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

Clark, Matt

Elsheikha, Hanny

Leija, Anaid Lugo

Marques, Leonel

Moradi, Emilia

Perez-Cota, Fernando

Smith, Richard J.

Sottile, Virginie

Symonds, Michael E.

Velickovic, Ksenija

Webb, Kevin F.

Repository@Nottingham

Cell imaging by sub-opticalwavelength phonon microscopyFernando Perez-Cota,1,∗ Richard J. Smith,1 Emilia Moradi,1,Anaid Lugo Leija,2, Ksenija Velickovic,2 Leonel Marques,1Kevin F. Webb,1 Michael E. Symonds,3 Virginie Sottile,2 Hanny Elsheikha,4and Matt Clark11Optics and photonics group, University of Nottingham, University Park, Nottingham, UK2 Wolfson STEM Centre, University of Nottingham, University Park, Nottingham, UK3 Early Life Research Unit, Division of Child Health, Obstetrics & Gynaecology,and Nottingham Digestive Disease Centre and Biomedical Research Centre,School of Medicine, University of Nottingham, UK4 School of Veterinary Medicine and Science, University of Nottingham,Sutton Bonington Campus, Leicestershire, UK∗ezzfap@nottingham.ac.ukAbstractThe mechanical properties of cells are largely unknown, but they play an important rolein cell function and behaviour. Living cells are particularly challenging for mechanicalimaging techniques as cells are very fragile and easy to damage unless the method isnon invasive. This paper presents recent developments that have enabled the use oflaser generated phonons (ultrasound) with sub-optical wavelengths to look inside livingcells. The use of phonons gives contrast from changes in the mechanical structure ofthe cell. The phonons are generated and detected with laser beams which allows highresolution images to be taken. Additionally because the time of flight of the phononscan be recorded it is possible to section the images with resolution that depends onthe phonon wavelength. This technique can provide a novel modality for the analysisof individual cell components in a non-destructive way.Mechanical cell imagingVariation in the mechanical properties of a cell such as stiffness often provide little or nooptical contrast when illuminated under visible light. However, the mechanical informationis directly related to specific cell characteristics such as cell mobility, adherence or division.Thus, there is an interest in measuring these properties with the aim of revealing underlyingmechanisms. Current methods for this purpose include acoustic microscopy1, atomic forcemicroscopy2, Brillouin microscopy3 and photoacoustic microscopy4 where the last two, forsingle cell applications, are still in their infancy. Given the dimensions of a cell, extractionof useful mechanical information is challenging – cells are very sensitive to environmentalstimulus and they are very difficult to measure without causing an undesired reaction. Inthat sense, conventional mechanical imaging systems often struggle while at the same timetheir resolution often remains below that of conventional optical microscopy. Despite this,mechanical characteristics of cells have produced important contributions. For instance, inthe field of cancer research, diseased cells where found to have different mechanical propertiescompared to healthy cells5.In this landscape, laser-generated phonons offers several advantages over existing tech-nologies. Unlike conventional acoustic microscopes6, it has the capacity to work at the highfrequencies necessary for high resolution imaging (∼5GHz). The obtained measurements areindependent of the optical absorption profile of the sample as in the case of opto-acoustics7.1The measurements are also taken through the cell (not just near the surface as in the caseof atomic force microscopy5). There is no need for chemical labels and, unlike Brillouinmicroscopy, where the Brillouin frequency is measured on a given optical volume,8;3, ad-ditional information such as sub-optical sectioning, attenuation9 and impedance10 can beobtained.The phonon microscopeIn a phonon microscope, a pulse of laser light is absorbed in a metal film to generate aphonon field. The sound is then detected by a second pulse of light in a pump-probeconfiguration11 which gives access to the GHz region without the need for extremely fastelectronics and detectors. The detected signal could be a traditional pulse-echo signal or aninterference signal, the so-called Brillouin oscillation12 – which is a direct measure of therefractive index and the speed of sound. Brillouin oscillations result from the interferenceof light scattered from the phonon wavefront with the unscattered light, (see figure 1a).As the wavefront travels into the sample, the phase of the scattered component changesand so the signal oscillates at a specific frequency, fB . If the phonon field travels fromone material to another the fB changes accordingly. The Brillouin frequency fB can bemeasured in three dimensions by applying signal processing techniques to the time-resolvedtrace to track the changes in frequency as the sound propagates through different materialsin the sample. The lateral resolution is determined by optical diffraction while the axialresolution is determined by the acoustic wavelength λacoustic ∼ λprobe/2n13 (see figure 1b).There are significant challenges to be addressed to allow this technology to be suitablefor live