Picosecond ultrasonics for elasticity-based imaging and characterisation of biological cells

Characterisation of the elasticity of biological cells is growing as a way to study cell biology. Cell mechanics are related to cell behaviour and potential applications offer great opportunities. Current methods to study cell mechanics are often limited as they may require contact or greater resolution. From the state of the art, the use of high frequency ultrasound (phonons) is one of the most promising since it offers label-free, high resolution and can be integrated on optical microscopes.


INTRODUCTION
The mechanics and elastic properties of cells have not been extensively explored. One of the main reasons for this is simply because the state of the art has not enabled strong biological conclusions to be made. Techniques often apply physical stress to the cell and lack sufficient resolution. This lack of suitable technology for cell imaging is not surprising given the difficulty of the problem: cells are small, exhibit little optical contrast, and are extremely delicate when isolated. Measuring elastic properties is also difficult since these can be modified by the cell itself depending on the situation. Despite this, practical demonstration of the characterisation of the elasticity of cells has generated interest, and has enabled practical applications and discovery. [1][2][3][4][5][6] Picosecond ultrasonics (PU) 7,8 is one of the most promising new methods for characterisation of elasticity of cells which combines conventional ultrasonics with Brillouin scattering. 9 These capabilities are unique in a single instrument which has enabled imaging of biological cells using sub-optical wavelength ultrasound. [10][11][12][13][14][15] In this contribution, we discuss recent advances in PU for the imaging characterisation of biological cells. In particular we pay particular attention to the limitations and promises of this technology.

CHARACTERISATION OF CELL ELASTICITY
Its possible to increase our understanding of normal and disrupted cell physiology by studying the relationship of the elastic properties of a cell, with known biological functions. For instance, differences in elasticity have been observed between cancer, normal, metastatic and benign cells. 16,17 The cytoskeleton serves as the underlying mechanical framework that enables cell function. Also, the actin and tubulin cytoskeleton is conserved between all eukaryotes 18 however, the evolution of the mechanical characteristics of the cytoskeleton under different processes is poorly understood.
Techniques for characterising elasticity can be roughly separated into contact and non-contact techniques. Contact techniques include methods such as micropipette aspiration, 19, 20 atomic force microscopy (AFM), 21 microfluidics 22 and optical tweezers. 23 In these methods, cells are placed under stress to observe (optically) a deformation.
Non-contact methods use strain pulses (ultrasound) to probe cell elasticity. These methods are desirable as it is generally accepted that cells do not exhibit disturbance by low power acoustics. Non-contact methods include scanning acoustic microscopy (SAM), 24 Brillouin microscopy (BM), 25 photoacoustic microscopy (PAM) 26 and phonon microscopy (PM). 27 The latter derives from picosecond ultrasonics and offers complementary features to Brillouin and acoustic microscopy.

PICOSECOND ULTRASONICS
Picosecond ultrasonics (PU) was pioneered in the mid 1980's with the first generation and detection of coherent phonons. The break-through came with the development of femtosecond pulsed lasers which enabled ability to work in the GHz regime. This was achieved by the application of a novel variation of the pump-probe method using a delay line. 7 Soon after, the detection of Brillouin scattering as an interferometric signal was reported. 9 This form of Brillouin scattering is now known as time-resolved Brillouin scattering (TRBS) and offers a measure of the sound velocity from a transparent and homogeneous medium. These lead to the characterisation at the bulk or thin-film scales of various materials. For instance, characterisation of metallic thin films, 28 semiconductors, 29 dielectrics 30 crystals 31 or liquids. 32 Initially, cell imaging seemed like an unlikely application as it required low acquisition speed, opaque transducers and relatively high optical powers. However, access to the Brillouin shift motivated the development of new technologies. This is because the Brillouin shift (f B ) provides a measure of the sound velocity (ν) at normal incidence and hence an indirect measure of the elasticity of the cell: where n is the refractive index of the medium and λ the optical probe wavelength. The sound velocity, an elastic property, then could be mapped with optical resolution which is a promising prospect since it enables non-contact characterisation of biological cells with elasticity-related contrast and optical resolution.
The first report of TRBS from a cell was reported as a single measurement from an onion cell 33 . Reports on other cell types cells 34 followed, however imaging remained difficult. Application of TRBS still posed several issues such as limited signal-to-noise ratio (SNR), low acquisition speed (only a few points per hour) and complex pump-probe system experimental arrangements.
The development of asynchronous optical sampling (ASOPS 35 ) improved acquisition rates to a few points per second by removing delay caused by moving parts. This enabled early high-quality images of biological cells using PU in fixed 3T3 fibroblast 10 and bone marrow 12 cells. However, living cells remained challenging until the development of cavity transducers and the use of high thermal-conductivity substrates. These innovations enabled the first report on living cells 36 and the early insights into specific biological questions using TRBS. 27 Elasticity-based contrast has many potential applications in health and biology that can be explored with PU. Its potential for super-optical resolution, the non-contact aspect of sound and label-free biocompatible operation makes it a highly promising technique. However, uptake of picosecond acoustics faces clear limitations that currently restricts its use to those developing it. These limitations include biocompatibility and acquisition speed which will be discussed in the following section.

