Search

Significance Volume regulation is key in maintaining important tissue functions, such as growth or healing. In this process, the role of efflux of cellular fluids is difficult to capture due to the lack of apt technologies. Here, we use a tool based on Brillouin light scattering (BLS) that uses the interaction of a laser light with inherent picosecond timescale density fluctuations in the sample. We induced gradual volume decrease in multicellular spheroids using osmotic perturbations. BLS revealed a nonlinear increase of the tissue compressibility due to the subsequent increased biopolymer crowding within the cells. Our findings should inspire research regarding volume regulation and cellular crowding in tissues and stimulate the emergence of models for cell mechanics at short timescales., Volume regulation is key in maintaining important tissue functions, such as growth or healing. This is achieved by modulation of active contractility as well as water efflux that changes molecular crowding within individual cells. Local sensors have been developed to monitor stresses or forces in model tissues, but these approaches do not capture the contribution of liquid flows to volume regulation. Here, we use a tool based on Brillouin light scattering (BLS) that uses the interaction of a laser light with inherent picosecond timescale density fluctuations in the sample. To investigate volume variations, we induced osmotic perturbations with a polysaccharide osmolyte, Dextran (Dx), and compress cells locally within multicellular spheroids (MCSs). During osmotic compressions, we observe an increase in the BLS frequency shift that reflects local variations in the compressibility. To elucidate these data, we propose a model based on a mixing law that describes the increase of molecular crowding upon reduction of the intracellular fluids. Comparison with the data suggests a nonlinear increase of the compressibility due to the dense crowding that induces hydrodynamic interactions between the cellular polymers.

Significance Plants need to constantly adapt to a changing environment. Adaptation includes responses to biotic and abiotic stress. Key elements determining the response to abiotic stress are the cell walls surrounding all plant cells and the phytohormone abscisic acid, which influence turgor pressure in plants. Turgor pressure in plant cells is much higher than in animal cells and a key driver of plant growth and development. Here, we investigate the mechanism regulating cell wall stiffness and coordinating changes in stiffness and turgor with abscisic acid production. We characterize key elements of the mechanism and dissect its mode of action. This knowledge will enable us to improve plant resistance to drought stress, which is necessary due to our changing environment., Plant cells can be distinguished from animal cells by their cell walls and high-turgor pressure. Although changes in turgor and the stiffness of cell walls seem coordinated, we know little about the mechanism responsible for coordination. Evidence has accumulated that plants, like yeast, have a dedicated cell wall integrity maintenance mechanism. It monitors the functional integrity of the wall and maintains integrity through adaptive responses induced by cell wall damage arising during growth, development, and interactions with the environment. These adaptive responses include osmosensitive induction of phytohormone production, defense responses, as well as changes in cell wall composition and structure. Here, we investigate how the cell wall integrity maintenance mechanism coordinates changes in cell wall stiffness and turgor in Arabidopsis thaliana. We show that the production of abscisic acid (ABA), the phytohormone-modulating turgor pressure, and responses to drought depend on the presence of a functional cell wall. We find that the cell wall integrity sensor THESEUS1 modulates mechanical properties of walls, turgor loss point, ABA biosynthesis, and ABA-controlled processes. We identify RECEPTOR-LIKE PROTEIN 12 as a component of cell wall integrity maintenance–controlling, cell wall damage–induced jasmonic acid (JA) production. We propose that THE1 is responsible for coordinating changes in turgor pressure and cell wall stiffness.

International audience; Biological composites offer self-healing properties, biocompatibility, high responsivity to external stimuli, and multifunctionality, due, in part, to their complex, hierarchical microstructure. Such materials can be inexpensively grown, and self-assembled from the bottom up, enabling democratized, sustainable manufacturing routes for micro- and nano-devices. While biological composites have been shown to incorporate rich photonic structures, their phononic properties have hitherto remained unexplored. In this study, we demonstrate that biological composites in the form of micron-thick decellularized onion cell scaffolds behave as an organic phononic material, with the presence of band gaps forbidding the propagation of elastic waves in select frequency ranges. We show that the onion cells’ phononic properties can be phenotypically tuned, and anticipate these findings will yield new biologically-derived, “green,” and genetically tailorable phononic materials.

Measuring the complex mechanical properties of biological objects has become a necessity to answer key questions in mechanobiology and to propose innovative clinical and therapeutic strategies. In this context, Brillouin light scattering (BLS) has recently come into vogue, offering quantitative imaging of the mechanical properties without labels and with a micrometer resolution. In biological samples, the magnitude of the spectral changes are typically of a few tens of MHz, and the ability of modern spectrometers to monitor such subtle changes needs to be evaluated. Moreover, the multiplicity of variations in optical arrangements, specific to each lab, requires to set a standard for the assessment of the characteristics of BLS systems. In this paper we propose a protocol to evaluate the precision and accuracy of two commercial spectrometers that is reproducible across labs. For a meaningful comparison, we coupled the spectrometers to the same microscope and to the same laser. We first evaluated the optimum acquisition time and laser power. We evaluated the precision using pure water samples. We determined the accuracy by probing water solutions with increasing concentration of salt and comparing it with theory. Following these quantifications, we applied the VIPA-based spectrometer to tumor spheroids engineered from different cell lines that possess different metastatic potentials and resistance to therapies. On these models, we detected significant changes in the linewidth suggesting that BLS measurements of the viscosity could be used as a read-out to distinguish different levels of drug resistance.

