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Microrheology, the study of fluids on micron length-scales, promises to reveal insights into cellular biology, including mechanical biomarkers of disease and the interplay between biomechanics and cellular function. Here a minimally-invasive passive microrheology technique is applied to individual living cells by chemically binding a bead to the surface of a cell, and observing the mean squared displacement of the bead at timescales ranging from milliseconds to 100s of seconds. Measurements are repeated over the course of hours, and presented alongside novel analysis to quantify changes in the cells' low-frequency elastic modulus and the cell's dynamics over the time window from around 0.01s to 10s. An analogy to optical trapping allows verification of the invariant viscosity of HeLa S3 cells under control conditions and after cytoskeletal disruption. Stiffening of the cell is observed during cytoskeletal rearrangement in the control case, and cell softening when the actin cytoskeleton is disrupted by Latrunculin B. These data correlate with conventional understanding that integrin binding and recruitment triggers cytoskeletal rearrangement. This is, to our knowledge, the first time that cell stiffening has been measured during focal adhesion maturation, and the longest time over which such stiffening has been quantified by any means., 16 pages, 6 figures

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.

Brillouin light scattering (BLS) is an emerging method for cell imaging and characterisation. It allows elasticity-related contrast, optical resolution and label-free operation. Phonon microscopy detects BLS from laser generated coherent phonon fields to offer an attractive route for imaging since, at GHz frequencies, the phonon wavelength is sub-optical. Using phonon fields to image single cells is challenging as the signal to noise ratio and acquisition time are often poor. However, recent advances in the instrumentation have enabled imaging of fixed and living cells. This work presents the first experimental characterisation of phonon-based axial resolution provided by the response to a sharp edge. The obtained axial resolution is up to 10 times higher than that of the optical system used to take the measurements. Validation of the results are obtained with various polymer objects, which are in good agreement with those obtained using atomic force microscopy. Edge localisation, and hence profilometry, of a phantom boundary is measured with accuracy and precision of approximately 60 nm and 100 nm respectively. Finally, 3D imaging of fixed cells in culture medium is demonstrated.

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.

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.

Characterization of the elasticity of biological cells is growing as a new way to gain insight into cell biology. Cell mechanics are related to most aspects of cellular behavior, and applications in research and medicine are broad. Current methods are often limited since they require physical contact or lack resolution. From the methods available for the characterization of elasticity, those relying on high frequency ultrasound (phonons) are the most promising because they offer label-free, high (even super-optical) resolution and compatibility with conventional optical microscopes. In this Perspective contribution, we review the state of the art of picosecond ultrasonics for cell imaging and characterization, particularly for Brillouin scattering-based methods, offering an opinion for the challenges faced by the technology. The challenges are separated into biocompatibility, acquisition speed, resolution, and data interpretation and are discussed in detail along with new results.

In this paper we present a method for characterizing the thickness, and more interestingly, the variation of thickness in polymer thin films (

Metallic nanoparticles have been widely used for the last two decades as their optical properties can be exploited in fields such as physics, medicine and biology. The scattering and absorption properties of these devices are dependent on the material, the surrounding media and the size. The latter is crucial for many applications and a reliable measurement is a must. However, the current applications have some limitations such as they are destructive, cannot provide the size of individual particles or they use high vacuums. In this paper, we propose the use of an optical microscope which allows the measurement of multiple metallic nanoparticles non-destructively providing the size of each single particle.

The optical resonance of metal nanoparticles can be used to enhance the generation and detection of their main vibrational mode. In this work, we show that this method allows the accurate characterisation of the particle’s size because the vibrational frequency of plasmonic nanoparticles only depends on their mechanical properties. Moreover, by a careful selection of the particle size and/or probe laser wavelength, the detected signal can be increased by a large factor (∼9 for the particles used in this work) under the same illumination conditions. Finally, we show experimentally that particles of different sizes inside the point spread function can be observed due to the differences in their vibrational states, which could provide a feasible route to super-resolution.

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.

Label-free imaging of living cells below the optical diffraction limit poses great challenges for optical microscopy. Biologically relevant structural information remains below the Rayleigh limit and beyond the reach of conventional microscopes. Super-resolution techniques are typically based on the non-linear and stochastic response of fluorescent labels which can be toxic and interfere with cell function. In this paper we present, for the first time, imaging of live cells using sub-optical wavelength phonons. The axial imaging resolution of our system is determined by the acoustic wavelength (λa = λprobe/2n) and not on the NA of the optics allowing sub-optical wavelength acoustic sectioning of samples using the time of flight. The transverse resolution is currently limited to the optical spot size. The contrast mechanism is significantly determined by the mechanical properties of the cells and requires no additional contrast agent, stain or label to image the cell structure. The ability to breach the optical diffraction limit to image living cells acoustically promises to bring a new suite of imaging technologies to bear in answering exigent questions in cell biology and biomedicine.

