Opto-acoustic thin-film transducers for imaging of Brillouin oscillations on living cells

In any given media, the speed of sound is considerably slower than speed of light, and the exploration of the acoustic regime in the GHz range gives access to very short acoustic wavelengths. Short acoustic wavelengths is an intriguing path for high resolution live-cell imaging. At low frequencies, ultrasound has proved to be a valuable tool for the mechanical characterisation and imaging of biological tissues. There is much interest in using high frequency ultrasound to investigate single cells due to its mechanical contrast mechanism. Mechanical characterisation of cells has been performed by a number of techniques, such as atomic force microscopy, acoustic microscopy or Brillouin microscopy. Recently, Brillouin oscillations measurements on vegetal and mammal cells have been demonstrated in the GHz range. In this thesis, a method to extend this technique, from the previously reported single point measurements and line scans, into a high resolution acoustic imaging tool is presented. A novel approach based around a three-layered metal-dielectric-metal film is used as a transducer to launch acoustic waves into the cell being studied. The design of this transducer and imaging system is optimised 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 by detecting in transmission rather than reflection. It also generates acoustic waves efficiently by a careful selection of materials and wavelengths. Facilitates optical detection in transmission due to simplicity of arrangement and aids to dissipate heat away from the cell. The design of the transducers and instrumentation is discussed and Brillouin frequency images (two and three dimensions) on phantom, fixed and living cells are presented

Opto-acoustic thin-film transducers for imaging of Brillouin oscillations on living cells

In any given media, the speed of sound is considerably slower than speed of light, and the exploration of the acoustic regime in the GHz range gives access to very short acoustic wavelengths. Short acoustic wavelengths is an intriguing path for high resolution live-cell imaging. At low frequencies, ultrasound has proved to be a valuable tool for the mechanical characterisation and imaging of biological tissues. There is much interest in using high frequency ultrasound to investigate single cells due to its mechanical contrast mechanism. Mechanical characterisation of cells has been performed by a number of techniques, such as atomic force microscopy, acoustic microscopy or Brillouin microscopy. Recently, Brillouin oscillations measurements on vegetal and mammal cells have been demonstrated in the GHz range. In this thesis, a method to extend this technique, from the previously reported single point measurements and line scans, into a high resolution acoustic imaging tool is presented. A novel approach based around a three-layered metal-dielectric-metal film is used as a transducer to launch acoustic waves into the cell being studied. The design of this transducer and imaging system is optimised 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 by detecting in transmission rather than reflection. It also generates acoustic waves efficiently by a careful selection of materials and wavelengths. Facilitates optical detection in transmission due to simplicity of arrangement and aids to dissipate heat away from the cell. The design of the transducers and instrumentation is discussed and Brillouin frequency images (two and three dimensions) on phantom, fixed and living cells are presented

Thin-film transducers for the detection and imaging of Brillouin oscillations in transmission on cultured cells

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

3D phonon microscopy with sub-micron axial-resolution

© 2021, The Author(s). 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

Optically excited nanoscale ultrasonic transducers

In order to work at higher ultrasonic frequencies, for instance, to increase the resolution, it is necessary to fabricate smaller and higher frequency transducers. This paper presents an ultrasonic transducer capable of being made at a very small size and operated at GHz frequencies. The transducers are activated and read optically using pulsed lasers and without physical contact between the instrumentation and the transducer. This removes some of the practical impediments of traditional piezoelectric architectures (such as wiring) and allows the devices to be placed immediately on or within samples, reducing the significant effect of attenuation which is very strong at frequencies above 1 GHz. The transducers presented in this paper exploit simultaneous optical and mechanical resonances to couple the optical input into ultrasonic waves and vice versa. This paper discusses the mechanical and optical design of the devices at a modest scale (a few lm) and explores the scaling of the transducers toward the sub-micron scale. Results are presented that show how the transducers response changes depending on its local environment and how the resonant frequency shifts when the transducer is loaded by a printed protein sample

Thin-film transducers for the detection and imaging of Brillouin oscillations in transmission on cultured cells

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

Cell imaging by phonon microscopy: sub-optical wavelength ultrasound for non-invasive imaging

The mechanical properties of cells play an important role in cell function and behavior. This paper presents recent developments that have enabled the use of laser-generated phonons (ultrasound) with sub-optical wavelengths to look inside living cells. The phonons reveal contrast from changes in the elasticity of the cell and can provide high resolution three dimensional images

Sound of nano

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. © 2013 Copyright SPIE

AAF architecture.

<p>(A) Model of AAF/I constructed based on the crystal structures of DSC subunits, AggAdsA and AggBdsA, and the crystal structure of the F1 antigen mini-fiber <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004404#ppat.1004404-Zavialov2" target="_blank">[25]</a> (cartoon diagram). The fiber contains a single copy of the AggB subunit (green) at the tip of a polymer of the AggA subunits (a fragment containing three AggA subunits is shown). The insert shows localization of conserved residues in the structure of the fiber. (B) Topology diagram of the AAF/I fiber. (C) Negative stain transmission electron micrographs of diluted AAF/II fimbriae isolated from enteroaggregative <i>E. coli</i> strain 042.</p