The structure of teeth can be altered by diet, age or diseases such as caries and sclerosis. It is very important to characterize their mechanical properties to predict and understand tooth decay, design restorative dental procedures, and investigate their tribological behavior. However, existing imaging techniques are not well suited to investigating the micromechanics of teeth, in particular at tissue interfaces. Here, we describe a microscope based on Brillouin light scattering (BLS) developed to probe the spectrum of the light scattered from tooth tissues, from which the mechanical properties (sound velocity, viscosity) can be inferred with a priori knowledge of the refractive index. BLS is an inelastic process that uses the scattering of light by acoustic waves in the GHz range. Our microscope thus reveals the mechanical properties at the micrometer scale without contact with the sample. BLS signals show significant differences between sound tissues and pathological lesions, and can be used to precisely delineate carious dentin. We also show maps of the sagittal and transversal planes of sound tubular dentin that reveal its anisotropic microstructure at 1 µm resolution. Our observations indicate that the collagen-based matrix of dentine is the main load-bearing structure, which can be considered as a fiber-reinforced composite. In the vicinity of polymeric tooth-filling materials, we observed the infiltration of the adhesive complex into the opened tubules of sound dentine. The ability to probe the quality of this interfacial layer could lead to innovative designs of biomaterials used for dental restorations in contemporary adhesive dentistry, with possible direct repercussions on decision-making during clinical work. Statement of Significance Mechanical properties of teeth can be altered by diet, age or diseases. Yet existing imaging modalities cannot reveal the micromechanics of the tooth. Here we developed a new type of microscope that uses the scattering of a laser light by naturally-occurring acoustic waves to probe mechanical changes in tooth tissues at a sub-micrometer scale without contact to the sample. We observe significant mechanical differences between healthy tissues and pathological lesions. The contrast in mechanical properties also reveals the microstructure of the polymer-dentin interfaces. We believe that this new development of laser spectroscopy is very important because it should lead to innovative designs of biomaterials used for dental restoration, and allow delineating precisely destructed dentin for minimally-invasive strategies.

International audience; In this work, a novel lipophilic 5-fluorouracil (5-FU) derivative was synthesised and encapsulated into lipid nanocapsules (LNC). 5-FU was modified with lauric acid to give a lipophilic mono-lauroyl-derivative (5-FU-C12, MW of about 342 g/mol, yield of reaction 70%). 5-FU-C12 obtained was efficiently encapsulated into LNC (encapsulation efficiency above 90%) without altering the physico-chemical characteristics of LNC. The encapsulation of 5-FU-C12 led to an increased stability of the drug when in contact with plasma being the drug detectable until 3 h following incubation. Cytotoxicity assay carried out using MTS on 2D cell culture showed that 5-FU-C12-loaded LNC had an enhanced cytotoxic effect on glioma (9L) and human colorectal (HTC-116) cancer cell line in comparison with 5-FU or 5-FU-C12. Then, HCT-116 tumour spheroids were cultivated and the reduction of spheroid volume was measured following treatment with drug-loaded LNC and drugs alone. Similar reduction on spheroids volume was observed following the treatment with drug-loaded LNC, 5-FU-C12 and 5-FU alone, while blank LNC displayed a reduction in cell viability only at high concentration. Globally, our data suggest that the encapsulation increased the activity of the 5-FU-C12. However, in-depth evaluations of LNC permeability into spheroids are needed to disclose the potential of these nanosystems for cancer treatment.

International audience; Photoacoustic imaging is a hybrid modality that is used to image biological tissues. Using multispectral optical excitation, a functional image is obtained due to the tissue specific optical absorption that depends on the wavelengths. To classify multispectral photoacoustic images, supervised methods are classically used. However, definition of the reference spectra is often difficult, and this choice can have a large impact on the classification results. A possible approach to build relevant reference spectra is to use spectrophotometry. This study aims at comparing absorbance measured by a spectrophotometer and multispectral photoacoustic signals of various coloured phantoms. We compare qualitatively the shape of the spectra obtained, using these two modalities for each sample. Our data suggest that spectrophotometry is a promising way to define reference spectra for classification of multispectral photoacoustic datasets.

International audience; The structure of tumors can be recapitulated as an elastic frame formed by the connected cytoskeletons of the cells invaded by interstitial and intracellular fluids. The low-frequency mechanics of this poroelastic system, dictated by the elastic skeleton only, control tumor growth, penetration of therapeutic agents, and invasiveness. The high-frequency mechanical properties containing the additional contribution of the internal fluids have also been posited to participate in tumor progression and drug resistance, but they remain largely unexplored. Here we use Brillouin light scattering to produce label-free images of tumor microtissues based on the high-frequency viscoelastic modulus as a contrast mechanism. In this regime, we demonstrate that the modulus discriminates between tissues with altered tumorigenic properties. Our micrometric maps also reveal that the modulus is heterogeneously altered across the tissue by drug therapy, revealing a lag of efficacy in the core of the tumor. Exploiting high-frequency poromechanics should advance present theories based on viscoelasticity and lead to integrated descriptions of tumor response to drugs.

