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.

Ge 2 Sb 2 Te 5 is a phase change material candidate to constitute the active element of future nonvolatile memory devices. The evolution of the thermal resistance at the interface between an aluminum thin layer and Ge2Sb2Te5 is studied using the time resolved pump probe technique from room temperature to 400 °C. The thermal resistance is influenced by the amorphous to crystalline phase change occurring in Ge2Sb2Te5. The decrease in the thermal resistance from the amorphous to the crystalline phase is well explained by the diffuse mismatch model asymptotic form for high temperature. The large increase of the interface thermal resistance between fcc and hcp crystalline states is explained by the fast and significant grain growth and species inter-diffusion during this second phase change. This leads to the formation of an interfacial layer whose chemical and mechanical intrinsic properties have been measured in order to model the thermal resistance in the hcp state.

A method for identifying thermal properties of a stack constituted from a metallic thin layer deposited on a silicon substrate is proposed. Data are acquired using the picoseconds time resolved pump-probe technique. A Bayesian technique based on the Monte Carlo Markov Chain is implemented in order to identify simultaneously the thermal conductivity of the layer, the thermal resistance at the interface between the layer and the substrate and the extension of the heat source at the initial time. It is demonstrated that, despite to the correlation between sensitivity functions on the investigated time range, each parameter is accurately identified.

The picosecond ultrasonics technique is used to investigate the viscoelastic properties of nucleus of fixed single osteoblast progenitor cells adhering on a titanium alloy substrate. A two-color probing picosecond ultrasonics and a fluorescence visualization setups were developed and combined to allow to distinguish subcomponents inside the cell under investigation. It opens the way for quantitative measurements of the viscoelastic properties of single cells and of their sub-micrometer thickness. It is shown that a blue probe, lambda = 400 nm, is preferable to a red probe, lambda = 800 nm, to perform these measurements with fixed sub-micrometer bone cells. 26 GHz acoustic frequencies are detected in cells as thin as 135 nm. A 1D analytical model of the acoustic generation and of the optical detection is used to describe the experimental results. The nucleus longitudinal elastic moduli (13-16 GPa) and dynamic viscosities (13-30 cP) are measured at high frequencies (GHz) from a time-frequency analysis of the experimental data of fixed single cells.

The time-resolved pump-probe technique has been implemented to study the temperature-dependent thermal boundary resistance (TBR) at Al-Ge2Sb2Te5 (GST) interface, which is between metal and phase change semiconducting material. This study is made possible due to the accurate knowledge of the thermal properties (thermal conductivity and specific heat in the 20-400 degrees C temperature range) of the GST layer. The measure of the acoustic oscillation damping permits characterization of the adhesion at the Al-GST interface, a quantity that can be related to the temperature-dependent TBR in the 25-300 degrees C range.

Resume La technique pompe sonde resolue en temps est utilisee pour etudier les proprietes thermiques et mecaniques d’une couche de Ge2Sb2Te5 (GST) deposee sous forme de film sur un substrat de silicium. Une couche d’aluminium de quelques nanometres d’epaisseur est deposee sur la couche de GST ; elle joue le role de transducteur optique et thermique pour l’experience pompe – sonde. Cette etude est realisee en fonction de la temperature de l’echantillon. La connaissance prealable des proprietes thermique de la couches de GST en fonction de la temperature (mesurees par radiometrie photothermique module) nous ont permit de nous interesser plus precisement aux caracteristiques thermiques de l’interface AL GST. L’experience pompe – sonde aux temps courts (quelques picosecondes permet aussi de reveler le comportement acoustique de l’interface et donc de mettre en evidence l’adhesion de la couche d’aluminium sur le GST et ceci dans le domaine 25°C – 400°C de temperature. Les resultats nous ont permis de trouver une augmentation tres significative de la resistance thermique d’interface en fonction de la temperature croissante. Cette variation resulte principalement du changement de phase amorphe cristallin et cristallin fcc vers cristallin hcp au fur et a mesure que la temperature augmente.

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.

Time Resolved Pump Probe (TRPP) technique has been implemented to study the thermal and mechanical properties of Ge2Sb2Te5 (GST) film deposited on a silicon substrate. According to the knowledge of the thermal properties of the GST layer, the temperature dependant Thermal Boundary Resistance (TBR) at the metal-GST interface is evaluated. Measuring the acoustic oscillation and more particularly its damping leads to characterize the adhesion at the metal – GST interface. This quantity can be efficiently related to the temperature dependent TBR in the 25°C – 400°C range. The TBR increases with temperature and follows the changes of the crystalline structure of materials. A linear relation between the acoustic reflection coefficient and the logarithm of the thermal boundary resistance is found.

A semi-analytical model is developed to calculate the elastic response of a gold submicronic particle embedded in a silica thin film, following a subpicosecond optical excitation. Real and imaginary parts of the transient reflectivity are then derived, accounting for elastooptic contribution in the particle and in the matrix, as well as particle and film surface motion. The major contribution comes from the particle surface displacement. Picosecond ultrasonics measurements in a reflection configuration on a single 430 nm diameter gold particle confirm the conclusions of the model and demonstrates the suitability of this technique to investigate the opto-acoustic response of such submicronic particles.

We present a novel ultrafast pump-probe system, allowing for a drastic reduction in acquisition time, typically a few tens of minutes for 20,000 frames. We present acoustic waves and heat transfer measurements in nanometric layers.

