Ultraviolet Resonant Raman Scattering of Electrolyte Solutions.

Saltwater stands as the most prevalent liquid on Earth. Consequently, substantial interest has been directed toward its characterization, both as an independent system and as a solvent for complex structures such as biomacromolecules. In the last few decades, special emphasis was placed on the investigation of the hydration properties of ions for the fundamental role they play in numerous chemical processes. In this study, we employed multi-wavelength Raman spectroscopy to examine the hydration shell surrounding bromide ions in solutions of simple electrolytes, specifically lithium bromide, potassium bromide, and cesium bromide, at two different concentrations. Cation-induced differences among electrolytes were observed in connection to their tendency to form ion pairs. An increased sensitivity to reveal the structure of the first hydration shell was evidenced when employing ultraviolet excitation in the 228-266 nm range, under resonance conditions with the charge transfer transition to the solvent peaked at about 200 nm. Other than a significant increase in the Raman cross-section for the OH stretching band when shifting from pure water to the solution, a larger enhancement for the Raman signal of the H-O-H bending mode over the stretching vibration was observed. Thus, the bending band plays a crucial role in monitoring the H-bond structure of water around the anions related to the charge distribution within the first hydration shell of anions, being an effective probe of hydration phenomena.

Brillouin-Raman micro-spectroscopy and machine learning techniques to classify osteoarthritic lesions in the human articular cartilage.

In this study, Brillouin and Raman micro-Spectroscopy (BRamS) and Machine Learning were used to set-up a new diagnostic tool for Osteoarthritis (OA), potentially extendible to other musculoskeletal diseases. OA is a degenerative pathology, causing the onset of chronic pain due to cartilage disruption. Despite this, it is often diagnosed late and the radiological assessment during the routine examination may fail to recognize the threshold beyond which pharmacological treatment is no longer sufficient and prosthetic replacement is required. Here, femoral head resections of OA-affected patients were analyzed by BRamS, looking for distinctive mechanical and chemical markers of the progressive degeneration degree, and the result was compared to standard assignment via histological staining. The procedure was optimized for diagnostic prediction by using a machine learning algorithm and reducing the time required for measurements, paving the way for possible future in vivo characterization of the articular surface through endoscopic probes during arthroscopy.

Microscale mechanochemical characterization of drying oil films by in situ correlative Brillouin and Raman spectroscopy.

Correlative Brillouin and Raman microspectroscopy (BRaMS) is applied for the in situ monitoring of the chemical and physical changes of linseed oil during polymerization. The viscoelastic properties of the drying oil throughout the phase transition were determined by Brillouin light scattering (BLS) and joined to the Raman spectroscopic information about the chemical process responsible for the oil hardening. A comparative study was then performed on an oil mock-up containing ZnO, one of the most common white pigments used in cultural heritage. The intriguing outcomes open new research perspectives for a deeper comprehension of the processes leading to the conversion of a fluid binder into a dry adhering film. The description of both chemical and structural properties of the polymeric network and their evolution are the basis for a better understanding of oil painting degradation. Last, as a feasibility test, BRaMS was applied to study a precious microfragment from J. Pollock's masterpiece Alchemy.

Hydration Dynamics of Model Peptides with Different Hydrophobic Character.

The multi-scale dynamics of aqueous solutions of the hydrophilic peptide N-acetyl-glycine-methylamide (NAGMA) have been investigated through extended frequency-range depolarized light scattering (EDLS), which enables the broad-band detection of collective polarizability anisotropy fluctuations. The results have been compared to those obtained for N-acetyl-leucinemethylamide (NALMA), an amphiphilic peptide which shares with NAGMA the same polar backbone, but also contains an apolar group. Our study indicates that the two model peptides induce similar effects on the fast translational dynamics of surrounding water. Both systems slow down the mobility of solvating water molecules by a factor 6-8, with respect to the bulk. Moreover, the two peptides cause a comparable far-reaching spatial perturbation extending to more than two hydration layers in diluted conditions. The observed concentration dependence of the hydration number is explained considering the random superposition of different hydration shells, while no indication of solute aggregation phenomena has been found. The results indicate that the effect on the dynamics of water solvating the amphiphilic peptide is dominated by the hydrophilic backbone. The minor impact of the hydrophobic moiety on hydration features is consistent with structural findings derived by Fourier transform infrared (FTIR) measurements, performed in attenuated total reflectance (ATR) configuration. Additionally, we give evidence that, for both systems, the relaxation mode in the GHz frequency range probed by EDLS is related to solute rotational dynamics. The rotation of NALMA occurs at higher timescales, with respect to the rotation of NAGMA; both processes are significantly slower than the structural dynamics of hydration water, suggesting that solute and solvent motions are uncoupled. Finally, our results do not indicate the presence of super-slow water (relaxation times in the order of tens of picoseconds) around the peptides investigated.