International audience; Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

International audience; MultiCellular Tumor Spheroids (MCTS), which mimic the 3-Dimensional (3D) organization of a tumor, are considered as better models than conventional cultures in 2-Dimensions (2D) to study cancer cell biology and to evaluate the response to chemotherapeutic drugs. A real time and quantitative follow-up of MCTS with simple and robust readouts to evaluate drug efficacy is still missing. Here, we evaluate the chemotherapeutic drug 5-Fluorouracil (5-FU) response on the growth and integrity of MCTS two days after treatment of MCTS and for three colorectal carcinoma cell lines with different cohesive properties (HT29, HCT116 and SW480). We found different sensitivity to 5-FU for the three CRC cell lines, ranging from high (SW480), intermediate (HCT116) and low (HT29) and the same hierarchy of CRC cell lines sensitivity is conserved in 2D. We also evidence that 5-FU has a strong impact on spheroid cohesion, with the apparition of a number of single detaching cells from the spheroid in a 5-FU dose- and cell line-dependent manner. We propose an innovative methodology for the chemosensitivity evaluation in 3D MCTS that recapitulates and regionalizes the 5-FU-induced changes within MCTS over time. These robust phenotypic read-outs could be easily scalable for high-throughput drug screening that may include different types of cancer cells to take into account tumor heterogeneity and resistance to treatment.

Probing the mechanical properties of plant cell wall is crucial to understand tissue dynamics. However, the exact symmetry of the mechanical properties of this anisotropic fiber-reinforced composite remains uncertain. For this reason, biologically relevant measurements of the stiffness coefficients on individual living cells are a challenge. For this purpose, we have developed the single-cell optoacoustic nanoprobe (SCOPE) technique, which uses laser-generated acoustic waves to probe the stiffness, thickness and viscosity of live single-cell subcompartments. This all-optical technique offers a sub-micrometer lateral resolution, nanometer in-depth resolution, and allows the non-contact measurement of the mechanical properties of live turgid tissues without any assumption of mechanical symmetry. SCOPE experiments reveal that single-cell wall transverse stiffness in the direction perpendicular to the epidermis layer of onion cells is close to that of cellulose. This observation demonstrates that cellulose microfibrils are the main load-bearing structure in this direction, and suggests strong bonding of microfibrils by hemicelluloses. Altogether our measurement of the viscosity at high frequencies suggests that the rheology of the wall is dominated by glass-like dynamics. From a comparison with literature, we attribute this behavior to the influence of the pectin matrix. SCOPE's ability to unravel cell rheology and cell anisotropy defines a new class of experiments to enlighten cell nano-mechanics.

International audience; 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.

The reflection of picosecond ultrasonic pulses from a cell-substrate interface is used to probe cell-biomaterial adhesion with a subcell resolution. We culture monocytes on top of a thin biocompatible Ti metal film, supported by a transparent sapphire substrate. Low-energy femtosecond pump laser pulses are focused at the bottom of the Ti film to a micron spot. The subsequent ultrafast thermal expansion launches a longitudinal acoustic pulse in Ti, with a broad spectrum extending up to 100 GHz. We measure the acoustic echoes reflected from the Ti-cell interface through the transient optical reflectance changes. The time-frequency analysis of the reflected acoustic pulses gives access to a map of the cell acoustic impedance Zc and to a map of the film-cell interfacial stiffness K simultaneously. Variations in Zc across the cell are attributed to rigidity and density fluctuations within the cell, whereas variations in K are related to interfacial intermolecular forces and to the nano-architecture of the transmembrane bonds.

International audience; By means of an ultrafast optical technique, we track focused gigahertz coherent phonon pulses in objects down to sub-micron in size. Infrared light pulses illuminating the surface of a single metal-coated silica fibre generate longitudinal-phonon wave packets. Reflection of visible probe light pulses from the fibre surface allows the vibrational modes of the fibre to be detected, and Brillouin optical scattering of partially transmitted light pulses allows the acoustic wavefronts inside the transparent fibre to be continuously monitored. We thereby probe acoustic focusing in the time domain resulting from generation at the curved fibre surface. An analytical model, supported by three-dimensional simulations, suggests that we have followed the focusing of the acoustic beam down to a~150-nm diameter waist inside the fibre. This work significantly narrows the lateral resolution for focusing of picosecond acoustic pulses, normally limited by the diffraction limit of focused optical pulses to~1 μm, and thereby opens up a new range of possibilities including nanoscale acoustic microscopy and nanoscale computed tomography.

International audience; Zero-group velocity (ZGV) Lamb modes are associated with sharp local acoustic resonances and allow, among other features, local measurement of Poisson's ratio. While the thermoelastic generation of Lamb waves in metal plates has been widely studied, the case of materials of low-optical absorption remains unexplored. In materials such as glasses, the generation of bulk elastic waves has been demonstrated to be sensitive to the refracted light distribution. In this paper, a detailed analysis of the effect of light refraction on the laser-based generation of ZGV Lamb modes is presented. Experiments are performed on a bare glass plate without the need for an additional layer for light absorption or reflection. Using an appropriate tilted volume source, it is shown that the laser-ultrasonic technique allows non-contact measurement of the Poisson's ratio.

By means of an ultrafast opto-acoustic technique we study the nanoindentation of thin chromium films on sapphire substrates using a ceramic ball bearing. Acoustic pulses at approximately 40 GHz returning from the film-indenter interface allow the film indentation profiles to be probed to sub-nanometer resolution over contact areas approximately 25 microm in radius. The deformation of the films during loading is thereby revealed. Furthermore, thermal wave imaging of the contact at megahertz frequencies is simultaneously achieved.

Conference of 42nd Annual Review of Progress in Quantitative Nondestructive Evaluation, QNDE 2015, Incorporating the 6th European-American Workshop on Reliability of NDE ; Conference Date: 26 July 2015 Through 31 July 2015; Conference Code:119404; International audience; Directivity patterns of an acoustic source generated by the absorption of a laser pulse at the interface between a transparent epoxy-resin half-space and an opaque CFRP isotropised half-space have been calculated using the reciprocity theorem. Longitudinal and shear radiation patterns of acoustic field emitted in the CFRP are compared in respect with the loading surface condition.