cell imaging: direct exposure of the cells to light and heat, low signal–to–noise ratio(SNR) and low acquisition speed. Exposure to light induces damage by absorption in thecell itself and by the heat generated by the absorption occurring in the generation film. LowSNR due to weak phonon-to-photon interactions is commonly compensated by large photonintensities. Low acquisition speeds given by a mechanical delay line means that exposure tolight is often unnecessarily long. All these factors decrement the ability to image living oreven fixed cells using conventional phonon techniques.Our solution to the challenges of cell phonon imaging are given by a novel opto-acoustictransducer, measurement configuration and electronic pump-probe method. A schematicof our microscope is shown in figure 1. A three-stack thin film transducer14 is speciallydesigned to absorb the pump beam while transmitting the probe beam. As both beamsapproach the transducer via the substrate, the cell is shielded, by the transducer itself, frompump light and the probe light is used efficiently. This manages the amount of light exposurefor the cells. For the substrate, we use sapphire due to its high thermal conductivity whichhelps to dissipate the heat generated at the transducer thus reducing thermal load appliedto the cells. The mechanical resonance of the transducer increases the acoustic amplitude atthe frequency of interest (∼ 5GHz) thus improving SNR. Finally, an asynchronous opticalsampling (ASOPS) pump-probe system produces an electronic sweep across two pulsedlasers eliminating the need for a delay line. This increases acquisition speed, in our case,to 1-2 measurements every second depending on averaging. The combination of all thoseallowed to image both fixed and living cells within biologically relevant time scales13.2Cell imaging using phononsFigure 2 shows two examples of cell imaging using phonons. Figures 2a–d show the imagesobtained from a fixed adipose cell differentiated from a stem cell. The optical image is shownin figure 2a where some fat droplets are clearly visible. Figures 2b–d show a subsection(560nm) of the result presented in figure 2b as it moves in the axial direction. Here eachpicture corresponds to a 400nm step forward in z starting at 600nm (lens NA is 0.42,and depth of focus ∼8µ). As the section moves, the fat droplet marked by a circle isrevealed to be approximately situated at 1 µm away from the substrate and its thicknessis approximately that of one section. Figures 2e and 2f show the example of a cardiac cellwhere the sarcomeres, a mechanically relevant structure, are clearly visible. Finally figure2g shows a representative experimental trace.Figure 3 shows further examples of this method. Figures 3a-b show the example ofan acanthamoeba where the voids in the amoeba are revealed to be cell medium. Thisparticular fact is more difficult to deduce with optical imaging as optical contrast does notgives enough information, even using fluorescence labels. Figures 3c-j show examples ofliving cells. Living 3T3 cells are shown on figures 3c–h. The acoustic image of the cellsshow good correlation with their optical counterparts, their resolution is limited in this caseby acquisition speed due to cell mobility. However, this opens the opportunity to image thecells dynamically. Figures 3i–j show a live adipose cell. Here the large fat droplets showgood contrast with respect to the rest of the cell. However, due to sound attenuation, theacoustical penetration depth is smaller than the optical depth of focus making the dropletsappear smaller in size.DiscussionWe have introduced a label free, high resolution acoustic live-cell imaging method usingphonons. This method has the ability to resolve an object based on the contrast of itsmechanical properties. The lateral resolution is limited by optical diffraction however theaxial resolution is limited by the acoustic wavelength. It was also shown that the method iscompatible with living cells. This technique offers a label-free alternative for live-cell imagingand mechanical characterisation. For the cell types presented in this paper, the change inthe measured Brillouin frequency within a cell is predominantly related to changes in theacoustic velocity. The ability to measure the response of the speed of sound on a living celldynamically, under a given stimulus, offers great potential to study the mechanical responseof cells.In principle, the highest axial resolution achievable using this method is λprobe/2n, whichis ∼280nm in cells at λprobe =780 nm. This is significantly better than the optical axialresolution of the optical system used to take the measurements, (NA of 0.42 and depth offocus of ∼ 8µm) and is higher than that achievable with a typical oil immersion confocalsystem. In practise the axial resolution is currently limited by SNR and contrast.Most other sectioning techniques require re-scanning at a different axial position, how-ever time-resolving the acoustic signal allows sectioning of the measured volume by post-processing. Therefore, the cell only receives one dose of light during the imaging process.The time to acquire the signal is ∼1–2 seconds per point due to averaging, which typicallyyields 5–10 voxels per point if the data is