Francesca Palombo

Application of Fourier transform infrared spectroscopic imaging to the study of effects of age and dietary l-arginine on aortic lesion composition in cholesterol-fed rabbits

Diet-induced atherosclerotic lesions in the descending thoracic segment of rabbit aorta were analysed ex vivo by micro-attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectroscopic imaging. The distribution and chemical character of lipid deposits within the arterial wall near intercostal branch ostia were assessed in histological sections from immature and mature rabbits fed cholesterol with or without l-arginine supplements. Previous studies have shown that both these properties change with age in cholesterol-fed rabbits, putatively owing to changes in the synthesis of nitric oxide (NO) from l-arginine. Immature animals developed lesions at the downstream margin of the branch ostium, whereas lipid deposition was observed at the lateral margins in mature animals. Dietary l-arginine supplements had beneficial effects in mature rabbit aorta, with overall disappearance of the plaques; on the other hand, they caused only a slight decrease of the lipid load in lesions at the downstream margin of the ostium in immature rabbits. ATR–FTIR imaging enabled differences in the lipid to protein density ratio of atherosclerotic lesions caused by age and diet to be visualized. Lipid deposits in immature rabbits showed higher relative absorbance values of their characteristic spectral bands compared with those in immature l-arginine-fed rabbits and mature rabbits. The multivariate methods of principal component analysis (PCA) and factor analysis (FA) were employed, and relevant chemical and structural information were obtained. Two distinct protein constituents of the intima–media layer at different locations of the wall were identified using the method of FA. This approach provides a valuable means of investigating the structure and chemistry of complex heterogeneous systems. It has potential for in vivo diagnosis of pathology.

Biomechanics of fibrous proteins of the extracellular matrix studied by Brillouin scattering.

Brillouin light scattering (BLS) spectroscopy is a technique that is able to detect thermally excited phonons within a material. The speed of propagation of these phonons can be determined from the magnitude of the Brillouin frequency shift between incident and scattered light, thereby providing a measure of the mechanical properties of the material in the gigahertz range. The mechanical properties of the extracellular matrices of biological tissues and their constituent biopolymers are important for normal tissue function and disturbances in these properties are widely implicated in disease. BLS offers the prospect of measuring mechanical properties on a microscopic scale in living tissues, thereby providing insights into structure–function relationships under normal and pathological conditions. In this study, we investigated BLS in collagen and elastin—the fibrous proteins of the extracellular matrix (ECM). Measurements were made on type I collagen in rat tail tendon, type II collagen in articular cartilage and nuchal ligament elastin. The dependence of the BLS spectrum on fibre orientation was investigated in a backscattering geometry using a reflective substrate. Two peaks, a bulk mode arising from phonon propagation along a quasi-radial direction to the fibre axis and a mode parallel to the surface, depending on sample orientation relative to the fibre axis, could be distinguished. The latter peak was fitted to a model of wave propagation through a hexagonally symmetric elastic solid, and the five components of the elasticity tensor were combined to give axial and transverse Youngs, shear and bulk moduli of the fibres. These were 10.2, 8.3, 3.2 and 10.9 GPa, and 6.1, 5.3, 1.9 and 8 GPa for dehydrated type I collagen and elastin, respectively. The former values are close to those previously reported. A microfocused BLS approach was also applied providing selection of single fibres. The moduli of collagen and elastin are much higher than those measured at lower frequency using macroscopic strains, and the difference between them is much less. We therefore believe, like previous investigators, that molecular-scale viscoelastic effects are responsible for the frequency dependence of the fibre biomechanics. Combining BLS with larger-scale mechanical testing methods therefore should, in the future, provide a means of following the evolution of mechanical properties in the formation of the complex structures found in the ECM.

Mechanical mapping with chemical specificity by confocal Brillouin and Raman microscopy.

We describe the first application of confocal Brillouin and Raman microscopy to ex vivo human epithelial tissue - Barretts oesophagus. This non-invasive label-free approach provides high-resolution mechanical mapping with chemical specificity, opening the route to a new integrated method for multiple biomedical and bioengineering applications, and potentially in vivo real-time diagnostics.

