Simultaneous laser ultrasonic measurement of sound velocities and thickness of plates using combined mode local acoustic spectroscopy.

Standard ultrasonic thickness measurements require the sound velocity of the sample to be known and vice versa. We present a method, which we have termed combined mode local acoustic spectroscopy (CoMLAS) for simultaneously determining a plate's thickness and sound velocities without requiring such a priori knowledge. It is based on a combination of three guided wave modes sustained by a plate at discrete frequencies, which we generate and detect using laser ultrasound. We use a pulsed laser that is shaped into a periodic line pattern on the sample's surface to generate elastic waves and measure the response at the pattern's center with a vibrometer. The surface acoustic wave mode produces an interference peak in the response spectrum at the frequency corresponding to the wavelength matching the pattern line spacing. By limiting the total size of the excitation pattern, we can simultaneously generate two zero-group-velocity plate resonances, providing two additional peaks in the spectrum. The plate's local thickness and longitudinal and transverse sound velocities are calculated from the peak frequencies. We demonstrate the feasibility of CoMLAS on steel and aluminum sheets with a thickness of around 2mm by resolving thickness steps and temperature-induced changes in the sound velocities. Using numerical simulations and control experiments, we provide insights into the method's accuracy and limitations. The choice of excitation pattern, the method's sensitivity, and the influence of sample inhomogeneity and anisotropy are discussed. CoMLAS does not require scanning mechanics and provides local plate properties. The results shown are achieved with low-energy lasers and signal averaging. Considerations on signal-to-noise ratio indicate that a realization with available lasers of higher energy will enable single-shot measurements. This qualifies the method for use on moving samples in an industrial environment.

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.

Determining longitudinal and transverse elastic wave attenuation from zero-group-velocity Lamb waves in a pair of plates.

A method for the determination of longitudinal and transverse bulk acoustic wave attenuation from measurements of the decay-rate of two independent zero-group-velocity resonances in a couple of matched plates is presented. A linear relation is derived, which links the bulk-wave attenuation coefficients to the decay-rate of plate-resonances. The relation is used to determine the acoustic loss of tungsten at GHz frequencies from noncontact laser-ultrasonic measurements in plates with thicknesses of about 1 µm. The longitudinal and transverse attenuation was found to amount to 1918 m-1 and 7828 m-1 at 2.16 GHz and 3265 m-1 and 12181 m-1 at 2.46 GHz. The presented approach is validated with calculated responses to a thermoelastic source, and the accuracy of the obtained attenuation values is estimated to be in the range of 10%.

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.

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.