Non-contact elastography methods in mechanobiology: a point of view.

In recent decades, mechanobiology has emerged as a novel perspective in the context of basic biomedical research. It is now widely recognized that living cells respond not only to chemical stimuli (for example drugs), but they are also able to decipher mechanical cues, such as the rigidity of the underlying matrix or the presence of shear forces. Probing the viscoelastic properties of cells and their local microenvironment with sub-micrometer resolution is required to study this complex interplay and dig deeper into the mechanobiology of single cells. Current approaches to measure mechanical properties of adherent cells mainly rely on the exploitation of miniaturized indenters, to poke single cells while measuring the corresponding deformation. This method provides a neat implementation of the everyday approach to measure mechanical properties of a material, but it typically results in a very low throughput and invasive experimental protocol, poorly translatable towards three-dimensional living tissues and biological constructs. To overcome the main limitations of nanoindentation experiments, a radical paradigm change is foreseen, adopting next generation contact-less methods to measure mechanical properties of biological samples with sub-cell resolution. Here we briefly introduce the field of single cell mechanical characterization, and we concentrate on a promising high resolution optical elastography technique, Brillouin spectroscopy. This non-contact technique is rapidly emerging as a potential breakthrough innovation in biomechanics, but the application to single cells is still in its infancy.

Brillouin and Raman Micro-Spectroscopy: A Tool for Micro-Mechanical and Structural Characterization of Cortical and Trabecular Bone Tissues.

Human bone is a specialized tissue with unique material properties, providing mechanical support and resistance to the skeleton and simultaneously assuring capability of adaptation and remodelling. Knowing the properties of such a structure down to the micro-scale is of utmost importance, not only for the design of effective biomimetic materials but also to be able to detect pathological alterations in material properties, such as micro-fractures or abnormal tissue remodelling. The Brillouin and Raman micro-spectroscopic (BRmS) approach has the potential to become a first-choice technique, as it is capable of simultaneously investigating samples' mechanical and structural properties in a non-destructive and label-free way. Here, we perform a mapping of cortical and trabecular bone sections of a femoral epiphysis, demonstrating the capability of the technique for discovering the morpho-mechanics of cells, the extracellular matrix, and marrow constituents. Moreover, the interpretation of Brillouin and Raman spectra merged with an approach of data mining is used to compare the mechanical alterations in specimens excised from distinct anatomical areas and subjected to different sample processing. The results disclose in both cases specific alterations in the morphology and/or in the tissue chemical make-up, which strongly affects bone mechanical properties, providing a method potentially extendable to other important biomedical issues.

Brillouin microscopy for the evaluation of hair micromechanics and effect of bleaching.

Brillouin microscopy is a new form of optical elastography and an emerging technique in mechanobiology and biomedical physics. It was applied here to map the viscoelastic properties of human hair and to determine the effect of bleaching on hair properties. For hair samples, longitudinal measurements (i.e. along the fibre axis) revealed peaks at 18.7 and 20.7 GHz at the location of the cuticle and cortex, respectively. For hair treated with a bleaching agent, the frequency shifts for the cuticle and cortex were 19.7 and 21.0 GHz, respectively, suggesting that bleaching increases the cuticle modulus and-to a minor extent-the cortex modulus. These results demonstrate the capability of Brillouin spectroscopy to address questions on micromechanical properties of hair and to validate the effect of applied treatments.

