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 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.

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.

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.

Practical way to avoid spurious geometrical contributions in Brillouin light scattering experiments at variable scattering angles.

In this Letter, we present a simple method to avoid the well-known spurious contributions in the Brillouin light scattering (BLS) spectrum arising from the finite aperture of collection optics. The method relies on the use of special spatial filters able to select the scattered light with arbitrary precision around a given value of the momentum transfer (Q). We demonstrate the effectiveness of such filters by analyzing the BLS spectra of a reference sample as a function of scattering angle. This practical and inexpensive method could be an extremely useful tool to fully exploit the potentiality of Brillouin acoustic spectroscopy, as it will easily allow for effective Q-variable experiments with unparalleled luminosity and resolution.

Concentration dependence of hydration water in a model peptide.

The molecular dynamics of aqueous solutions of a model amphiphilic peptide is studied as a function of concentration by broad-band light scattering experiments. Similarly to protein aqueous solutions, a considerable retardation, of about a factor 6-8, of hydration water dynamics with respect to bulk water is found, showing a slight dependence on solute concentration. Conversely, the average number of water molecules perturbed by the presence of peptide, i.e. the hydration number, appears to be strongly modified by adding solute. Its behaviour, decreasing upon increasing concentration, can be interpreted considering the random close-to-contact condition experienced by solute particles. Overall, the present findings support the view of a "long range" effect of peptides on the surrounding water, extending beyond the first two hydration shells.

Depolarized light scattering and dielectric response of a peptide dissolved in water.

The density and orientational relaxation of bulk water can be separately studied by depolarized light scattering (DLS) and dielectric spectroscopy (DS), respectively. Here, we ask the question of what are the leading collective modes responsible for polarization anisotropy relaxation (DLS) and dipole moment relaxation (DS) of solutions involving mostly hydrophobic solute-water interfaces. We study, by atomistic molecular dynamics simulations, the dynamics and structure of hydration water interfacing N-Acetyl-leucine-methylamide dipeptide. The DLS response of the solution is consistent with three relaxation processes: bulk water, rotations of single solutes, and collective dipole-induced-dipole polarizability of the solutes, with the time-scale of 130-200 ps. No separate DLS response of the hydration shell has been identified by our simulations. Density fluctuations of the hydration layer, which largely contribute to the response, do not produce a dynamical process distinct from bulk water. We find that the structural perturbation of the orientational distribution of hydration waters by the dipeptide solute is quite significant and propagates ∼3-5 hydration layers into the bulk. This perturbation is still below that produced by hydrated globular proteins. Despite this structural perturbation, there is little change in the orientational dynamics of the hydration layers, compared to the bulk, as probed by both single-particle orientational dynamics and collective dynamics of the dipole moment of the shells. There is a clear distinction between the perturbation of the interfacial structure by the solute-solvent interaction potential and the perturbation of the interfacial dynamics by the corresponding forces.

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.

Combining Multiple Spectroscopic Techniques to Reveal the Effects of Staphylococcus aureus Infection on Human Bone Tissues.

Osteomyelitis (OM) and periprosthetic joint infections (PJIs) are major public health concerns in Western countries due to increased life expectancy. Infections usually occur due to bacterial spread through fractures, implants, or blood-borne transmission. The pathogens trigger an inflammatory response that hinders bone tissue regeneration. Treatment requires surgical intervention, which involves the precise removal of infected tissue, wound cleansing, and local and systemic antibiotic administration. Staphylococcus aureus (SA) is one of the most common pathogens causing infection-induced OM and PJIs. It forms antimicrobial-resistant biofilms and is frequently found in healthcare settings. In this proof-of-concept, we present an approach based on multiple spectroscopic techniques aimed at investigating the effects of SA infection on bone tissue, as well as identifying specific markers useful to detect early bacterial colonization on the tissue surface. A cross-section of a human femoral diaphysis, with negative-culture results, was divided into three parts, and the cortical and trabecular regions were separated from each other. Two portions of each bone tissue type were infected with SA for one and seven days, respectively. Multiple techniques were used to investigate the impact of the infection on bone tissue, Brillouin-Raman microspectroscopy and attenuated total reflection Fourier transform infrared spectroscopy were employed to assess and develop a new noninvasive diagnostic method to detect SA by targeting the bone of the host. The results indicate that exposure to SA infection significantly alters the bone structure, especially in the case of the trabecular type, even after just one day. Moreover, Raman spectral markers of the tissue damage were identified, indicating that this technique can detect the effect of the pathogens' presence in bone biopsies and pave the way for potential application during surgery, due to its nondestructive and contactless nature.