The mechanical properties of cells play a key role in several fundamental biological processes, such as migration, proliferation, differentiation and tissue morphogenesis. The complexity of the inner cell composition and the intricate meshwork formed by transmembrane cell-substrate interactions demands a non-invasive technique to probe cell mechanics and cell adhesion at a subcell scale. In this paper we review the use of laser-generated GHz acoustic waves—a technique called picosecond ultrasonics (PU)—to probe the mechanical properties of single cells. We first describe applications to vegetal cells and biomimetic systems. We show how these systems can be used as simple models to understand more complex animal cells. We then present an opto-acoustic bio-transducer designed for in vivo measurements in physiological conditions. We illustrate the use of this transducer through the simultaneous probing of the density and compressibility of Allium cepa cells. Finally, we demonstrate that this technique can quantify animal-cell adhesion on metallic surfaces by analyzing the acoustic pulses reflected off the cell-metal interface. This innovative approach allows investigating quantitatively cell mechanics without fluorescent labels or mechanical contact to the cell.

Techniques that can probe the thermal properties of cells are used in many applications ranging from cryogenic preservation to hyperthermia therapy, and provide powerful tools to investigate diseased conditions. The structural complexity of cells, however, requires innovative modalities operating at a subcell scale. We developed a label-free, non-ionizing technique based on a thermoelastic lens. With this device, we captured images of single cells with a ∼2 μm resolution based on thermal properties as the contrast mechanism. To investigate the thermorheological behaviour of cells, we present simultaneous acoustic imaging using an inverted opto-acoustic microscope. Acoustic impedances extracted from the acoustic images support the effusivity obtained from the thermal images. This technique should provide diagnostic tools at the single cell scale.

The interior of the cell nucleus is comparable to a solid network bathed in an interstitial fluid. From the extrapolation of low frequency data, it is expected that such network should dictate the response of the nucleus to mechanical stress at high frequencies, described by unique elastic moduli. However, none of the existing techniques that can probe the mechanical properties of cells can exceed the kHz range, and the mechanics of the nuclear network remain poorly understood. We use laser-generated acoustic waves to probe remotely the stiffness and viscosity of nuclei in single cells in the previously unexplored GHz range with a ∼100 nm axial resolution. The probing of cells at contrasted differentiation stages, ranging from stem cells to mature cells originating from different tissues, demonstrates that the mechanical properties of the nuclear network are common across various cell types. This points to an asymptotically increasing influence of a solid meshwork of connected chromatin fibers.

Mechanical contacts are crucial to systems in engineering, electronics and biology. The microscopic nature of the contacting surfaces determines how they mesh on the nanoscale. There is thus much interest in methods that can map the actual area of two surfaces in contact-the real contact area during the loading or unloading phases. We address this problem using an ultrafast optical technique to generate non-equilibrium electrons that diffuse across a nanoscale mechanical contact between two thin gold films deposited on sapphire. We image this process in the contact and near-contact regions to micron resolution in situ using transient optical reflectivity changes on femtosecond time scales. By use of a model of the ultrashort-time electron dynamics, we account for an up to similar to 40%dropin the transient optical reflectivity change on contact. We thereby show how the real contact area of a nanoscale contact can be mapped. Applications include the probing of microelectronic mechanical devices.

International audience; The symmetry of a thermoelastic source resulting from laser absorption can be broken when the direction of light propagation in an elastic half-space is inclined relatively to the surface. This leads to an asymmetry of the directivity patterns of both compressional and shear acoustic waves. In contrast to classical surface acoustic sources, the tunable volume source allows one to take advantage of the mode conversion at the surface to control the directivity of specific modes. Physical interpretations of the evolution of the directivity patterns with the increasing light angle of incidence and of the relations between the preferential directions of compres-sional-and shear-wave emission are proposed. In order to compare calculated directivity patterns with measurements of normal displacement amplitudes performed on plates, a procedure is proposed to transform the directivity patterns into pseudo-directivity patterns representative of the experimental conditions. The comparison of the theoretical with measured pseudo-directivity patterns demonstrates the ability to enhance bulk-wave amplitudes and to steer specific bulk acoustic modes by adequately tuning light refraction.

In laser ultrasonics, when focusing strongly the pump beam, a three-dimensional (3D) diffracted acoustic field can be generated. Considering weakly absorbing materials, the 3D interaction of this elastic perturbation with the optical detection beam must be taken into account. A semi-analytical model is proposed to describe such phenomena in reflectometric measurements. Once the acoustooptic interaction has been partially linearized, the resulting inhomogeneous differential system is solved using the matricant method. Good agreement is found comparing the calculated results with the experimental data in an aluminum film.

Gigahertz acoustic waves propagating on the surface of a metal halfspace are detected using different all-optical detection schemes, namely, deflectometry and beam distortion detection techniques. Both techniques are implemented by slightly modifying a conventional reflectometric setup. They are then based on the measurement of the reflectivity change but unlike reflectometric measurements, they give access to the sample surface displacement. A semi-analytical model, taking into account optical, thermal, and mechanical processes responsible for acoustic waves generation, allows analyzing the physical content of the detected waveforms.

