Understanding the mechanical properties of cells and their relation to different physiological conditions is essential to the study of fundamental processes such as proliferation, migration, or differentiation.

The group has pioneered the applications of ultrafast opto-acoustics to probe and image single biological cells. We have demonstrated that the coherent generation of GHz acoustic waves using ultrashort laser pulses, namely picosecond ultrasonics, can efficiently be used to perform single cell ultrasonography. This unconventional cell imaging modality is label free and relies on the organelles mechanical properties as the contrast mechanism.

The technique allows probing the contact between cell and biomaterial with a micron in plane resolution and yields access to the local cell compressibility, remotely. The unequalled capabilities and the foreseen applications suggest tremendous potentialities for single-cell biology.

We used sub-picosecond light pulses to launch high-frequency ultrasound in cells. The dual detection of acoustic echoes and of the time-domain Brillouin scattering allowed us to map remotely and in a single run experiment: the cell adhesion, thickness, storage modulus and mass density, all with a micron resolution. This dual picosecond opto-acoustic microscopy was demonstrated with the multiple imaging of a mitotic macrophage-like cell. The novel modality is compatible with simultaneous standard fluorescence imaging.

Femtosecond lasers for pump (red) and probe (green) were inserted in a standard microscope (left). Acoustic signal from probe 1 allows measurement of the Brillouin frequency shift and allows mapping the cell thickness remotely (top). Acoustic reflectivity measured with probe 2 gives access to the cell adhesion stiffness and to cell acoustic impedance (bottom). By combining data from both arms we mapped the mass density in the nucleus of a mitotic macrophage (right).

Related publications:

• L. Liu et al.Biophotonics 12(8), e201900045 (2019)

Cell morphological analysis has long been used in cell biology and physiology for abnormality identification, early cancer detection, and dynamic change analysis under specific environmental stresses. We demonstrated that picosecond ultrasonic microscopy can be used to achieve the remote mapping of cell 3D morphology quantitatively with an in-plane resolution limited by optics and an out-of-plane accuracy down to a tenth of the optical wavelength. For this, we tracked GHz coherent acoustic phonons and their resonance harmonics and mapped the 3D morphology of an entire osteosarcoma cell. The resulting image complies with the image obtained by standard atomic force microscopy, and both revealed very close roughness mean values. In addition, while scanning macrophages and monocytes, we demonstrated an enhanced contrast of thickness mapping by taking advantage of the detection of high-frequency resonance harmonics.

Thickness mapping of macrophages (left) and monocytes (right). (a,b) White light top-view images. (c,d) Maps of the acoustic resonances fR measured in the recorded PU signals. (e,f) quantitative 3D cell morphologies.

Related publications:

• L. Liu et al.Scient. Rep. 9, 6409 (2019)

We developed an inverted pulsed opto-acoustic microscope (iPOM) operating in the 10 to 100 GHz range. These frequencies allow mapping quantitatively cell structures as thin as 10 nm and resolving the fibrillar details of cells. Using this non-invasive all-optical system, we produce high-resolution images based on mechanical properties as the contrast mechanisms, and we can observe the stiffness and adhesion of single migrating stem cells.

However, manipulating biological media in physiological conditions is often a technical challenge when using a laser-based setup. We have therefore designed 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 (see figure 1). In particular, by exploiting the acoustic reflection coefficient at the sample-transducer interface and the photoacoustic interaction inside the transparent sample, we have probed simultaneously the density and compressibility of a single vegetal cell.

Absorption of fs light pulses in a thin metal layer launches longitudinal acoustic pulses with ps dynamics. Coherent phonons propagate through the transducer, are reflected at the metal-cell interface and are optically detected. Scanning laser pulses at the metal interface we image the inhomogeneous reflection of sound by the cell.

Raw acoustic images of the nuclear region and of the lamellipodium of a migrating human mesenchymal stem cell (hMSC) after fixation. The very high acoustic contrast reveals the structure of the nucleus, the actin network and the fine details of the complexity of the adhesion sites at the edge of the lamellipodium. Processing of acoustic signals allows mapping interfacial stiffness and acoustic impedance with same micron resolution. Click on the links to watch nucleus and lamellipodium videoscopy on a ps time frame.