PHONON MICROSCOPY: THE LIMITATIONS AND OPPORTUNITIES
Our approach to PU (TRBS) is a method we call phonon microscopy 10, 27, 36 (see Fig. 1). In this method, the signal is collected from the transmitted light through a custom opto-acoustic cavity transducer. 10 This configuration enabled 3D reconstruction of the Brillouin frequency using cell phantoms, 10 live cell imaging 36 and early investigations of cell processes. 37 Figure 1a shows a simplified schematic of the phonon microscope. Two ASOPS 35 synchronised pulsed lasers (pump and probe, 390 and 780nm respectively) are delivered to the sample using an inverted microscope. The incident light is converted into a strain pulse by an opto-acoustic transducer made out of gold and indium tin oxide (ITO). 10 The transmitted light, which carries the signal, is collected by a second objective. The contrast in This method exposes the sample to heat, potentially damage light doses and requires long acquisition times (few points per second). Although these are strong barriers, they are not fundamental and can be overcome in the future.

Biocompatibility
Non-invasiveness is one of the promises of acoustic imaging as contrast is intrinsic and therefore does not require labels. Using relatively long probing wavelengths (red to near infrared) minimize photodamage. However, heat is a by-product of sound generation and a source of damage. Maximising transmittivity of the probe light in phonon microscopy is desired however, it causes leakage of the pump beam (390nm) which also limits biocompatibility. Figure 1b shows fibroblast cells exposed to 390nm pulsed laser light. Cell damage, visualised by the use of propidium iodide, is rapid at higher intensities. We estimate that only 5mW/min (300mJ,with λ=390nm, 150fs pulses, 100MHz repetition rate and NA=0.6) is required to kill a living 3T3 fibroblast cell. However, 40mW/min (1.5J) of 780nm (light blue) did not cause any significant rise of the PI signal over longer timescales.
It is possible to replace the usual glass substrate with materials that exhibit greater thermal conductivity. Figure 1c shows a finite-element method (FEM) simulation of the steady-state temperature rise in the imaging volume. Under typical experimental conditions 10 for glass, sapphire and diamond. Sapphire reduces the temperature rise to only 5 o C while diamond practically removes any temperature rise. While sapphire and diamond coverslips are expensive, they can be easily be reused. A configuration with near infrared (NIR) wavelengths for pump and probe and high-thermal conductivity substrate is then suitable for PU imaging of living cells. Figure 1d shows Brillouin frequency and fluorescence measured from cells before and after death using all-NIR wavelengths (780nm) and sapphire coverslips. Cell death clearly affected the measured Brillouin frequency which exhibited a sharp transition at the moment of death. If biocompatibility is not considered, it is likely that unintended variations in the elastic properties of the specimens will be measured. Figure 1e shows the live/dead assay for the cells under study, which corroborates cell death at approximately 2.5Hrs. The surrounding cells are also dead discarding the measurement process as the cause of death.

