spectroscopy of isotope mixed crystalline water ice.

Mid-infrared spectra have been measured for crystalline water ice aerosols of widely varied H/D isotopic composition. Particles with diameters ranging from 10-200 nm were generated via rapid collisional cooling with a cold buffer gas over a range of temperatures from 7-200 K. In near isotopically pure ices, the νL band position is slightly red-shifted with increasing temperature whilst in the ν2 region apparently anomalous shifts in peak maxima are explained by the contribution of a broad 2νL band of H2O and a 3νL band of D2O together with ν2 intensity that is particularly weak in low temperature crystalline ice. The hydrogen bonded OH (or OD) oscillator bands of near pure H2O (or D2O) ices are blue-shifted with temperature, with a gradient very similar to that of the corresponding band in isotope diluted samples, HOD in D2O (or H2O). It implies that this observed temperature trend is predominantly due to the intrinsic change in local hydride stretch potential energy, rather than to changes in intermolecular coupling. However, it is also observed that the narrow hydride stretch bands of an isotope diluted sample rapidly develop sub-band structure as the oscillator concentration increases, evidence of strong intermolecular coupling and a high degree of delocalisation. Anomalous blue-shifts in the OD stretch profile as D2O concentration grows is attributable to Fermi resonance with 2ν2 of D2O, in much closer proximity than the corresponding H2O levels. Theoretical results from a mixed quantum/classical approach are used to validate these findings in the hydride stretching region. Theory qualitatively reproduces the experimental trends as a function of temperature and isotopic variance.

Conformational steering in dicarboxy acids: the native structure of succinic acid.

Succinic acid, a dicarboxylic acid molecule, has been investigated spectroscopically with computational support to elucidate the complex aspects of its conformational composition. Due to the torsional freedom of the carbon backbone and hydroxy groups, a large number of potentially plausible conformers can be generated with an indication that the gauche conformer is favored over the trans form. The microwave and millimeter wave spectra have been analyzed and accurate spectroscopic constants have been derived that correlate best with those of the lowest energy gauche conformer. For an unambiguous conformational identification measurements were extended to the monosubstituted isotopologues, precisely determining the structural properties. Besides bond distances and angles, particularly the dihedral angle has been determined to be 67.76(11)°, confirming the anomalous tendency of the methylene units to favor gauche conformers when a short aliphatic segment is placed between two carbonyl groups.

Synchrotron-FTIR microspectroscopy enables the distinction of lipid accumulation in thraustochytrid strains through analysis of individual live cells.

The superior characteristics of high photon flux and diffraction-limited spatial resolution achieved by synchrotron-FTIR microspectroscopy allowed molecular characterization of individual live thraustochytrids. Principal component analysis revealed distinct separation of the single live cell spectra into their corresponding strains, comprised of new Australasian thraustochytrids (AMCQS5-5 and S7) and standard cultures (AH-2 and S31). Unsupervised hierarchical cluster analysis (UHCA) indicated close similarities between S7 and AH-7 strains, with AMCQS5-5 being distinctly different. UHCA correlation conformed well to the fatty acid profiles, indicating the type of fatty acids as a critical factor in chemotaxonomic discrimination of these thraustochytrids and also revealing the distinctively high polyunsaturated fatty acid content as key identity of AMCQS5-5. Partial least squares discriminant analysis using cross-validation approach between two replicate datasets was demonstrated to be a powerful classification method leading to models of high robustness and 100% predictive accuracy for strain identification. The results emphasized the exceptional S-FTIR capability to perform real-time in vivo measurement of single live cells directly within their original medium, providing unique information on cell variability among the population of each isolate and evidence of spontaneous lipid peroxidation that could lead to deeper understanding of lipid production and oxidation in thraustochytrids for single-cell oil development.

Comparison of FTIR transmission and transfection substrates for canine liver cancer detection.

FTIR spectroscopy is a widely used technique that provides insights into disease processes at the molecular level. Due to its numerous advantages it is becoming an increasingly powerful tool for the study of biological materials and has the potential to become an excellent diagnostic method, especially considering the low cost of transflection substrates. However, questions about the usefulness of the transflection measurement mode due to the complicated nature of physical processes occurring during the measurement and in particular the Electric Field Standing Wave (EFSW) effect have been raised. In this paper we present a comparison of the two most common FT-IR measurement modes: transmission and transfection using healthy and pathologically altered tissue (histiocytic sarcoma). We found that the major differences between normal and cancerous tissue were associated with changes DNA and carbohydrate content. In particular we identified a band at 964 cm(-1) assigned to a nucleic acid phosphodiester backbone mode, which appeared more pronounced in cancerous tissue irrespective of the substrate. We applied Principal Component Analysis, Unsupervised Hierarchical Cluster Analysis and k-means clustering to transmission and transflection substrates and found that both measurement modes were equally capable of discrimination normal form cancerous tissue. Moreover, the differences between spectra from cancerous and normal tissue were significantly more important than the ones arising from the measurement modes.

The limits of rovibrational analysis: the severely entangled ν1 Polyad vibration of dichlorodifluoromethane in the greenhouse IR window.

