Predicting nanocarriers' efficacy in 3D models with Brillouin microscopy.

Thanks to their unique nanoscale properties, nanomedicines can overcome some of the shortcomings of conventional therapies. For better predictive screening, it is important to assess their performance in three-dimensional (3D) multicellular tumour spheroids (MCTS) that can recapitulate the physiological barriers found in real tumours. Today, the evaluation of drug delivery nanosystems in MCTS is mainly explored by means of microscopy techniques that are invasive and require fluorescent labels which modify the composition and fate of the carriers. In recent years, a new quantitative microscopy technique based on Brillouin light scattering (BLS) has been proposed that uses the interaction of laser light with picosecond timescale density fluctuations in the sample. Because it is label-free, all-optical and non-destructive, BLS has gained interest in the pharmaceutical and biomedical fields. In this work, we implemented a fast BLS spectrometer and used the Brillouin frequency shift at the center of the MCTS as a quantitative readout for drug efficacy. We first investigated the ability of this setup to quantify drug efficacy in MCTS grown in classical multiwell plates and concluded that the low number of samples available in the multiwells limits the statistical significance of the results. To improve the throughput, we then combined the microscope with agarose microwells designed to fabricate a large number of MCTS and test 50 MCTS in less than a minute. Using this platform, we assessed the efficacy of polymeric nanoparticles (NPs) loaded with a platinum derivative anticancer drug (dichloro(1,2-diaminocyclohexane)platinum(II)) in reducing the growth of colorectal cancer cells (HCT-116) in MCTS. We observe a time- and dose-dependent decrease in the frequency shift, revealing the progressive loss of mechanical integrity in the MCTS. These results demonstrate that BLS probing of MCTS grown in agarose microwells is a promising tool for high-throughput screening of nanocarriers in 3D models.

Probing molecular crowding in compressed tissues with Brillouin light scattering.

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.

THESEUS1 modulates cell wall stiffness and abscisic acid production in Arabidopsis thaliana.

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.

Micromechanical imaging of dentin with Brillouin microscopy.

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.

Evaluation of commercial virtually imaged phase array and Fabry-Pérot based Brillouin spectrometers for applications to biology.

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.

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

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

High-Frequency Mechanical Properties of Tumors Measured by Brillouin Light Scattering.

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.

Drug delivery to tumours using a novel 5-FU derivative encapsulated into lipid nanocapsules.

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.

Opto-acoustic microscopy reveals adhesion mechanics of single cells.

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.

In-depth phenotypic characterization of multicellular tumor spheroids: Effects of 5-Fluorouracil.

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.

Optical tracking of picosecond coherent phonon pulse focusing inside a sub-micron object.

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.

Effect of refracted light distribution on the photoelastic generation of zero-group velocity Lamb modes in optically low-absorbing plates.

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.

Probing single-cell mechanics with picosecond ultrasonics.

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.

Universality of the network-dynamics of the cell nucleus at high frequencies.

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.

Nanoscale mechanical contacts mapped by ultrashort time-scale electron transport.

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 ~40% drop in 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.

Transverse mechanical properties of cell walls of single living plant cells probed by laser-generated acoustic waves.

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.

Remote opto-acoustic probing of single-cell adhesion on metallic surfaces.

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.

Acoustic beam steering by light refraction: illustration with directivity patterns of a tilted volume photoacoustic source.

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 compressional- 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.

Beam distortion detection and deflectometry measurements of gigahertz surface acoustic waves.

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.

Effect of laser beam incidence angle on the thermoelastic generation in semi-transparent materials.

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.