At low frequencies ultrasound is a valuable tool to mechanically characterize and image biological tissues. There is much interest in using high-frequency ultrasound to investigate single cells. Mechanical characterization of vegetal and biological cells by measurement of Brillouin oscillations has been demonstrated using ultrasound in the GHz range. This paper presents a method to extend this technique from the previously reported single-point measurements and line scans into a high-resolution acoustic imaging tool. Our technique uses a three-layered metal-dielectric-metal film as a transducer to launch acoustic waves into the cell we want to study. The design of this transducer and measuring system is optimized to overcome the vulnerability of a cell to the exposure of laser light and heat without sacrificing the signal-to-noise ratio. The transducer substrate shields the cell from the laser radiation, efficiently generates acoustic waves, facilitates optical detection in transmission, and aids with heat dissipation away from the cell. This paper discusses the design of the transducers and instrumentation and presents Brillouin frequency images on phantom, fixed, and living cells.

Ultrasound is widely used for imaging, measurement and diagnostics in the MHz region and is perhaps most familiar as a medical or non-destructive imaging or measurement tool. In the MHz frequency range the wavelength is typically measured in microns and is many times longer than the wavelength of visible light, limiting its resolution to objects much larger than the nano-scale. It is possible to perform ultrasonic imaging and measurement at much higher frequencies, in the GHz region. Here the acoustic wavelength is typically less than that of light permitting the higher resolutions than optical microscopy and the ability to probe micro and nano-scale objects. At these high frequencies ultrasonics has much to offer the nano-world as a powerful diagnostic tool: it could be used in circumstances where optical microscopy, electron microscopy and probe microscopy cannot, such as inside living objects. Despite the potential that ultrasonics offers for imaging and measurement at the micro and nano-scale, performing ultrasonics at the nano-scale is hampered by many problems that render the techniques typically used in the MHz region impractical. In this paper we discuss some of the practical problems standing in the way of nano-ultrasonics and some of the solutions, especially the use of pico-second laser ultrasonics and the development of nano-ultrasonic transducers and their application to ultrasonic imaging inside living cells.

Simplified schematic of an all-fiber adaptive interferometer in a two-wave mixing (TWM) configuration is applied for detection of mechanical vibrations. This experiment used OFS-Fitel single-mode Er-doped optical fiber HG980. To improve the adaptive properties of the interferometer more, optical pumping of the Er-doped fibers (at 980 nm) could be done, or rare-earth doping elements with shorter spontaneous relaxation times of the metastable level could be utilized.

Transient two-wave mixing in Er-doped optical fibers with saturable absorption was shown to be nearly two times more efficient in linear interferometric configuration with significantly different powers of recording waves than in symmetric recording arrangement.

We report results of transient two-wave mixing (TWM) in Er-doped fibers with saturable absorption in a linear configuration of an adaptive interferometric vibrometer with essentially different powers of recording waves. The TWM signal modulation depth detected in the weak (reflected) wave was shown to be twice as strong as in the symmetric configuration with equal recording powers. In accordance with theoretical predictions, the experimentally observed TWM signal amplitude grew continuously with the fiber optical density in the whole investigated range of alpha(0)L approximately 0.2-4. At the recording wavelength 1492 nm it proved to be quite close to the theoretical limit of approximately alpha(0)L/2 for weakly absorbing fibers, and in 1-m-long fiber of high optical density reached maximal value of approximately 0.8. The TWM response time went down with the recording light power and for P(0) approximately 10 mW was in the submillisecond region.

At GHz frequencies the wavelength of ultrasound becomes comparable or smaller than the wavelength of light and so offers an intriguing way to image at the small scale. In this paper we report on new developments in pico-second laser ultrasonics that have resulted in the 3 dimensional imaging of live cells using elastic waves with shorter-than-optical wavelengths.

Mechanical imaging and characterisation of biological cells has been a subject of interest for the last twenty years. Ultrasonic imaging based on the scanning acoustic microscope (SAM) and mechanical probing have been extensively reported. Large acoustic attenuation at high frequencies and the use of conventional piezo-electric transducers limit the operational frequency of a SAM. This limitation results in lower resolution compared to an optical microscope. Direct mechanical probing in the form of applied stress by contacting probes causes stress to cells and exhibits poor depth resolution. More recently, laser ultrasound has been reported to detect ultrasound in the GHz range via Brillouin oscillations on biological cells. This technique offers a promising new high resolution acoustic cell imaging technique. In this work, we propose, design and apply a thin-film based opto-acoustic transducer for the detection in transmission of Brillouin oscillations on cells. The transducer is used to generate acoustic waves, protect the cells from laser radiation and enhance signal-to-noise ratio (SNR). Experimental traces are presented in water films as well as images of the Brillouin frequency of phantom and fixed 3T3 fibroblast cells.