Five intense bands of dichlorodifluoromethane (CFC-12, or R12) in the infrared atmospheric window help make it a major greenhouse contributor. These include the ν1 fundamental at 1101.4 cm(-1) and the ν2 + ν3 combination at 1128.6 cm(-1). High-resolution spectra measured using the Australian Synchrotron Far-Infrared beamline were analyzed, and transitions of C(35)Cl2F2 were assigned to ν1, ν2 + ν3, and the ν3 + 2ν5 combination at 1099.7 cm(-1). The (v3 = 1; v5 = 2) state couples indirectly to v1 = 1 via Fermi resonances linking both states with v2 = v3 = 1. The v1 = 1 rotational levels are further riddled with perturbations and avoided crossings due to Coriolis resonance with the upper vibrational states of ν2 + ν9 at 1102.4 cm(-1) and (indirectly) ν2 + ν7 at 1105.8 cm(-1). A global treatment of all these states fits the observed line positions and satisfactorily accounts for the significant intensity of ν2 + ν3. Spectral simulations elucidate resonance perturbations that affect the distribution of IR absorption in the CF stretch region, and consequently the global warming potential of R12. Combination levels derived from rovibrational analysis lead to reassessment of the gas phase wavenumber values for the ν3 (458.6 cm(-1)), ν7 (437.7 cm(-1)) and ν9 (436.9 cm(-1)) fundamentals of C(35)Cl2F2, consistent with a cold, vapor phase far IR spectrum and previously published solid state spectra. B3LYP and MP2 anharmonic frequency calculations provide further support. At the MP2/aug-cc-pVTZ level, the root mean square (r.m.s.) error for unscaled anharmonic fundamentals is 6.2 cm(-1), decreased to 1.7 cm(-1) if only considering the seven lowest wavenumber modes, and integrated band intensities according with experimental literature values. Smaller basis sets produce band strengths that are too high. Low-resolution band assignments are reported for C(35)Cl(37)ClF2, C(37)Cl2F2, and (13)C(35)Cl2F2.

Red blood cells polarize green laser light revealing hemoglobin's enhanced non-fundamental Raman modes.

In general, the first overtone modes produce weak bands that appear at approximately twice the wavenumber value of the fundamental transitions in vibrational spectra. Here, we report the existence of a series of enhanced non-fundamental bands in resonance Raman (RR) spectra recorded for hemoglobin (Hb) inside the highly concentrated heme environment of the red blood cell (RBC) by exciting with a 514.5 nm laser line. Such bands are most intense when detecting parallel-polarized light. The enhancement is explained through excitonic theory invoking a type C scattering mechanism and bands have been assigned to overtone and combination bands based on symmetry arguments and polarization measurements. By using malaria diagnosis as an example, we demonstrate that combining the non-fundamental and fundamental regions of the RR spectrum improves the sensitivity and diagnostic capability of the technique. The discovery will have considerable implications for the ongoing development of Raman spectroscopy for blood disease diagnoses and monitoring heme perturbation in response to environmental stimuli.

Monitoring UVR induced damage in single cells and isolated nuclei using SR-FTIR microspectroscopy and 3D confocal Raman imaging.

SR-FTIR in combination with Principal Component Analysis (PCA) was applied to investigate macromolecular changes in a population of melanocytes and their extracted nuclei induced by environmentally relevant fluxes of UVR (Ultraviolet Radiation). Living cells and isolated cellular nuclei were investigated post-irradiation for three different irradiation dosages (130, 1505, 15,052 Jm(-2) UVR, weighted) after either 24 or 48 hours of incubation. DNA conformational changes were observed in cells exposed to an artificial UVR solar-simulator source as evidenced by a shift in the DNA asymmetric phosphodiester vibration from 1236 cm(-1) to 1242 cm(-1) in the case of the exposed cells and from 1225 cm(-1) to 1242 cm(-1) for irradiated nuclei. PCA Scores plots revealed distinct clustering of spectra from irradiated cells and nuclei from non-irradiated controls in response to the range of applied UVR radiation doses. 3D Raman confocal imaging in combination with k-means cluster analysis was applied to study the effect of the UVR radiation exposure on cellular nuclei. Chemical changes associated with apoptosis were detected and included intra-nuclear lipid deposition along with chromatin condensation. The results reported here demonstrate the utility of SR-FTIR and Raman spectroscopy to probe in situ DNA damage in cell nuclei resulting from UVR exposure. These results are in agreement with the increasing body of evidence that lipid accumulation is a characteristic of aggressive cancer cells, and are involved in the production of membranes for rapid cell proliferation.

Detection of an en masse and reversible B- to A-DNA conformational transition in prokaryotes in response to desiccation.

The role that DNA conformation plays in the biochemistry of cells has been the subject of intensive research since DNA polymorphism was discovered. B-DNA has long been considered the native form of DNA in cells although alternative conformations of DNA are thought to occur transiently and along short tracts. Here, we report the first direct observation of a fully reversible en masse conformational transition between B- and A-DNA within live bacterial cells using Fourier transform infrared (FTIR) spectroscopy. This biospectroscopic technique allows for non-invasive and reagent-free examination of the holistic biochemistry of samples. For this reason, we have been able to observe the previously unknown conformational transition in all four species of bacteria investigated. Detection of this transition is evidence of a previously unexplored biological significance for A-DNA and highlights the need for new research into the role that A-DNA plays as a cellular defence mechanism and in stabilizing the DNA conformation. Such studies are pivotal in understanding the role of A-DNA in the evolutionary pathway of nucleic acids. Furthermore, this discovery demonstrates the exquisite capabilities of FTIR spectroscopy and opens the door for further investigations of cell biochemistry with this under-used technique.

Detection and quantification of early-stage malaria parasites in laboratory infected erythrocytes by attenuated total reflectance infrared spectroscopy and multivariate analysis.

New diagnostic modalities for malaria must have high sensitivity and be affordable to the developing world. We report on a method to rapidly detect and quantify different stages of malaria parasites, including ring and gametocyte forms, using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR) and partial least-squares regression (PLS). The absolute detection limit was found to be 0.00001% parasitemia (<1 parasite/µL of blood; p < 0.008) for cultured early ring stage parasites in a suspension of normal erythrocytes. Future development of universal and robust calibration models can significantly improve malaria diagnoses, leading to earlier detection and treatment of this devastating disease.