Predicting the Refractive Index of Tissue Models Using Light Scattering Spectroscopy.

In this work, we report the application of Raman microspectroscopy for analysis of the refractive index of a range of tissue phantoms. Using both a custom-developed setup with visible laser source and a commercial microspectrometer with near infrared laser, we measured the Raman spectra of gelatin hydrogels at various concentrations. By building a calibration curve from measured refractometry data and Raman scattering intensity for different vibrational modes of the hydrogel, we were able to predict the refractive indices of the gels from their Raman spectra. This work highlights the importance of a correlative approach through Brillouin-Raman microspectroscopy for the mechano-chemical analysis of biologically relevant samples.

Transition across a sharp interface: Data from Raman and Brillouin imaging spectroscopy.

Brillouin and Raman imaging are powerful techniques for the investigation of complex materials and they are widely used in material science and biophysics [1], [2], [3], [4], [5], [6], [7]. When dealing with microstructures, the results interpretation requires an accurate understanding of the interaction processes in presence of acoustic and chemical boundaries between different materials [8], [9], [10], [11], [12], [13], [14], [15]. The data here reported are obtained while scanning with sub-micron resolution the sharp interfaces between vitreous-SiO2/Water and Polyethylene (PET)/Glycerol. Molecular and acoustic vibrations were observed by means of a recently developed micro-spectrometer, which acquires simultaneously Raman and Brillouin spectra on the same point with high spatial and spectral resolution [3]. Two external optic configurations were adopted in order to evidence the dependency of the measurements on the optical scattering volume. The evolution of the detected phonon modes, propagating and not propagating, is obtained by a direct observation of the raw data for the two interfaces, which present different acoustic mismatch. These experimental records can be exploited by researchers employing Raman and Brillouin imaging to discuss the resolution limit of the techniques and to compare the effect of different experimental set-ups. Moreover, thanks to their high spectral resolution they can be useful to researchers working on acoustic phonon transport at interfaces to model the dependency of transmission of long wavelength phonons on the acoustic mismatch.

Viscoelastic properties of biopolymer hydrogels determined by Brillouin spectroscopy: A probe of tissue micromechanics.

Many problems in mechanobiology urgently require characterization of the micromechanical properties of cells and tissues. Brillouin light scattering has been proposed as an emerging optical elastography technique to meet this need. However, the information contained in the Brillouin spectrum is still a matter of debate because of fundamental problems in understanding the role of water in biomechanics and in relating the Brillouin data to low-frequency macroscopic mechanical parameters. Here, we investigate this question using gelatin as a model system in which the macroscopic physical properties can be manipulated to mimic all the relevant biological states of matter, ranging from the liquid to the gel and the glassy phase. We demonstrate that Brillouin spectroscopy is able to reveal both the elastic and viscous properties of biopolymers that are central to the structure and function of biological tissues.

Ketoprofen poly(lactide-co-glycolide) physical interaction studied by Brillouin spectroscopy and molecular dynamics simulations.

The performances of poly(lactic-co-glycolic acid) drug delivery systems are affected by the molecular interactions established between the drug and the polymer matrix as well as by the physical state of the drug embedded. Indeed, the drug may induce polymer plasticization with a drastic change in the release kinetics and medicinal product performances. The aim of this study was to better understand the interactions between poly(lactic-co-glycolic acid) and ketoprofen, the latter known to plasticize hydrophilic and hydrophobic polymers. Ketoprofen interacts with poly(lactic-co-glycolic acid) exerting a maximum plasticizing effect at weight fractions around 0.25. Higher ketoprofen amounts form heterogeneous mixtures with the non-soluble molecules dispersed in the matrix as crystals or amorphous domains, depending on the preparation method. Unexpectedly, the amorphous ketoprofen dispersed in the poly(lactic-co-glycolic acid) matrix is remarkably stable. H-bonding seems responsible for the glass transition temperature reduction and the limited solubility. Brillouin spectroscopy and molecular dynamics simulation data suggest that ketoprofen solubility increases with temperature and non-polar interactions are responsible for this phenomenon.

Correlative Brillouin and Raman spectroscopy data acquired on single cells.