International audience; When a laser beam is absorbed in a semi-transparent material, a volume acoustic source is created owing to penetration of the laser beam inside the material and to thermoelastic transduction. Many experimental and theoretical studies have been conducted to better understand this ultrasound generation process with normal laser light incidence on the material surface. The purpose of this paper is to analyze the effects of the asymmetry caused by oblique incidence of a laser line source on the generation of acoustic waves in semi-transparent isotropic materials. Experiments on a glass plate demonstrate that such an obliquely incident laser light strongly affects bulk acoustic waves generation. Compressional and shear waves are enhanced and the loss of symmetry of the acoustic source causes asymmetrical behavior of the acoustic waves. Surprisingly, compressional-wave amplitude decreases whereas shear-wave amplitude increases in the region where the electromagnetic energy is refracted. This feature is explained by semi-analytical calculations.

We use femtosecond laser pulses absorbed in a metallic transducer, namely the picosecond ultrasonics technique, for the remote optical generation and detection of GHz acoustic frequencies in single cells by pump-probe sampling. Samples are MC3T3 cells adhering on a TiAl4V alloy substrate. Both pump and probe beams are focused at the cell/transducer interface. The pump absorption yields a temperature rise in the absorbing substrate and a picosecond acoustic pulse is generated through the thermoelastic effect. The probe beam is partially reflected from the metallic interface and partially scattered by the acoustic wavefront propagating in the transparent cell. The change of reflectivity of the cell is measured as a function of the pump-probe time delay. Interferences arise from the two probe contributions causing the so-called Brillouin oscillations. Optical phase variations due to acoustic-induced changes in cell thickness are simultaneously measured. The result of a semi-analytical calculation is fitted to the experimental data. Acoustic frequencies are detected at 30 GHz in the nucleus of single osteoblast cells.

International audience; The purpose of this paper is to study the effect of a non-normal optical penetration due to an obliquely incident laser source. It is shown that the loss of symmetry due to such a penetration influences specific bulk acoustic modes. For a given detection position, increase in shear wave amplitude is obtained by orienting the incident laser source. 1. Introduction Since the first quantitative approaches in the 60's [1], a large number of studies has been conducted to better understand the generation of laser-generated acoustic waves. Models for the acoustic generation in the thermoelastic regime, taking into account the optical penetration of the source [2] or the effect of source width [3], were developed. A more general approach based on Green's function formalism also included thermal diffusion in the medium [4]. These works have dealt with the modeling of a circular spot of laser illumination. However, modeling of the acoustic field generated by a line source is of interest, since the signal-to-noise ratio may be increased in this experimental situation [5]. All the works cited above assume a normal incidence of the laser beam with respect to the illuminated surface of the sample. The purpose of this paper is to analyse the effects of oblique incidence on acoustic waves generated by a line-focused laser source. We present a theoretical model accounting for the effects of optical penetration, finite width of the beam and pulse duration of the laser source. The absorbed energy density is first calculated by solving Maxwell's equations. Then the thermoelastic equations are solved to obtain the displacement field through a semi-analytical calculation based on a double Fourier transform in space and time. It is shown that oblique incidence increases shear wave amplitude. Changes of the longitudinal waveforms are also discussed.

Using an ultrafast optical technique we measure coherent phonon-pulse reflection from---and heat flow across---a mechanical contact of nanoscale thickness between a thin metal film and a spherical dielectric indenter. Picosecond phonon wave packets at $\ensuremath{\sim}50\text{ }\text{GHz}$ returning from this interface probe the pressure distribution, the contact area, and the indentation profile to subnanometer resolution, revealing the film deformation in situ. These measurements and simultaneous thermal-wave imaging at $\ensuremath{\gtrsim}1\text{ }\text{MHz}$ are consistent with significant enhancement of phonon transport across the near-contact nanogap.

A diffracted picosecond ultrasonic field is generated by a 100 fs laser pulse focused onto a spot with diameter less than $1\text{ }\ensuremath{\mu}\text{m}$. As the pulse duration is shorter than the relaxation time of the electrons, the microscopic evolutions must be accounted for in metals. Following the absorption of the laser energy transported by the photons, electrons diffuse in all directions and simultaneously transfer their energy to the crystal lattice through collisions with phonons. The diffraction of the resulting electronic and phononic sources launches a three-dimensional (3D) ultrasonic field, containing bulk and surface waves. This microscopic generation process is here described with a 3D two-temperature model. A combination of Hankel and Fourier transforms allows the analytical solving of the temperature and elastic fields in the frequency--wave number domain. As expected, the highest frequencies composing the acoustic signal are affected by the quasi-instantaneous expansion of the electronic source, yet a theoretical analysis also demonstrates the influence of ultrafast electron dynamic on surface waves and on the low acoustic frequencies generated by diffraction. This conclusion is revealed by the study of the Rayleigh wave in a $2.1\text{\ensuremath{-}}\ensuremath{\mu}\text{m}$-thick gold film deposited on a semi-infinite silicon substrate. The good agreement between the calculated spatiotemporal signals and the experimental results supports the analytical predictions.

International audience; The three-dimensional 3D photoelastic interaction involved in the detection mechanism of picosecond ultrasonics is investigated in micrometric metallic films. In pump-probe experiments, the laser source beam is focused to a spot size of less than 1 μm. A 3D diffracted acoustic field is generated at high frequencies of several tens of gigahertz, containing longitudinal and shear waves altogether. Their propagation changes the dielectric permittivity tensor and the material becomes optically heterogeneous. Consequently, the detection process is modeled through the propagation of the laser probe beam in a material with dielectric properties varying in all directions. Thus, the solution of Maxwell's equations leads to a differential system, the source term of which is proportional to the acoustic field itself. In the frame of small perturbation theory, the latter is decomposed into a continuous sum of monochromatic plane waves. The scattered electromagnetic field is described using the matricant, and the ensuing analytical solution then allows analyzing the 3D photoelastic interaction. The contribution of acoustic diffraction and shear wave detection to the reflectometric signal is put into relief. Good agreement with experiments performed in a 1 μm thick aluminum film is found.