Acquisition speed
Modulation depths of the detected PU signals are on the order of 10 −4 -10 −6 . Low modulation depth leads to averaging which in turn increases acquisition time. Improving then signal-to-noise ratio (SNR) can thus result in either greater image quality or faster acquisition.
Currently, there are two main transducer typologies: single layer 11,12,33 and three layer cavity-like transducers where the absorption is provided by either gold (Au) 10 or titanium (Ti). 13 We consider that the transducer and its substrate, in any given PU configuration, is the key to enhancing signal efficiency and managing thermal and photon loads. The state of the art in transducer design is at an early stage and there are many opportunities for improvement. A coupled optical-thermal-stress model 8 allows us to use the amplitude of the TRBS signal for the design of the transducer (see Fig. 2a). Having a measure of transmittance at pump (T pump ), probe (T probe ) and relative Brillouin signal amplitude (A f B in water), it is possible to produce a figure of merit (OAT ) for our transducer performance using UV(390nm) and NIR(780nm) wavelengths for pump and probe respectively: where SN R ∝ T probe and U V dose ∝ T pump /SN R 2 . The resultant figure of merit (OAT ) shown in Fig.  2b indicates best performance at approximately dimensions of 10nm, and 150nm for the Au and ITO layers respectively.
Acquisition speed is central to the usability of PU in a practical setting and key to the future adoption of the technology. In pump-probe, the time resolution is given by the pulse length of the lasers which allows bandwidth of up to a terahertz (if using femtosecond pulses). However, this ability comes with a compromise in acquisition speed. The need for time-reconstruction and averaging means that a 10ns event takes millions of times longer to acquire.
There are several paths to improve this problem: enhancing signal amplitude so less averages are needed or using a parallel/wide-field method. Each one of these has the potential to improve acquisition speed significantly, while carrying its own set of challenges. Enhancing signal amplitude requires increasing signal efficiency alongside managing thermally and photon induced damage (as discussed above). Parallel illumination aims to deliver simultaneously multiple points at the sample to increase acquisition speed. 38 This gives rise to technical complexity and increased cost of instrumentation due to the need to deliver, align and collect light from multiple tightly focused laser spots. High power lasers, additional conditioning optics, high well-depth cameras (due to low modulation depths) and multi-channel acquisition cards are required. Nevertheless, acquisition speed might increase linearly with each additional measuring spot.

Applications
We are exploring several cell types with applications in healthcare and biology. The first one, cardiac muscle cells (see Fig. 2c), is an intriguing cell type in terms of mechanics. Muscle cells contain a structure of fibres (sarcomeres) which are the functional unit of contraction. The striations (∼2.2µm) in the PU mechanical signal correspond to their periodicity which are clearly visible in this image. Heart disease is one of the main causes of death in western countries and the elastic behaviour of cardiac cells might provide useful insights into both normophysiology and disease. Figure 2d shows a cell infected with Toxoplasma gondii: a cell-infecting parasite. As it invades the host cell, it forms a vacuole membrane from the membrane of the host cell rendering it resistant to the host immune system. As the parasite is compositionally different to the host cell, it generates significant contrast when imaging using a elasticity-related method. In terms of elasticity, the mechanics of infection and the influence of the parasite on the mechanics of the host cell are poorly understood. Finally, Fig. 2e shows a stem cell differentiating into an adipose cell. This type of cell has the ability to store energy in the body in the form of fat. Stem cells are responsible for healing the body and are the key to regenerative medicine. This is due to the fact that stem cells can transform to other cell types to replace damaged cells. Stem cell therapies are presently inhibited in their clinical translation, by the paucity of label-free biomarkers to confirm their state of differentiation, for fear of introducing harmful features, including tumorigenic mutations into patients. Differentiation as a process is poorly-understood from a mechanical perspective. Here we demonstrate that elasticity offers intriguing prospects as the long-sought label-free differentiation biomarker, since the differentiation of stem cells generate various mechanical features such as cytoskeletal remodeling, formation of fat droplets, or even sarcomeres.

CONCLUSION
In summary, characterisation of cell elasticity can enable discovery in various areas of biology where picosecond ultrasonics is poised to help realise these discoveries. However, limitations of the technology need to be addressed in order to be broadly available. Also, a critical mass of experimental data and physical models are needed to realise the full potential of the technology.