The distribution of chemical species and the mechanical modulation inside a single cell or tissue are of fundamental importance to characterize their physiological activity or their pathological conditions [1-4]. Here we analyse these properties by means of label free, non invasive, spectroscopic methods. In particular, we use a recently developed micro-spectrometer, which acquires simultaneously Raman and Brillouin spectra on the same point with subcellular resolution [5]. The techniques ability to analyse the chemical composition and the mechanical properties of single cells has been tested on NIH/3T3 murine fibroblast cells grown in adhesion on silicon substrates. Here we report the data acquired from fixed cells after their oncogenic transformation. Mechanical and chemical evolution is evident by direct inspection of raw data. Sharing our experimental records can be valuable for researchers interested in the analysis of single cells by Raman and Brillouin spectroscopy in order: i) to compare data acquired by different set-ups and ii) to correctly model the fitting functions.

Meso-Raman approach for rapid yeast cells identification.

An increasing effort is currently devoted to developing Raman spectroscopy for identification of microorganisms. Micro-Raman setups are typically used for this purpose with the limit that the intra-species and inter-species spectral variability are comparable, thus limiting the identification capability. To overcome this limit a meso-Raman approach is here implemented. Thin films of planktonic cells are analyzed throughout the collection of back-scattered light providing a Raman signal already averaged over tens of cells. The collecting of unpolarized (VU) and depolarized (HV) Raman signals increased the spectral information obtainable from the data, demonstrating the ability of the principal component analysis to differentiate the most common Candida species, namely C. glabrata, C. albicans, C. parapsilosis and C. tropicalis. The proposed method can contribute to bring Raman spectroscopy closer to its potential clinical use for fast identification of yeast cells.

Trehalose-induced slowdown of lysozyme hydration dynamics probed by EDLS spectroscopy.

We use extended depolarized light scattering spectroscopy to study the dynamics of water in a lysozyme-trehalose aqueous solution over a broad time scale, from hundreds to fractions of picoseconds. We provide experimental evidence that the sugar, present in the ternary solution in quantity relevant for biopreservation, strongly modifies the solvation properties of the protein. By comparing aqueous solutions of lysozyme with and without trehalose, we show that the combined action of sugar and protein produces an exceptional dynamic slowdown of a fraction of water molecules around the protein, which become more than twice slower than in the absence of trehalose. We speculate that this ultraslow water may be caged between the sugar and protein surface, consistently with a water entrapment scenario. We also demonstrate that the dynamics of these water molecules gets slower and slower upon cooling. On the basis of these findings, we believe such ultraslow water close to the lysozyme is likely to be involved in the mechanism of bioprotection.

Brillouin Light Scattering: Applications in Biomedical Sciences.

Brillouin spectroscopy and imaging are emerging techniques in analytical science, biophotonics, and biomedicine. They are based on Brillouin light scattering from acoustic waves or phonons in the GHz range, providing a nondestructive contactless probe of the mechanics on a microscale. Novel approaches and applications of these techniques to the field of biomedical sciences are discussed, highlighting the theoretical foundations and experimental methods that have been developed to date. Acknowledging that this is a fast moving field, a comprehensive account of the relevant literature is critically assessed here.

Brillouin Light Scattering Microspectroscopy for Biomedical Research and Applications: introduction to feature issue.

There has been a marked revival of interest in brillouin light scattering spectroscopy/microscopy over the last decade in regards to applications related to all optically studying the mechanical problems associated with systems of biological and medical interest. This revival has been driven by advancements in spectrometer design, together with mounting evidence of the critical role that mechanical properties can play in biological processes as well as the onset of diverse diseases. This feature issue contains a series of papers spanning some of the latest developments in the field of Brillouin light scattering spectroscopy and microscopy as applied to systems of biomedical interest.

Morpho-mechanics of human collagen superstructures revealed by all-optical correlative micro-spectroscopies.