sectioned.Mechanical imaging of cells is an important field of research. However, the challengespresented to measure cells mechanically are significant. The method presented here provides3a new tool for mechanical imaging and characterisation of cells which is also the only newalternative to the photon for three dimensional imaging of live cells since the invention ofthe optical microscope.1 AcknowledgementsThis work was supported by the Engineering and Physical Sciences Research Council [grantnumbers EP/K021877/1, EP/G061661/1] (EPSRC). F.P.C and A.L.L. acknowledge Cona-cyt for sponsoring his studies. K.V. Aknowledge CASCADE and Marie curie for supportinghis studies.References[1] J A Hildebrand, D Rugar, R N Johnston, and C F Quate. Acoustic microscopy ofliving cells. Proceedings of the National Academy of Sciences of the United States ofAmerica, 78(3):1656–60, March 1981.[2] Pierre Parot, Yves F Dufreˆne, Peter Hinterdorfer, Christian Le Grimellec, Daniel Nava-jas, Jean-Luc Pellequer, and Simon Scheuring. Past, present and future of atomic forcemicroscopy in life sciences and medicine. Journal of molecular recognition : JMR,20(6):418–31, January 2007.[3] Giuliano Scarcelli, William J Polacheck, Hadi T Nia, Kripa Patel, Alan J Grodzin-sky, Roger D Kamm, and Seok Hyun Yun. Noncontact three-dimensional mappingof intracellular hydromechanical properties by Brillouin microscopy. Nature Methods,12(12):1132–1134, October 2015.[4] Eric M. Strohm, Michael J. Moore, and Michael C. Kolios. Single Cell PhotoacousticMicroscopy: A Review. IEEE Journal of Selected Topics in Quantum Electronics,22(3):137–151, May 2016.[5] M. Lekka, J. Lekki, M. Marszaek, P. Golonka, Z. Stachura, B. Cleff, and A.Z.Hrynkiewicz. Local elastic properties of cells studied by SFM. Applied Surface Science,141(3-4):345–349, March 1999.[6] Jerzy Litniewski and Ju¨rgen Bereiter-Hahn. Measurements of cells in culture by scan-ning acoustic microscopy. Journal of Microscopy, 158(1):95–107, April 1990.[7] Eric M. Strohm, Michael J. Moore, and Michael C. Kolios. High resolution ultrasoundand photoacoustic imaging of single cells. Photoacoustics, 4(1):36–42, 2016.[8] F Palombo, M Madami, N Stone, and D Fioretto. Mechanical mapping with chemicalspecificity by confocal Brillouin and Raman microscopy. The Analyst, 139(4):729–33,February 2014.[9] F. Yang, T. J. Grimsley, S. Che, G. A. Antonelli, H. J. Maris, and A. V. Nurmikko.Picosecond ultrasonic experiments with water and its application to the measurementof nanostructures. Journal of Applied Physics, 107(10):103537, May 2010.4[10] T Dehoux, M Abi Ghanem, O F Zouani, J-M Rampnoux, Y Guillet, S Dilhaire, M-CDurrieu, and B Audoin. All-optical broadband ultrasonography of single cells. Scientificreports, 5:8650, January 2015.[11] C. Thomsen, H. Grahn, H. Maris, and J. Tauc. Surface generation and detection ofphonons by picosecond light pulses. Physical Review B, 34(6):4129–4138, September1986.[12] C. Thomsen, H.T. Grahn, H.J. Maris, and J. Tauc. Picosecond interferometric tech-nique for study of phonons in the brillouin frequency range. Optics Communications,60(1):55–58, 1986.[13] Fernando Pe´rez-Cota, Richard J. Smith, Emilia Moradi, Leonel Marques, Kevin F.Webb, and Matt Clark. High resolution 3D imaging of living cells with sub-opticalwavelength phonons. Scientific Reports, 6(1):39326, December 2016.[14] Fernando Pe´rez-Cota, Richard J. Smith, Emilia Moradi, Leonel Marques, Kevin F.Webb, and Matt Clark. Thin-film optoacoustic transducers for subcellular Brillouinoscillation imaging of individual biological cells. Applied Optics, 54(28):8388, September2015.5Figure 1: Simplified diagram of three dimensional phonon imaging of cells. In (a), a lightpulse (pump) is used to thermoelastically generate a coherent acoustic field which is probedby a second light beam (probe, shown in angle for clarity). The interference of the direct(solid) and scattered (dashed) probe beams induces an oscillation in the detected probe lightintensity. The frequency of this oscillation fB is a function of the speed of sound. As thephonon field travels from material 1 to another material 2 (frequency change is exaggeratedfor clarity), the detected frequency changes from fB1 to fB2 as graphically shown in (b). Asthe spatial position z is related to the time information by the speed of sound, it is possibleto section the optical volume by post-processing without the need of further acquisition,mechanical positioning or change of focal position.6Figure 2: Phonon imaging of fixed cells. Images (a–d) show a stem cell differentiated intoan adipose cell. (a) is an optical picture, (b–d) Show 800nm sections extracted by post-processing, here a fat droplet is contained within a single section. (e–f) Example of acousticimaging of a cardiac cell. (e) Optical image, (f) Acoustic image, where the sarcomerestructures are clearly visible. (g) Representative detected signal.7Figure 3: Laser ultrasound imaging of fixed and living cells. Images (a–b) showsAcanthamoeba. The holes in the Acanthamoeba clearly show the same Brillouin frequencyas the medium. (c–h) Live 3T3 fibroblasts. where the resultant image shows good correla-tion. (i–j). Live adipose cell. The large fat droplets show good contrast compared to therest of the cell.8