International audience; The three-dimensional (3D) photoelastic interaction involved in the detection mechanism of picosecond ultrasonics is investigated in micrometric metallic films. In pump-probe experiments, the laser source beam is focused to a spot size less than 1 µm. A 3D diffracted acoustic field is generated at high frequencies of several tens of gigahertz, containing longitudinal and shear waves altogether. Their propagation changes the dielectric permittivity tensor and the material becomes optically heterogeneous. Consequently, the detection process is modeled through the propagation of the laser probe beam in a material with dielectric properties varying in all directions. Thus, the solving of Maxwell equations leads to a differential system, the source term of which is proportional to the acoustic field itself. The scattered electromagnetic field is described using the matricant and the ensuing analytical solution allows then analyzing the 3D photoelastic interaction. Good agreement with experiments performed in a 1 µm thick aluminum film is found.

An optical grating has been introduced in a picosecond ultrasonics experiment, in order to vary continuously the duration of the laser beam pulse from 0.1to150ps. The evolution of the measured signal has been observed and analyzed through the comparison with a theoretical approach based on a two-temperature model. The latter allows matching the acoustic echoes together with the thermal background and the coincidence peak, for each pulse duration and at any time scale. The broadening of the acoustic echoes and the disappearing of its Brillouin component, along with the diminishing of the thermal coincidence peak, have been demonstrated when increasing the pulse duration. For a constant incident pulse energy, the efficiency of acoustic generation is optimum for the shortest pulses. Nevertheless, for longer pulses designed to obtain thermal conditions below the ablation threshold, acoustic generation could be enhanced.

We present a time resolved spectroscopy for the picosecond ultrasonics technique. Detection of picosecond coherent phonons using a wavelength continuum generation in a photonic crystal fiber with femtosecond laser pulses is introduced. Measurements are performed on two samples made of tungsten and gallium arsenide for selected wavelengths of probe pulses within a bandwidth of 250nm with an 825nm center wavelength. Characteristic changes of the shape of the acoustic signal due to the selection of the probe wavelength are shown.

Probing the mechanical properties of the nucleus is essential to understanding whole-cell mechanics under different physiological conditions. The nucleus has a complex composition yielding intricate rheological behavior that is appealing for measurements over a wide frequency range. However none of the existing techniques can exceed the kHz range. Moreover, the invasive nature of most of these techniques hampers the study of cell mechanics evolution during biological processes under physiological conditions. Here, we report the use of laser-generated GHz acoustic waves to probe the stiffness and viscosity of nuclei in single live cells. We demonstrate that the stiffness and viscosity reflect the compressional dynamics of the nuclear components. Furthermore we reveal the existence of a universal nuclear stiffness equal to 15 GPa in adult mammalian cells. We also emphasize the importance of Poisson's ratio in the describing the dynamic mechanical behavior of cells. Accordingly, we postulate that at GHz acoustical frequencies, anharmonic processes might occur in the cell in addition to thermally activated absorption processes already considered at low frequencies. On this basis, we demonstrate that the mechanical properties of the internal structure of the cell nucleus probed by GHz acoustic waves correlate with a specific gene expression pattern during cell differentiation. We suggest that the induction of differentiation synchronizes the stiffness of the cell nuclei. This analysis is supported by the observation of a new stress fiber organization around the nucleus and of an increased number of focal adhesions throughout the entire cell. The method described here is therefore capable of probing in a non-invasive manner the nanomechanical behavior of single live cell nuclei. This approach should open new areas in the investigation of physiological processes under biological conditions.

Cells have a complex composition that yields an intricate rheological behavior, appealing for measurements over a wide frequency range. However the existing techniques cannot exceed the kHz range, and rely for most on injected or contacting functionalized microprobes. Here, we report on an innovative non-contact optoacoustic probing of the mechanical properties of single cells at GHz acoustic frequencies under physiological conditions.We culture cells on a biocompatible Ti6Al4V metal alloy. Low-energy femtosecond laser pulses are focused at the cell-Ti6Al4V interface to a sub-µm spot. The ensuing ultrafast thermal dilatation of the metal launches a high-frequency sound pulse in the cell. Acoustic propagation is measured remotely with an ultrafast laser probe through Brillouin light scattering. This all-optical non-invasive technique offers a broad frequency range, extending up to 1 THz, a sub-µm lateral resolution and a nanometer in-depth resolution.For illustration, we here concentrate on the cell nucleus. Using the laser-generated GHz acoustic waves, we probe the stiffness and viscosity of the nuclei of various cell types. We demonstrate for the first time that, at GHz frequencies, the nucleus stiffness tends to a unique value, much larger than that observed at kHz frequencies. We demonstrate that this universal stiffness reflects the compressional dynamics of the nucleus components.We also show that the evolution of the mechanical properties of the nucleus during cell differentiation is correlated with a specific gene expression pattern. In the frame of soft glass rheology, we project the GHz mechanical properties of the differentiated nuclei to the kHz range. Comparison with the kHz data obtained by alternative techniques suggests the existence of a new absorption process appearing at GHz frequencies.This innovative technique defines a new class of experiments to enlighten cell mechanics in physiological conditions at a subcell scale.