Assessing Mechanochemical Properties of Acrylonitrile Butadiene Styrene (ABS) Items in Cultural Heritage Through a Multimodal Spectroscopic Approach.

A multimodal spectroscopic approach is proposed to correlate the mechanical and chemical properties of plastic materials in art and design objects, at both surface and subsurface levels, to obtain information about their conservation state and to monitor their degradation. The approach was used to investigate the photo-oxidation of acrylonitrile butadiene styrene (ABS), a plastic commonly found in many artistic and design applications, using ABS-based LEGO bricks as model samples. The modifications of the chemical and viscoelastic properties of ABS during photoaging were monitored by correlative Brillouin and Raman microspectroscopy (BRaMS), combined with portable and noninvasive broad-range external reflection infrared (IR) spectroscopy and nuclear magnetic resonance (NMR) relaxometry, directly applicable in museums. BRaMS enabled combined measurements of Brillouin light scattering and Raman spectroscopy in a microspectroscopic setup, providing for the coincident probe of the chemical and mechanical changes of ABS at the sample surface. NMR relaxometry allowed for noninvasive measurements of relaxation times and depth profiles which are directly related to the molecular mobility of the material. Complementary chemical information was acquired by external reflection IR spectroscopy. The simultaneous probe of the chemical and mechanical properties by this multimodal spectroscopic approach enabled us to define a decay model of ABS in terms of compositional changes and variation of stiffness and rigidity occurring with photodegradation. The knowledge acquired on LEGO samples has been used to rate the conservation state of ABS design objects noninvasively investigated by external reflection Fourier transform IR spectroscopy and NMR relaxometry offered by the MObile LABoratory (MOLAB) platform of the European Research Infrastructure of Heritage Science.

Volume properties and spectroscopy: a terahertz Raman investigation of hen egg white lysozyme.

The low frequency depolarized Raman spectra of 100 mg/ml aqueous solutions of hen egg white lysozyme (HEWL) have been collected in the 25-85 °C range. Short and long exposures to high temperatures have been used to modulate the competition between the thermally induced reversible and irreversible denaturation processes. A peculiar temperature evolution of spectra is evidenced under prolonged exposure of the protein solution at temperatures higher than 65 °C. This result is connected to the self-assembling of polypeptide chains and testifies the sensitivity of the technique to the properties of both protein molecule and its surrounding. Solvent free spectra have been obtained after subtraction of elastic and solvent components and assigned to a genuine vibrational contribution of hydrated HEWL. A straight similarity is observed between the solvent-free THz Raman feature and the vibrational density of states as obtained by molecular dynamics simulations; according to this, we verify the relation between this spectroscopic observable and the effective protein volume, and distinguish the properties of this latter respect to those of the hydration shell in the pre-melting region.

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.

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.

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.

Extended frequency range depolarized light scattering study of N-acetyl-leucine-methylamide-water solutions.

We have studied the influence of the amphiphilic model peptide N-acetyl-leucine-methylamide (NALMA) on the dynamics of water using extended frequency range depolarized light scattering (EDLS), between 0.3 GHz and 36 THz. This technique allowed us to separate solute from solvent dynamics and bulk from hydration water, providing both characteristic times and relative fractions. In the temperature range 5-65 °C, a retardation factor from 9 to 7 is found for water hydrating NALMA. Moreover, in the same range, a hydration number from 62 to 50 is observed, corresponding to more than two hydration layers. This strong perturbation suggests the existence of a collective effect of amphiphilic molecules on surrounding water molecules.

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.

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.