Related publications:

• T. Dehoux et al.Sci. Rep. 5, 8650 (2015)

We have demonstrated the ability of the picosecond ultrasonic technique to image non-specific contacts of single animal cells with metallic substrates. Monocytes were cultured on top of an opto-acoustic bio-transducer. Low-energy femtosecond pump laser pulses were focused at the bottom of the Ti film to a micron spot. The subsequent ultrafast thermal expansion launched a longitudinal acoustic pulse in Ti, with a broad spectrum extending up to 100 GHz. We measured the acoustic echoes reflected from the film/cell interface through the transient optical reflectance changes. The time-frequency analysis of the reflected acoustic pulses with a wavelet transform gives access to a map of the acoustic impedance of the cell and of the stiffness of the film-cell interface.

a) schematic view of the sample. (b) White-light image of the monocyte and line scanned across the cell (c) Measured (black plain line) and theoretical (red dashed line) reflection coefficient vs frequency at three positions in the first cell (8, 14 and 20 µm). These positions are indicated in Fig. (d) with red dots. (d) Acoustic reflection coefficient along the cell contact area at frequencies 30 GHz (black) and 60 GHz (grey).

Related publications:

• M. Abi Ghanem et al.J. Biophoton. 7, 453 (2014)

Absorption of fs laser pulses in the metal launches an acoustic wavefront in the fixed cell. Its propagation along the cell thickness is detected through the Brillouin acousto-optic interaction.[1] The time resolved so-called Brillouin oscillations yield remote measurements of the cell thickness and of the sound velocity and attenuation in the GHz range for vegetal and animal cells.[2]

(a) Experimental configuration for Brillouin scattering detection. (b) Transient optical reflectivity measured for the intranuclear region of a single osteosarcoma cell.

We have probed the stiffness and viscosity of nuclei in single animal 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.[3] This pointed to an asymptotically increasing influence of a solid meshwork of connected chromatin fibres.
We have developed alternative optical configurations. In particular, the acoustic wave was launched at the bottom of a thin metal film and the reflectivity was measured through the cell. By both 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.[4]

>We probed the mechanical properties of vegetal live cells sub-compartiments. Experiments with onion cells revealed that single-cell wall transverse stiffness in the direction perpendicular to the epidermis layer is close to that of cellulose.[5] This observation demonstrated that cellulose micro-fibrils are the main load-bearing structure in this direction, and suggested strong bonding of micro-fibrils by hemicelluloses. Altogether measurement of the viscosity at such high frequencies suggest that the rheology of the wall is dominated by glass-like dynamics, a behaviour attributed to the influence of the pectin matrix.

(a) Experimental configuration for acoustic reflection coefficient measurements. (b) Transient optical reflectivity measured (plain line) in a thin wall (800 nm) of a vegetal cell. Theoretical calculations are plotted with dotted lines. (c) Longitudinal sound velocity for the cell vacuole (left n = 13) and cell wall (right n = 8).

Related publications :

• [1] C. Rossignol et al., Appl. Phys. Lett.93, 123901 (2008)
• [2] M. Ducousso et al., Eur. Phys. J. Appl. Phys. 61, 11201 (2013)
• [3] O. F. Zouani et al., Soft Matter 10, 8737-8743 (2014)
• [4] T. Dehoux and B. Audoin, J. Appl. Phys 112, 124702 (2012)
• [5] A. Gadalla et al., Planta 239, 1129 (2014)

Soft polymer micro-objects are of great interest for their biological cell-mimicking properties their encapsulation capacities, their use as contrast agents for ultrasonic imaging, as well as their use in self-healing materials. All the same, their compressibility, viscosity and shell thickness play an important role in the adhesion involved in specific tissues targeting, in the drug release rate, or in their echogenicity. However, at submicrometer scales, controlling these properties is challenging and requires elaborate characterization methods.

In particular, a large body of work has been devoted to microcapsules due to their potential in the food and pharmaceutical industries. Microcapsules are mostly used at MHz frequencies, and their individual rheological behavior is usually inferred from their collective response by modelling. Correlating the input of these models with the mechanics of single capsules at higher frequencies is therefore of the utmost importance to complete the description of their intricate dynamics in view of improving their design and modelling.

Using an ultrafast optical technique (Fig. x, left) 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 (Fig. x, right). A time-frequency analysis allows determination of the thickness 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, signature of thermally activated processes in glasses.

Related publications :

• T. Dehoux et al.Soft Matter 8, 2586 (2012)