Recently, the coherent generation of GHz acoustic waves using ultrashort laser pulses has demonstrated the ability to probe the sound velocity in vegetal cells and in cell-mimicking soft micro-objects with micrometer resolution, opening tremendous potentialities for single-cell biology. However, manipulating biological media in physiological conditions is often a technical challenge when using a laser-based setup. In this article, we present a new opto-acoustic bio-transducer composed of a thin metal film sputtered on a transparent heat sink that allows reducing importantly the laser-induced cellular stresses, and offers a wide variety of optical configurations. In particular, by exploiting the acoustic reflection coefficient at the sample-transducer interface and the photoacoustic interaction inside the transparent sample, the density and compressibility of the sample can be probed simultaneously. Using an ad hoc signal analysis based on Hilbert and wavelet transforms, these quantities are measured accur...

International audience; Laser ultrasonic techniques allow the remote analysis of adhesion mechanisms at imperfect interfaces up to GHz frequencies. However, the sensitivity of interface waves to the properties of the contact is not very well known. In the present work, the mechanical boundary conditions are described considering that the contacting solid half-spaces are connected by tangential and normal springs. Such a modeling implies a discontinuity of the displacement field across the interface. To identify the relative amplitudes of the different types of interface waves-skimming, leaky Rayleigh (LR) and Stoneley (St) waves-a semi-analytical time domain model describing the thermoelastic laser generation is derived. The results illustrate the influence of the boundary conditions on the attenuation of the LR wave and on the existence of the St wave. In addition, a single compact and elegant dispersion equation is presented to investigate the behaviour of the interface waves propagating along a generalized imperfect boundary. Such analysis reveals the existence of a cutoff frequency f(c) close to which the St wave behaves like a skimming transverse wave. A scaled analysis demonstrates that two master curves suffice to describe the dispersion of LR and St waves and that f(c) is inversely proportional to the tangential interfacial spring constant. [http://dx.doi.org/10.1063/1.4733949]

Using an ultrafast optical technique we probe coherent phonon propagation inside a single microcapsule composed of a nanometric polymer shell made of poly(lactide-co-glycolide) (PLGA) encapsulating a liquid perfluorooctyl bromide (PFOB) core. Longitudinal storage and loss moduli are measured simultaneously in the transparent shell and core at frequencies ∼18 and ∼4 GHz, respectively, using time-resolved Brillouin spectroscopy. A time-frequency analysis allows determination of the thicknesses of several capsule shells ranging from 620 down to 80 nm. Comparison with lower frequency data shows a weak power-law frequency dependence of phonon attenuation in the PLGA shell, the signature of thermally activated processes in glasses.

Biological objects are exquisitely sensitive to temperature variations and their mechanical characterization is often a challenge when using the picosecond ultrasonics technique. To reduce the laser-induced temperature rise, we place single biological cells on a thin metal transducer and we focus the laser beam that generates the acoustic waves at frequencies ≤ 150 GHz on the rear side of the transducer. The acoustic waves propagate through the transducer and are partially transmitted to the cell to create the so-called Brillouin oscillations. The frequency of these oscillations provides a direct measurement of the sound velocity. The simultaneous measurement of the acoustic reflection coefficient at the transducer/cell interface allows the determination of both the density and the compressibility of the cell.

The measurement of the mechanical properties of single biological cells with a nanometer depth resolution using only coherent light is proposed. A pump-probe set-up based on an ultrafast laser (100 fs pulses) is used to excite and detect acoustic frequencies in the GHz range. Experiments are performed on single fixed mouse MC3T3 cells adhering on titanium alloy substrate. Using two different probe wavelengths, the contributions to the optical detection resulting from the cell interface displacements and from interactions between acoustic waves and the laser light are identified. Semi-analytical calculations allow the determination of acoustic celerities and thicknesses in cells thinner than 150 nm.

This Erratum corrects an error from an earlier Letter [Opt. Lett. 34, 3740 (2009)] in the calculation of the mode frequencies of a silica microsphere for angular mode number l=0.

We investigate the contact between a thin metal film and a ball bearing indented to nanometre depths using picosecond ultrasonics. The area of contact is acoustically imaged to micron spatial resolution using GHz acoustic pulses produced by ultrashort pulsed optical excitation of the film through a transparent substrate. In particular, acoustic echoes are detected through transient optical reflectance changes that are monitored by probe optical pulses. In this way we image the acoustic reflection coefficient at the interface between the film and the indenter. In addition, by imaging the arrival time of the acoustic echoes we determine the penetration profile of the indenter to nanometre precision. Furthermore, imaging the transient thermoreflectance gives a different means for measuring the contact area through spatial variations in thermal diffusion. We thus demonstrate that picosecond ultrasonics and thermoreflectance provide powerful tools for the non‐contact evaluation of mechanical contacts. These te...

Surface waves propagation in micro-layers is very useful for the evaluation of mechanical properties in anisotropic materials. In the present work, they are detected without any transducer by means of beam distortion detection technique in gold and highly ordered poly-graphite (HOPG) micro-layers. The surface wave velocity in HOPG is evaluated as 20 km/s, value close to Rayleigh wave velocity in diamond. The typical frequency (~10 GHz) of surface wave in HOPG is extremely high. Fast diffusion of electrons plays an important role in the process of sound excitation in gold. For this reason, a two-temperature model has been applied to describe the three-dimensional thermoelastic generation of bulk and surface acoustic waves. Good coincidence between experimental and calculated wavefronts is obtained.