In every biological tissue, morphological and topological properties strongly affect its mechanical features and behaviour, so that ultrastructure, composition and mechanical parameters are intimately connected. Overall, it is their correct interplay that guarantees the tissue functionality. The development of experimental methods able to correlate these properties would open new opportunities both in the biological and the biomedical fields. Here, we report a correlative study intended to map supramolecular morphology, biochemical composition and viscoelastic parameters of collagen by all-optical microscopies. In particular, using human corneal tissue as a benchmark, we correlate Second-Harmonic Generation maps with mechanical and biochemical imaging obtained by Brillouin and Raman micro-spectroscopy. The study highlights how subtle variations in supramolecular organization originate the peculiar mechanical behavior of different subtypes of corneal lamellae. The presented methodology paves the way to the non-invasive assessment of tissue morpho-mechanics in biological as well as synthetic materials.

Brillouin-Raman mapping of natural fibers with spectral moment analysis.

Brillouin spectroscopy has emerged as a novel analytical tool for biophotonic research and applications. It operates on a microscopic scale and in the GHz spectral range, providing a new spatial and frequency window for the analysis of the materials elasticity. Here we investigate spectral moments calculation as a means of analysing Brillouin and Raman spectra, providing rapid access to peak intensity and frequency shift, with robust application to fast scanning measurements. This work demonstrates the potential of the method, especially in the case of micro-structured samples, typical of bio-medical applications.

Hyperspectral analysis applied to micro-Brillouin maps of amyloid-beta plaques in Alzheimer's disease brains.

A recent investigation on the architecture and chemical composition of amyloid-ß (Aß) plaques in ex vivo histological sections of an Aß-overexpressing transgenic mouse hippocampus has shed light on the infrared light signature of cell-activation related biomarkers of Alzheimer's disease. A correlation was highlighted between the biomechanical properties detected by Brillouin microscopy and the molecular make-up of Aß plaques provided by FTIR spectroscopic imaging and Raman microscopy (with correlative immunofluorescence imaging) in this animal model of the disease. In the Brillouin spectra of heterogeneous materials such as biomedical samples, peaks are likely the result of multiple contributions, more or less overlaid on a spatial and spectral scale. The ability to disentangle these contributions is very important as it may give access to discrete components that would otherwise be buried within the Brillouin peak envelope. Here, we applied an unsupervised non-negative matrix factorization method to analyse the spontaneous Brillouin microscopy maps of Aß plaques in transgenic mouse hippocampal sections. The method has already been proven successful in decomposing chemical images and is applied here for the first time to acoustic maps acquired with a Fabry-Perot Brillouin microscope. We extracted and visualised a decrease in tissue rigidity from the core through to the periphery of the plaque, with spatially distinct components that we assigned to specific entities. This work demonstrates that it is possible to reveal the structure and mechanical properties of Aß plaques, with details visualized by the projection of the mechanical contrast into a few relevant channels.

Yeast Biofilm as a Bridge Between Medical and Environmental Microbiology Across Different Detection Techniques.

Medical and environmental microbiology have two distinct, although very short, histories stemming, the first from the pioneering works of Sommelweiss, Pasteur, Lister and Koch, the second mainly from the studies of Bejerink and Winogradsky. These two branches of microbiology evolved and specialized separately producing distinct communities and evolving rather different approaches and techniques. The evidence accumulated in recent decades indicate that indeed most of the medically relevant microorganisms have a short circulation within the nosocomial environment and a larger one involving the external, i.e. non-nosocomial, and the hospital environments. This evidence suggests that the differences between approaches should yield to a convergent approach aimed at solving the increasing problem represented by infectious diseases for the increasingly less resistant human communities. Microbial biofilm is one of the major systems used by these microbes to resist the harsh conditions of the natural and anthropic environment, and the even worse ones related to medical settings. This paper presents a brief outline of the converging interest of both environmental and medical microbiology toward a better understanding of microbial biofilm and of the various innovative techniques that can be employed to characterize, in a timely and quantitative manner, these complex structures. Among these, micro-Raman along with micro-Brillouin offer high hopes of describing biofilms both at the subcellular and supercellular level, with the possibility of characterizing the various landscapes of the different biofilms. The possibility of adding a taxonomic identification of the cells comprising the biofilm is a complex aspect presenting several technical issues that will require further studies in the years to come.