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

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Perez-Cota, Fernando and Smith, Richard J. and Moradi, Emilia and Leija, Anaid Lugo and Velickovic, Ksenija and Marques, Leonel and Webb, Kevin F. and Symonds, Michael E. and Sottile, Virginie and Elsheikha, Hanny and Clark, Matt (2017) Cell imaging by phonon microscopy: sub-optical wavelength ultrasound for non-invasive imaging. Imaging & Microscopy, 2017 (3). ISSN 1439-4243 Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/46556/1/iandm%20%28002%29.pdfCopyright and reuse: The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions.This article is made available under the University of Nottingham End User licence and may be reused according to the conditions of the licence.  For more details see: http://eprints.nottingham.ac.uk/end_user_agreement.pdfA note on versions: The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the repository url above for details on accessing the published version and note that access may require a subscription.For more information, please contact eprints@nottingham.ac.ukCell imaging by sub-opticalwavelength phonon microscopyFernando Perez-Cota,1,∗ Richard J. Smith,1 Emilia Moradi,1,Anaid Lugo Leija,2, Ksenija Velickovic,2 Leonel Marques,1Kevin F. Webb,1 Michael E. Symonds,3 Virginie Sottile,2 Hanny Elsheikha,4and Matt Clark11Optics and photonics group, University of Nottingham, University Park, Nottingham, UK2 Wolfson STEM Centre, University of Nottingham, University Park, Nottingham, UK3 Early Life Research Unit, Division of Child Health, Obstetrics & Gynaecology,and Nottingham Digestive Disease Centre and Biomedical Research Centre,School of Medicine, University of Nottingham, UK4 School of Veterinary Medicine and Science, University of Nottingham,Sutton Bonington Campus, Leicestershire, UK∗ezzfap@nottingham.ac.ukAbstractThe mechanical properties of cells are largely unknown, but they play an important rolein cell function and behaviour. Living cells are particularly challenging for mechanicalimaging techniques as cells are very fragile and easy to damage unless the method isnon invasive. This paper presents recent developments that have enabled the use oflaser generated phonons (ultrasound) with sub-optical wavelengths to look inside livingcells. The use of phonons gives contrast from changes in the mechanical structure ofthe cell. The phonons are generated and detected with laser beams which allows highresolution images to be taken. Additionally because the time of flight of the phononscan be recorded it is possible to section the images with resolution that depends onthe phonon wavelength. This technique can provide a novel modality for the analysisof individual cell components in a non-destructive way.Mechanical cell imagingVariation in the mechanical properties of a cell such as stiffness often provide little or nooptical contrast when illuminated under visible light. However, the mechanical informationis directly related to specific cell characteristics such as cell mobility, adherence or division.Thus, there is an interest in measuring these properties with the aim of revealing underlyingmechanisms. Current methods for this purpose include acoustic microscopy1, atomic forcemicroscopy2, Brillouin microscopy3 and photoacoustic microscopy4 where the last two, forsingle cell applications, are still in their infancy. Given the dimensions of a cell, extractionof useful mechanical information is challenging – cells are very sensitive to environmentalstimulus and they are very difficult to measure without causing an undesired reaction. Inthat sense, conventional mechanical imaging systems often struggle while at the same timetheir resolution often remains below that of conventional optical microscopy. Despite this,mechanical characteristics of cells have produced important contributions. For instance, inthe field of cancer research, diseased cells where found to have different mechanical propertiescompared to healthy cells5.In this landscape, laser-generated phonons offers several advantages over existing tech-nologies. Unlike conventional acoustic microscopes6, it has the capacity