Measurement of drug and macromolecule diffusion across atherosclerotic rabbit aorta ex vivo by attenuated total reflection–Fourier transform infrared imaging

Diffusion of two model drugs-benzyl nicotinate and ibuprofen-and the plasma macromolecule albumin across atherosclerotic rabbit aorta was studied ex vivo by attenuated total reflection-Fourier transform infrared (ATR-FTIR) imaging. Solutions of these molecules were applied to the endothelial surface of histological sections of the aortic wall that were sandwiched between two impermeable surfaces. An array of spectra, each corresponding to a specific location in the section, was obtained at various times during solute diffusion into the wall and revealed the distribution of the solutes within the tissue. Benzyl nicotinate in Ringers solution showed higher affinity for atherosclerotic plaque than for apparently healthy tissue. Transmural concentration profiles for albumin demonstrated its permeation across the section and were consistent with a relatively low distribution volume for the macromolecule in the middle of the wall. The ability of albumin to act as a drug carrier for ibuprofen, otherwise undetected within the tissue, was demonstrated by multivariate subtraction image analysis. In conclusion, ATR-FTIR imaging can be used to study transport processes in tissue samples with high spatial and temporal resolution and without the need to label the solutes under study.

Molecular dynamics of liquid acetone determined by depolarized Rayleigh and low-frequency Raman scattering spectroscopy.

Slow to ultrafast dynamics of liquid acetone at variable temperature was investigated by depolarized Rayleigh and low-frequency Raman scattering spectroscopy, in the region 0-200 cm(-1). A detailed analysis was performed on the spectra and corresponding time responses, and a consistent view of the molecular dynamics of this dipolar solvent was obtained. The effects of temperature on the spectra were interpreted, and distinct dynamical processes identified. At very low frequencies, or long time scales, acetone dynamics is characterized by a slow diffusive reorientation obeying the Stokes-Einstein-Debye hydrodynamic theory only in the limit of subslip boundary conditions. An alternative model based on the microviscosity concept proved to be able to reproduce this correlation time and its temperature dependence. A comparative analysis of collective and single-molecule reorientational times, these latter estimated from intramolecular Raman spectra, led to an orientational correlation parameter g(2) of unity, which denotes a statistical disorder of molecular polarizability tensors. A fast local restructuring process is putatively responsible for an additional contribution at subpicosecond time scales often referred to as intermediate response in other molecular liquids. The high frequency portion of the dynamical susceptibility showed the signature of librational intermolecular motions, giving rise to an ultrafast decay of the time correlation function of polarizability anisotropy. The overall approach, which provided valuable information on dynamics, structure and molecular interactions of neat acetone, will be applied to acetone electrolytic solutions.

Tuning the Hydrophobic Interaction: Ultrafast Optical Kerr Effect Study of Aqueous Ionene Solutions

The molecular origin of the hydrophobic effect continues to be widely studied. Here we design an experiment to tune independently hydrophilic and hydrophobic interactions through the study of a series of aqueous ionene solutions. The dynamics of these solutions are probed using the ultrafast optical Kerr effect, which measures polarizability anisotropy relaxation. Analysis of these data yields information on both structural dynamics within the water hydrogen-bonded network and the low frequency intermolecular bending and stretching H-bond modes. In all cases the ionene solute retards the structural dynamics compared to bulk water. However, the effect is small and cannot be assigned specifically to water-hydrophobe interactions. There is no evidence for a dramatic slowdown of the water dynamics observed by the optical Kerr effect when water is in the solvation shell of a hydrophobic group. The low frequency spectrum was recorded as a function of ionene concentration. Again the effect of the solute was small, and could be assigned mainly to the effect of anion solvation.

