Label-free Brillouin endo-microscopy for the quantitative 3D imaging of sub-micrometre biology.

This report presents an optical fibre-based endo-microscopic imaging tool that simultaneously measures the topographic profile and 3D viscoelastic properties of biological specimens through the phenomenon of time-resolved Brillouin scattering. This uses the intrinsic viscoelasticity of the specimen as a contrast mechanism without fluorescent tags or photoacoustic contrast mechanisms. We demonstrate 2 µm lateral resolution and 320 nm axial resolution for the 3D imaging of biological cells and Caenorhabditis elegans larvae. This has enabled the first ever 3D stiffness imaging and characterisation of the C. elegans larva cuticle in-situ. A label-free, subcellular resolution, and endoscopic compatible technique that reveals structural biologically-relevant material properties of tissue could pave the way toward in-vivo elasticity-based diagnostics down to the single cell level.

Classification of cancer cells at the sub-cellular level by phonon microscopy using deep learning.

There is a consensus about the strong correlation between the elasticity of cells and tissue and their normal, dysplastic, and cancerous states. However, developments in cell mechanics have not seen significant progress in clinical applications. In this work, we explore the possibility of using phonon acoustics for this purpose. We used phonon microscopy to obtain a measure of the elastic properties between cancerous and normal breast cells. Utilising the raw time-resolved phonon-derived data (300 k individual inputs), we employed a deep learning technique to differentiate between MDA-MB-231 and MCF10a cell lines. We achieved a 93% accuracy using a single phonon measurement in a volume of approximately 2.5 µm3. We also investigated means for classification based on a physical model that suggest the presence of unidentified mechanical markers. We have successfully created a compact sensor design as a proof of principle, demonstrating its compatibility for use with needles and endoscopes, opening up exciting possibilities for future applications.

Parallel imaging with phonon microscopy using a multi-core fibre bundle detection.

In this paper, we show a proof-of-concept method to parallelise phonon microscopy measurements for cell elasticity imaging by demonstrating a 3-fold increase in acquisition speed which is limited by current acquisition hardware. Phonon microscopy is based on time-resolved Brillouin scattering, which uses a pump-probe method with asynchronous optical sampling (ASOPS) to generate and detect coherent phonons. This enables access to the cell elasticity via the Brillouin frequency with sub-optical axial resolution. Although systems based on ASOPS are typically faster compared to the ones built with a mechanical delay line, they are still very slow to study real time changes at the cellular level. Additionally, the biocompatibility is reduced due to long light exposure and scanning time. Using a multi-core fibre bundle rather than a single channel for detection, we acquire 6 channels simultaneously allowing us to speed-up measurements, and open a way to scale-up this method.

Polarization-Sensitive Super-Resolution Phononic Reconstruction of Nanostructures.

In this paper, we show for the first time the polarization-sensitive super-resolution phononic reconstruction of multiple nanostructures in a liquid environment by overcoming the diffraction limit of the optical system (1 µm). By using time-resolved pump-probe spectroscopy, we measure the acoustic signature of nanospheres and nanorods at different polarizations. This enables the size, position, and orientation characterization of multiple nanoparticles in a single point spread function with the precision of 5 nm, 3 nm, and 1.4°, respectively. Unlike electron microscopy where a high vacuum environment is needed for imaging, this technique performs measurements in liquids at ambient pressure, ideal to study the insights of living specimens. This is a potential path toward super-resolution phononic imaging where the acoustic signatures of multiple nanostructures could act as an alternative to fluorescent labels. In this context, phonons also offer the opportunity to extract information about the mechanical properties of the surrounding medium as well as access to subsurface features.

Design of a resonant Luneburg lens for surface acoustic waves.

In this work we employ additive manufacturing to print a circular array of micropillars on an aluminium slab turning its top surface into a graded index metasurface for surface acoustic waves (SAW). The graded metasurface reproduces a Luneburg lens capable of focusing plane SAWs to a point. The graded index profile is obtained by exploiting the dispersion properties of the metasurface arising from the well-known resonant coupling between the micropillars (0.5 mm diameter and variable length ∼3 mm) and the surface waves propagating in the substrate. From the analytical formulation of the metasurface's dispersion curves, a slow phase velocity mode is shown to arise from the hybridisation of the surface wave with the pillar resonance. This is used to compute the radial height profile corresponding to the refractive index given by Luneburg's equation. An initial validation of the lens design, achieved through ray theory, shows that ray trajectories have a strong frequency dependence, meaning that the lens will only work on a narrow band. An ultrasonic experiment at 500 kHz where plane SAWs are generated with a piezoelectric transducer and a laser scanner measures the out of plane displacement on the metasurface, validates the actual lens performance and the manufacturing technique. Finally, comparison between the ray analysis and experimental results offers insight into the behaviour of this type of metasurface especially in the proximity of the acoustic bandgaps and highlights the possibility for acoustic shielding.