to work at the highfrequencies necessary for high resolution imaging (∼5GHz). The obtained measurements areindependent of the optical absorption profile of the sample as in the case of opto-acoustics7.1The measurements are also taken through the cell (not just near the surface as in the caseof atomic force microscopy5). There is no need for chemical labels and, unlike Brillouinmicroscopy, where the Brillouin frequency is measured on a given optical volume,8;3, ad-ditional information such as sub-optical sectioning, attenuation9 and impedance10 can beobtained.The phonon microscopeIn a phonon microscope, a pulse of laser light is absorbed in a metal film to generate aphonon field. The sound is then detected by a second pulse of light in a pump-probeconfiguration11 which gives access to the GHz region without the need for extremely fastelectronics and detectors. The detected signal could be a traditional pulse-echo signal or aninterference signal, the so-called Brillouin oscillation12 – which is a direct measure of therefractive index and the speed of sound. Brillouin oscillations result from the interferenceof light scattered from the phonon wavefront with the unscattered light, (see figure 1a).As the wavefront travels into the sample, the phase of the scattered component changesand so the signal oscillates at a specific frequency, fB . If the phonon field travels fromone material to another the fB changes accordingly. The Brillouin frequency fB can bemeasured in three dimensions by applying signal processing techniques to the time-resolvedtrace to track the changes in frequency as the sound propagates through different materialsin the sample. The lateral resolution is determined by optical diffraction while the axialresolution is determined by the acoustic wavelength λacoustic ∼ λprobe/2n13 (see figure 1b).There are significant challenges to be addressed to allow this technology to be suitablefor live cell imaging: direct exposure of the cells to light and heat, low signal–to–noise ratio(SNR) and low acquisition speed. Exposure to light induces damage by absorption in thecell itself and by the heat generated by the absorption occurring in the generation film. LowSNR due to weak phonon-to-photon interactions is commonly compensated by large photonintensities. Low acquisition speeds given by a mechanical delay line means that exposure tolight is often unnecessarily long. All these factors decrement the ability to image living oreven fixed cells using conventional phonon techniques.Our solution to the challenges of cell phonon imaging are given by a novel opto-acoustictransducer, measurement configuration and electronic pump-probe method. A schematicof our microscope is shown in figure 1. A three-stack thin film transducer14 is speciallydesigned to absorb the pump beam while transmitting the probe beam. As both beamsapproach the transducer via the substrate, the cell is shielded, by the transducer itself, frompump light and the probe light is used efficiently. This manages the amount of light exposurefor the cells. For the substrate, we use sapphire due to its high thermal conductivity whichhelps to dissipate the heat generated at the transducer thus reducing thermal load appliedto the cells. The mechanical resonance of the transducer increases the acoustic amplitude atthe frequency of interest (∼ 5GHz) thus improving SNR. Finally, an asynchronous opticalsampling (ASOPS) pump-probe system produces an electronic sweep across two pulsedlasers eliminating the need for a delay line. This increases acquisition speed, in our case,to 1-2 measurements every second depending on averaging. The combination of all thoseallowed to image both fixed and living cells within biologically relevant time scales13.2Cell imaging using phononsFigure 2 shows two examples of cell imaging using phonons. Figures 2a–d show the imagesobtained from a fixed adipose cell differentiated from a stem cell. The optical image is shownin figure 2a where some fat droplets are clearly visible. Figures 2b–d show a subsection(560nm) of the result presented in figure 2b as it moves in the axial direction. Here eachpicture corresponds to a 400nm step forward in z starting