Viscoelasticity of amyloid plaques in transgenic mouse brain studied by Brillouin microspectroscopy and correlative Raman analysis

Amyloidopathy is one of the most prominent hallmarks of Alzheimers disease (AD), the leading cause of dementia worldwide, and is characterized by the accumulation of amyloid plaques in the brain parenchyma. The plaques consist of abnormal deposits mainly composed of an aggregation-prone protein fragment, β-amyloid 1-40/1-42, into the extracellular matrix. Brillouin microspectroscopy is an all-optical contactless technique that is based on the interaction between visible light and longitudinal acoustic waves or phonons, giving access to the viscoelasticity of a sample on a subcellular scale. Here, we describe the first application of micromechanical mapping based on Brillouin scattering spectroscopy to probe the stiffness of individual amyloid plaques in the hippocampal part of the brain of a β-amyloid overexpressing transgenic mouse. Correlative analysis based on Brillouin and Raman microspectroscopy showed that amyloid plaques have a complex structure with a rigid core of β-pleated sheet conformation (β-amyloid) protein surrounded by a softer ring-shaped region richer in lipids and other protein conformations. These preliminary results give a new insight into the plaque biophysics and biomechanics, and a valuable contrast mechanism for the study and diagnosis of amyloidopathy.

Dynamics of formamide ionic solutions investigated by ultrafast optical Kerr effect.

Molecular dynamics of formamide solutions of alkali metal halide salts were investigated using the time-resolved ultrafast optical Kerr effect (OKE) to observe the effects of ion solvation on the dynamics of a nonaqueous high-permittivity H-bonding solvent. The picosecond orientational and ultrafast intermolecular dynamics of liquid formamide as a function of concentration of NaI and KI are compared with the temperature effect on the pure solvent. The effect of a range of other salts at fixed concentration is also recorded. Transient OKE and corresponding low-frequency (THz) Raman spectra of the solutions revealed differences in the solvent dynamics caused by ion solvation. Increasing concentrations of NaI and KI have the effect of slowing down the diffusive reorientation and reducing the librational frequencies of formamide, with cation-related effects observed on the THz Raman spectrum. These effects are discussed in terms of an ion perturbation of the H-bonding structure in the solution. This approach provides a valuable means of investigating the dynamics, structure, and interactions in complex, interacting systems.

Elucidating the association of water in wet 1-octanol from normal to high temperature by near- and mid-infrared spectroscopy.

Near- (NIR) and mid-infrared (MIR) absorption spectra of pure and water-saturated 1-octanol were measured along the liquid-gas coexistence curve from ambient temperature and pressure up to 300 degrees C and 10 MPa. Density of the mixture as a function of temperature was assessed by spectral analysis in the NIR region. Two distinct regimes of temperature were identified in the evolution of solvent-subtracted spectra: at normal conditions and up to 180 degrees C, water is organized in multimeric H-bonded aggregates, while at higher temperature, mainly dimers and monomers exist. Water-water and alcohol-alcohol H-bonding interactions play a major role in wet octanol at low temperature, with resulting microheterogeneity and water segregation at a molecular level; on the other hand, water-alcohol interactions are relevant at higher temperature, as also revealed by the estimated density. At low temperature, water dissolved in octanol shows features which are comparable to those of interfacial water obtained by vibrational sum frequency (VSF) spectroscopy, while at high temperature the spectra generally reproduce those of water in the supercritical phase. Overall spectral assignment was supported by ab initio calculations at the MP2 level on small water clusters.

High-Performance Versatile Setup for Simultaneous Brillouin-Raman Microspectroscopy

S. Corezzi acknowledges financial support from MIUR-PRIN (Project No. 2012J8X57P). S. Caponi acknowledges support from PAT (Provincia Autonoma di Trento) (GP/PAT/2012) “Grandi Progetti 2012” Project “MaDEleNA.” P. S., A. M., M. P. acknowledge financial support from Centro Nazionale Trapianti (Project: “Studio di cellule per uso clinico umano, con particolare riferimento a modelli cellulari (liposomi) e linee cellulari in interazione con crioconservanti e con materiali biocompatibili”). L. C. and S. Caponi acknowledge financial support from Consiglio Nazionale delle Ricerche-Istituto Officina dei Materiali. F. P. acnowledges support from the UK Engineering and Physical Sciences Research Council (Grant No. EP/M028739/1 (F. P.)). The authors acknowledge Jacopo Scarponi for valuable help in setting up the hardware and software system for simultaneous Raman and BLS measurements.