The optoacoustic response of a single submicron (430 nm) gold particle embedded in a silica thin film is experimentally revealed by femtosecond pump-probe experiments. A semianalytical model is developed to calculate the transient reflectivity accounting for optical index changes in both media and for particle and film surface displacements. The displacement of the particle-film interface turns out to be the major contribution to the measured signal. The amplitude of the acoustical component of the transient reflectivity is modulated by the depth at which the particle is buried in the film.

A 100 fs laser pulse passes through a single transparent cell and is absorbed at the surface of a metallic substrate. Picosecond acoustic waves are generated and propagate through the cell in contact with the metal. Interaction of the high frequency acoustic pulse with a probe laser light gives rise to Brillouin oscillations. The measurements are thus made with lasers for both the opto-acoustic generation and the acousto-optic detection, and acoustic frequencies as high as 11 GHz can be detected, as reported in this paper. The technique offers perspectives for single cell imaging. The in-plane resolution is limited by the pump and probe spot sizes, i.e. approximately 1 microm, and the in-depth resolution is provided by the acoustic frequencies, typically in the GHz range. The effect of the technique on cell safety is discussed. Experiments achieved in vegetal cells illustrate the reproducibility and sensitivity of the measurements. The acoustic responses of cell organelles are significantly different. The results support the potentialities of the hypersonic non-invasive technique in the fields of bio-engineering and medicine.

We have previously demonstrated [1,2] that a connection exists between Colored Picosecond Ultrasonic (CPU) experiments and electronic structure of metallic thin films. Indeed, a strong change of the detected acoustic echoes is observed when the laser is tuned around an interband transition wavelength. This connection suggests that CPU can be an useful tool for measuring interband transitions in thin metallic films. Surprisingly, by doing such a measurement on a series of ultra-thin Aluminium films, we observed a significant shift of the transition (from 880 to 970 nm) as the film thickness is reduced (from 400 to 120 A). We will discuss the origin of the phenomenon and propose some applications to the characterization of ultra-thin metallic films. [1] A. Devos and C. Lerouge, Phys. Rev. Lett. 86, 2669 (2001) [2] A. Devos and A. Le Louarn, Phys. Rev. B 68, 045405 (2003)

Picosecond ultrasonics is a non-destructive method for measuring mechanical properties such as velocity or stiffness coefficients for nanometric materials [1]. This technique uses femtosecond laser pulses for generating and detecting acoustics waves from GHz to THz. Its resolution is about nanometers in depth and a few micrometers laterally. For transparent materials it allows generating the so-called Brillouin oscillations, which frequencies are determined by the material sound velocity and the light beam wavelength. In this paper this technique is applied to biological cells. Measurements suggest promising perspectives for the imaging inside a single living cell; frequency content is from 5 to 20 GHz. A theoretical model based on Fourier heat and acoustic wave equations has been developed. These equations include heat diffusion and acoustic propagation respectively. It permits numerical simulations in time domain. First experimental results on animal and vegetal cells are presented and confronted with these calculated waveforms. [1] C. Thomsen, H. T. Grahn, H. J. Maris, J. Tauc, Phys. Rev. B 34, 4129, 1986

Ultrasonics signals at frequencies 5.7±0.1 and 6.8±0.1GHz are measured in two organelles of a single vegetal cell in vitro with a picosecond ultrasonic technique. Using standard values for cell optical index, ultrasound velocities of 1.6±0.1 and 2.0±0.1μm∕ns are measured from several signals recorded in the vacuole and in the nucleus of a single Allium cepa cell, respectively. A 1μm lateral and 0.25μm depth resolution is attained.

Having emerged in the 1980s, the laser ultrasonics technique with its non-contact generation and detection process overpasses the difficulties of coupling piezoelectric transducers with curved surfaces. To date, the authors [1] have been interested in acoustic generation for cylinders opaque at a given laser wavelength and for the acoustic source located at the cylinder surface. In this presentation, assuming point focusing of the laser pulses, we propose a three-dimensional (3D) semi-analytical model for acoustic waves generation and propagation in a partly transparent isotropic cylinder. First, the radial displacement at any position on the free surface is derived, in a 3D Fourier domain, for an inner point source. The response to a volume-source distribution along a radius is obtained as a convolution of the above Green function with the corresponding source distribution caused by optical absorption. Three inverse transforms are then applied to obtain the radial displacement at the cylinder surface. Picosecond ultrasonics experiments are performed on different micrometric fibers and compared with calculated waveforms for different optical absorptive properties. References [1] Y.D. Pan, C. Rossignol and B. Audoin, Appl. Phys. Lett. 82, 4379 (2003).

Picosecond ultrasonic experiments are widely used as pump-probe technique using longitudinal acoustic pulses and measuring time dependence of the signal. We present an experiment which combines an interferometer with the control of the pump-probe spatial overlap. Such set-up can perform a new kind of bulk phonon imaging based on ultrafast optical generation and detection scheme. When ultrafast acoustic longitudinal wave propagate in crystals they generate coherent transverse acoustic wave by diffraction. Transverse waves are known to give rise to phonon focusing patterns. The imaging of such patterns in time and space allowed us to partially reconstruct the wave front surface of bulk acoustic wave in crystals. The data may be viewed as 2D slices of the 3D acoustic wavefront revealing the basic anisotropy of the solid with unprecedented micronic lateral and in-depth resolution. Measurements are carried out in a 356 μm-thick GaAs sample as an example, but many different experimental conditions are compatible with the present set-up. This technique provides a new way to perform phonon energy and phonon polarisation dependent experiment with high frequency phonons.