at 600nm (lens NA is 0.42,and depth of focus ∼8µ). As the section moves, the fat droplet marked by a circle isrevealed to be approximately situated at 1 µm away from the substrate and its thicknessis approximately that of one section. Figures 2e and 2f show the example of a cardiac cellwhere the sarcomeres, a mechanically relevant structure, are clearly visible. Finally figure2g shows a representative experimental trace.Figure 3 shows further examples of this method. Figures 3a-b show the example ofan acanthamoeba where the voids in the amoeba are revealed to be cell medium. Thisparticular fact is more difficult to deduce with optical imaging as optical contrast does notgives enough information, even using fluorescence labels. Figures 3c-j show examples ofliving cells. Living 3T3 cells are shown on figures 3c–h. The acoustic image of the cellsshow good correlation with their optical counterparts, their resolution is limited in this caseby acquisition speed due to cell mobility. However, this opens the opportunity to image thecells dynamically. Figures 3i–j show a live adipose cell. Here the large fat droplets showgood contrast with respect to the rest of the cell. However, due to sound attenuation, theacoustical penetration depth is smaller than the optical depth of focus making the dropletsappear smaller in size.DiscussionWe have introduced a label free, high resolution acoustic live-cell imaging method usingphonons. This method has the ability to resolve an object based on the contrast of itsmechanical properties. The lateral resolution is limited by optical diffraction however theaxial resolution is limited by the acoustic wavelength. It was also shown that the method iscompatible with living cells. This technique offers a label-free alternative for live-cell imagingand mechanical characterisation. For the cell types presented in this paper, the change inthe measured Brillouin frequency within a cell is predominantly related to changes in theacoustic velocity. The ability to measure the response of the speed of sound on a living celldynamically, under a given stimulus, offers great potential to study the mechanical responseof cells.In principle, the highest axial resolution achievable using this method is λprobe/2n, whichis ∼280nm in cells at λprobe =780 nm. This is significantly better than the optical axialresolution of the optical system used to take the measurements, (NA of 0.42 and depth offocus of ∼ 8µm) and is higher than that achievable with a typical oil immersion confocalsystem. In practise the axial resolution is currently limited by SNR and contrast.Most other sectioning techniques require re-scanning at a different axial position, how-ever time-resolving the acoustic signal allows sectioning of the measured volume by post-processing. Therefore, the cell only receives one dose of light during the imaging process.The time to acquire the signal is ∼1–2 seconds per point due to averaging, which typicallyyields 5–10 voxels per point if the data is sectioned.Mechanical imaging of cells is an important field of research. However, the challengespresented to measure cells mechanically are significant. The method presented here provides3a new tool for mechanical imaging and characterisation of cells which is also the only newalternative to the photon for three dimensional imaging of live cells since the invention ofthe optical microscope.1 AcknowledgementsThis work was supported by the Engineering and Physical Sciences Research Council [grantnumbers EP/K021877/1, EP/G061661/1] (EPSRC). F.P.C and A.L.L. acknowledge Cona-cyt for sponsoring his studies. K.V. Aknowledge CASCADE and Marie curie for supportinghis studies.References[1] J A Hildebrand, D Rugar, R N Johnston, and C F Quate. Acoustic microscopy ofliving cells. Proceedings of the National Academy of Sciences of the United States ofAmerica, 78(3):1656–60, March 1981.[2] Pierre Parot, Yves F Dufreˆne, Peter Hinterdorfer, Christian Le Grimellec, Daniel Nava-jas, Jean-Luc Pellequer, and Simon Scheuring. Past, present and future of atomic forcemicroscopy in life sciences and medicine. Journal of molecular recognition : JMR,20(6):418–31, January 2007.