We use ultrashort optical pulses to excite and detect vibrations of single silica spheres with a diameter of 5 microm placed at the surface of an acoustically mismatched substrate. In addition to the photoelastic detection of picosecond longitudinal acoustic pulses propagating inside the bulk, we detect gigahertz acoustic resonances of the sphere through probe beam defocusing. The mode frequencies are in close accord with those calculated from the elastic vibrations of a free sphere. We also record a resonant enhancement in the amplitude of specific modes of two touching spheres.

The mechanical properties of biological tissue or a cell are often linked to their phenotype. Some pathologies stem from or trigger an evolution of the mechanical properties of the cells or the tissues. It is the case of cancer whose development causes an increase of tissue rigidity and a decrease of cell rigidity. To better understand those changes and their potential link to the evolution of the disease, physicists measure mechanical properties of model systems fabricated with cancer cells through different means. It is within this framework that this thesis is situated. Multicellular Tumor Spheroids were chosen as a model system for this study. Those aggregates can be grown in vitro and are well-suited to integration into physical measurements experimental setups. Their structure also ensure they keep some important properties observed in tumours, especially from the mechanical point of view. Acoustic waves were chosen as a characterization tool for this study. They allow direct non-invasive readout of the mechanical moduli of the tissue without any phototoxicity issue.For this study a Laser Ultrasonics setup was designed and built. A pulsed laser source was used to generate acoustic pulsed with a bandwidth of approximately 100 MHz. To detect the acoustic signal after propagation in the spheroid an interferometer was used.These laser ultrasonics measurement allowed us to extract mechanical parameters from the multicellular tumor spheroids fabricated from colorectal cancer cells. This way the sound velocity and acoustic attenuation from spheroids have been extracted and compared to results in the litterature. This has allowed us to discuss the current modelling hypothesis for the mechanical response of those system, Les propriétés mécaniques d'un tissu biologique ou d'une cellule sont très souvent liés à leur phénotype. Et certaines pathologies entrainent ou découlent directement d'une évolution des propriétés mécaniques des cellules ou des tissus. C'est notamment le cas du cancer dont le développement entraine une augmentation de la rigidité des tissus tout en étant provoquant une baisse de celle des cellules affectées. Afin de mieux comprendre ces changements et leur lien potentiel avec l'évolution de la maladie, les physiciens mesurent par divers moyens les propriétés mécaniques de différents systèmes modèles fabriqués à partir de cellules cancéreuses. C'est dans ce cadre que s'inscrit ce travail de thèse. Le choix de système de modèle s'est porté sur les sphéroïdes multicellulaires tumoraux. Ces agrégats de cellules cancéreuses peuvent être cultivés in vitro et se prêtent assez bien à la mise en place sur des montages de mesures physiques tout en conservant par le biais de leurs structure un certain nombre de propriétés observées chez les tumeurs notamment sur le plan mécanique. Les ondes acoustiques sont l'outil de caractérisation mécanique utilisé dans cette étude. Elles permettent de remonter directement aux modules mécaniques du tissu de manière non-invasive et en évitant tout problème de phototoxicité. Pour cette étude un montage ultrason laser a été conçu et mis en place. Une source laser pulsée a été utilisée pour générer des impulsions acoustiques dont la bande passante est de l'ordre de la 100 de MHz. Pour détecter le signal acoustique après propagation dans le sphéroïde un interféromètre a été utilisé. Ces mesures ultrasons laser ont permis d'extraire les paramètres mécaniques de sphéroïdes multicellulaires tumoraux fabriqués à partir de cellules de cancer colorectal. Ainsi la vitesse du son et l'atténuation acoustique de ces sphéroïdes à des fréquences de l'ordre du MHz ont pu être extraites et comparés aux résultats présents dans la littérature afin de discuter les hypothèses de modélisation de ces objets

The purpose of this work is to develop a method to image optoacoustic sources in solid media. To analyze the influence of the source geometry on laser-generated acoustic waves and thus qualitatively predict if the image of the source can be obtained with sufficient accuracy, the directivity of an acoustic source is considered first. In order to quantify more precisely this influence, the complete resolution of the propagation of acoustic waves generated by this source is then achieved. The analysis of the effect of oblique incidence of a laser beam in the photoacoustic generation process is proposed as an illustration. The second part of this thesis is devoted to the inverse problem resolution, which provides the image of the initial acoustic source. A backpropagation algorithm is then implemented. It is based on the time-reversal principle and the Huygens' principle, and simulates, in a solid medium, the propagation of the measured acoustic waves back to the initial position of the acoustic source., L'objectif de ces travaux est le développement d'une méthode d'imagerie de sources optoacoustiques en milieu solide. Afin d'analyser l'influence de la géométrie de la source sur les ondes acoustiques qu'elle génère et ainsi prévoir qualitativement si l'image de la source peut être obtenue avec une précision suffisante, la directivité d'une source acoustique est d'abord considérée. Pour quantifier plus précisément cette influence, la résolution complète de la propagation des ondes générées par cette source est ensuite menée. L'analyse de l'influence de l'incidence oblique d'un faisceau laser lors de la génération photoacoustique est proposée en illustration. La seconde partie de ce manuscrit est consacrée à la résolution du problème inverse qui permet d'obtenir l'image de la source initiale. Un algorithme de rétropropagation est alors mis en place. Il est basé sur les principes du retournement temporel et de Huygens, et simule la propagation, en milieu solide, des ondes mesurées vers la position initiale de la source acoustique.