Characterising the size and shape of metallic nano-structures by their acoustic vibrations.

The characterisation of metallic nano-structures is of great importance as their optical properties are strongly dependent on their size and shape. Inaccurate size or shape characterisation can result in misleading measurements in applications such as bio-imaging and sensing. Characterisation techniques such as dynamic light scattering, electron microscopy or atomic force microscopy are commonly used; however, performing sub-surface measurements (inside semi-transparent objects) or in liquid media are very challenging. Here, we use time-resolved pump-probe spectroscopy to characterise the size and shape of metallic nano-structures in a water surrounding medium by using their vibrational modes. We show that this technique can achieve size measurements with a precision of 3 nm for the largest nano-structures which are in agreement with electron microscopy images. Furthermore, we demonstrate the ability to probe individual nano-structures despite being located in the same optical point spread function (PSF). Combining the high precision and sub-optical measurements provided by this technique with the ability to insert metallic nano-structures inside biological samples might open a way to perform 3D characterisation measurements.

Tailored elastic surface to body wave Umklapp conversion.

Elastic waves guided along surfaces dominate applications in geophysics, ultrasonic inspection, mechanical vibration, and surface acoustic wave devices; precise manipulation of surface Rayleigh waves and their coupling with polarised body waves presents a challenge that offers to unlock the flexibility in wave transport required for efficient energy harvesting and vibration mitigation devices. We design elastic metasurfaces, consisting of a graded array of rod resonators attached to an elastic substrate that, together with critical insight from Umklapp scattering in phonon-electron systems, allow us to leverage the transfer of crystal momentum; we mode-convert Rayleigh surface waves into bulk waves that form tunable beams. Experiments, theory and simulation verify that these tailored Umklapp mechanisms play a key role in coupling surface Rayleigh waves to reversed bulk shear and compressional waves independently, thereby creating passive self-phased arrays allowing for tunable redirection and wave focusing within the bulk medium.

Apparent attenuation by opto-acoustic defocus in phonon microscopy.

Understanding the mechanical properties of biological cells is a challenging problem for the life sciences partly because there are limited methods for mapping elasticity with high resolution. Phonon microscopy is a form of Brillouin light scattering which uses coherent phonons for imaging with elasticity-related contrast, phonon resolution and without labels. It can measure material properties such as sound velocity, acoustic impedance and attenuation. To use it as a contrast mechanism in microscopy, high numerical aperture (NA) lenses are key to high resolution. However, increasing NA induces apparent attenuation, a premature decay of the detected signal. To reduce signal decay and quantify the sound attenuation coefficient in cells, it is necessary to understand the mechanisms that affect signal decay. Here we define opto-acoustic defocus as a signal decay mechanism and propose methods to achieve quantitative sound attenuation measurements, and to optimise in-depth imaging at high resolution which is crucial for cell imaging.

Time resolved Brillouin fiber-spectrometer.

This report introduces a novel time resolved Brillouin spectrometer, consisting of an opto-acoustic transducer which resides on the tip of a single-mode optical fiber of arbitrary length with 125 µm outer diameter and 5 µm sensing diameter. Demonstrated here are proof of concept spectroscopic measurements - shifts in Brillouin frequency - with sensitivities of 41±3MHz/%wt and 2.5±0.6 MHz/°C for changes in water-salinity and water-temperature, respectively, and an interpolated frequency resolution of 9±2 MHz. The technique benefits from low-cost raw materials, scalable fabrication, scalable pixel density, easy alignment, and data acquisition speeds down to 0.4 s: traits which make this compatible with in vivo applications.

Super-resolution imaging using nano-bells.

In this paper we demonstrate a new scheme for optical super-resolution, inspired, in-part, by PALM and STORM. In this scheme each object in the field of view is tagged with a signal that allows them to be detected separately. By doing this we can identify and locate each object separately with significantly higher resolution than the diffraction limit. We demonstrate this by imaging nanoparticles significantly smaller than the optical resolution limit. In this case the "tag" we have used is the frequency of vibration of nanoscale "bells" made of metallic nanoparticles whose acoustic vibrational frequency is in the multi-GHz range. Since the vibration of the particles can be easily excited and detected and the frequency is directly related to the particle size, we can separate the signals from many particles of sufficiently different sizes even though they are smaller than, and separated by less than, the optical resolution limit. Using this scheme we have been able to localise the nanoparticle position with a precision of ~3 nm. This has many potential advantages - such nanoparticles are easily inserted into cells and well tolerated, the particles do not bleach and can be produced easily with very dispersed sizes. We estimate that 50 or more different particles (or frequency channels) can be accessed in each optical point spread function using the vibrational frequencies of gold nanospheres. However, many more channels may be accessed using more complex structures (such as nanorods) and detection techniques (for instance using polarization or wavelength selective detection) opening up this technique as a generalized method of achieving super-optical resolution imaging.