[3] Giuliano Scarcelli, William J Polacheck, Hadi T Nia, Kripa Patel, Alan J Grodzin-sky, Roger D Kamm, and Seok Hyun Yun. Noncontact three-dimensional mappingof intracellular hydromechanical properties by Brillouin microscopy. Nature Methods,12(12):1132–1134, October 2015.[4] Eric M. Strohm, Michael J. Moore, and Michael C. Kolios. Single Cell PhotoacousticMicroscopy: A Review. IEEE Journal of Selected Topics in Quantum Electronics,22(3):137–151, May 2016.[5] M. Lekka, J. Lekki, M. Marszaek, P. Golonka, Z. Stachura, B. Cleff, and A.Z.Hrynkiewicz. Local elastic properties of cells studied by SFM. Applied Surface Science,141(3-4):345–349, March 1999.[6] Jerzy Litniewski and Ju¨rgen Bereiter-Hahn. Measurements of cells in culture by scan-ning acoustic microscopy. Journal of Microscopy, 158(1):95–107, April 1990.[7] Eric M. Strohm, Michael J. Moore, and Michael C. Kolios. High resolution ultrasoundand photoacoustic imaging of single cells. Photoacoustics, 4(1):36–42, 2016.[8] F Palombo, M Madami, N Stone, and D Fioretto. Mechanical mapping with chemicalspecificity by confocal Brillouin and Raman microscopy. The Analyst, 139(4):729–33,February 2014.[9] F. Yang, T. J. Grimsley, S. Che, G. A. Antonelli, H. J. Maris, and A. V. Nurmikko.Picosecond ultrasonic experiments with water and its application to the measurementof nanostructures. Journal of Applied Physics, 107(10):103537, May 2010.4[10] T Dehoux, M Abi Ghanem, O F Zouani, J-M Rampnoux, Y Guillet, S Dilhaire, M-CDurrieu, and B Audoin. All-optical broadband ultrasonography of single cells. Scientificreports, 5:8650, January 2015.[11] C. Thomsen, H. Grahn, H. Maris, and J. Tauc. Surface generation and detection ofphonons by picosecond light pulses. Physical Review B, 34(6):4129–4138, September1986.[12] C. Thomsen, H.T. Grahn, H.J. Maris, and J. Tauc. Picosecond interferometric tech-nique for study of phonons in the brillouin frequency range. Optics Communications,60(1):55–58, 1986.[13] Fernando Pe´rez-Cota, Richard J. Smith, Emilia Moradi, Leonel Marques, Kevin F.Webb, and Matt Clark. High resolution 3D imaging of living cells with sub-opticalwavelength phonons. Scientific Reports, 6(1):39326, December 2016.[14] Fernando Pe´rez-Cota, Richard J. Smith, Emilia Moradi, Leonel Marques, Kevin F.Webb, and Matt Clark. Thin-film optoacoustic transducers for subcellular Brillouinoscillation imaging of individual biological cells. Applied Optics, 54(28):8388, September2015.5Figure 1: Simplified diagram of three dimensional phonon imaging of cells. In (a), a lightpulse (pump) is used to thermoelastically generate a coherent acoustic field which is probedby a second light beam (probe, shown in angle for clarity). The interference of the direct(solid) and scattered (dashed) probe beams induces an oscillation in the detected probe lightintensity. The frequency of this oscillation fB is a function of the speed of sound. As thephonon field travels from material 1 to another material 2 (frequency change is exaggeratedfor clarity), the detected frequency changes from fB1 to fB2 as graphically shown in (b). Asthe spatial position z is related to the time information by the speed of sound, it is possibleto section the optical volume by post-processing without the need of further acquisition,mechanical positioning or change of focal position.6Figure 2: Phonon imaging of fixed cells. Images (a–d) show a stem cell differentiated intoan adipose cell. (a) is an optical picture, (b–d) Show 800nm sections extracted by post-processing, here a fat droplet is contained within a single section. (e–f) Example of acousticimaging of a cardiac cell. (e) Optical image, (f) Acoustic image, where the sarcomerestructures are clearly visible. (g) Representative detected signal.7Figure 3: Laser ultrasound imaging of fixed and living cells. Images (a–b) showsAcanthamoeba. The holes in the Acanthamoeba clearly show the same Brillouin frequencyas the medium. (c–h) Live 3T3 fibroblasts. where the resultant image shows good correla-tion. (i–j). Live adipose cell. The large fat droplets show good contrast compared to therest of the cell.8

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

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