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Opto-acoustic profiling of a cell-mimicking film with L < λ/2. (a) Time-resolved waveforms along the scan line that covers both PMMA and bare Ti (inset). (b) One typical measurement shows the waveform is dominated by surface motion. (c) Its time-frequency spectrum shows a frequency component of long lifetime around 5 GHz attributed to acoustic resonances. (d) Using resonance frequencies and sound velocity measured in thicker film, one can reconstruct the thickness profile of the entire scan line.
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Cell morphological analysis has long been used in cell biology and physiology for abnormality identification, early cancer detection, and dynamic change analysis under specific environmental stresses. This work reports on the remote mapping of cell 3D morphology with an in-plane resolution limited by optics and an out-of-plane accuracy down to a te...
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... now illustrate the second scenario, where the film thickness is less than the spatial extent of one wavelength λ B of the CAP that gives rise to the Brillouin acousto-optic interaction. Figure 2(a) presents the waveforms recorded along a scan line across a thin PMMA film of 150 nm in thickness. The region covered with film and the bare Ti region can still be discriminated from the map of waveforms, and correspond well with the optical microscopic picture (inset). ...
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... expected, short-time oscillations tend to dominate the signals in this case. Figure 2(b) shows one representative waveform in which Brillouin oscillations are only present fragmentally at the very beginning and can barely be identified. This observation is confirmed in the time-frequency spectrum (SI) shown in Fig. 2(c) where a frequency component with a lifetime up to 1 ns is observed around 5 GHz, as a result of the step motions of the interfaces at frequency f R . ...
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... microscopic picture (inset). As expected, short-time oscillations tend to dominate the signals in this case. Figure 2(b) shows one representative waveform in which Brillouin oscillations are only present fragmentally at the very beginning and can barely be identified. This observation is confirmed in the time-frequency spectrum (SI) shown in Fig. 2(c) where a frequency component with a lifetime up to 1 ns is observed around 5 GHz, as a result of the step motions of the interfaces at frequency f R . Keeping in mind that this frequency is that of an acoustic quarter wave plate and using the sound velocity measured in the thicker film, one gets the local thin film thickness of L = v/4f ...
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... observed around 5 GHz, as a result of the step motions of the interfaces at frequency f R . Keeping in mind that this frequency is that of an acoustic quarter wave plate and using the sound velocity measured in the thicker film, one gets the local thin film thickness of L = v/4f R . Using the resonance frequency profile shown on the right axis in Fig. 2(d) we measured the entire thickness profile of the thin film, as indicated on the left axis of the figure. The average thickness is calculated at 145 ± 27 nm, which is again consistent with the reference value, 150 ± 10 nm, measured using the stylus ...
Citations
... Moreover, the label-free characteristic of the method, enables a quick thickness characterization unlike fluorescence microscopy, where the measurement does not require much time, but involves a long sample preparation. Furthermore, the high accuracy achievable also between materials with similar acoustic properties, can provide an advantage with respect to other methods which need high mismatch-in terms of refractive index or acoustic properties-between the investigated structures and their environments [44,45]. This method can be applied regardless of the Brillouin acquisition system (VIPA, TFP, SBS), as long as it is possible: (i) to measure a variation in the frequency shift of the Brillouin peak during the transition between two materials, when they are characterized by similar Brillouin shift and/or ii) to measure the variation in the Brillouin scattering intensity when a given material leaves the scattering volume. ...
Brillouin spectroscopy has recently attracted attention as a powerful tool for the characterization of the mechanical properties of heterogeneous materials, particularly in the biological and biomedical domains. This study investigates the procedure to use Brillouin data to provide relevant morphological parameters of micro-structured samples. When acquiring Brillouin spectra at the interface between two regions of the sample, the spectrum shows signatures of both regions. This feature can be used to precisely identify the position of the interfaces by analyzing the evolution of the fitting parameters of the Brillouin spectra acquired by performing a linear scan across the interface. This concept has been demonstrated by measuring the thickness of adherent HEK293T cells. The results are validated using fluorescence microscopy, showing an excellent agreement. The present analysis showcases the wealth of information present in the Brillouin spectrum and the potentiality of Brillouin spectroscopy not only for mechanical characterization but also for label-free, high-resolution imaging of sample morphology. The study introduces the possibility of correlating mechanical properties and shape of biological samples using a single technique.
... The collection of Brillouin scattering techniques has also enabled significant growth in several techniques used for the high-resolution characterisation of elasticity 21,24,25 . Different modalities of spontaneous 24,26 , stimulated 27 and time-resolved [28][29][30] Brillouin scattering have demonstrated great potential for three-dimensional elasticity mapping at optical 31 ...
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.
... 17 Indeed, for 3D morphology study, PU has an obvious advantage over the commonly used AFM based approach as it involves no active physical contact with cells. 18 However, few results refer to living cells. 19 Indeed, immersion is a sine qua non condition for studying and characterizing live cells as they need to be in a nutritive thermostated solution. ...
... Consequently, we rejected this hypothesis and assume a sound velocity fixed at v N . 24 This would result in a thickness discontinuity in thickness of 20% between nucleus and cytoplasm which is consistent with what was found in Ref. 18 where the thickness jump was corroborated using AFM measurements. ...
... The standard deviation of thickness values obtained in the nucleus characterizes the roughness of the cell. 18 The uncertainty range given for the cytoplasm corresponds to the overestimation of the thickness due to the downward shift in frequency compared to f 1 ¼ 3v c =(4L). The mean values measured without and with nanoparticles are similar, attesting that the sediment of nanoparticles does not flatten the cell. ...
In this work, we show that the use of silica nanoparticles improves the imaging and 3D-morphological measurement down to nanometer thicknesses of fixed cells in solution with picosecond ultrasonics (PU). Synchronized ultrafast fs-laser pulses are used to generate coherent acoustic phonons (CAPs) that evoke the Brillouin light scattering and enable the recording of the time-resolved Brillouin oscillations along with the propagation of the acoustic nanopulses through a thin transparent cell in solution. Silica nanoparticles, whose size matches the phonon wavelength at the frequency of the Brillouin scattering in the solution, are used to strongly scatter the CAPs in the solution. Suppressing the Brillouin signature of the surrounding liquid, this protocol improves significantly the PU imaging and makes it possible to measure the mechanical properties of a transparent cell, including the thin peripheral region where the thickness is less than the Brillouin wavelength, equal to half the probe light wavelength in the cell, and where crucial interaction of the cell with its surroundings occurs. We present experimental evidence of the considerable improvement in the cartography of the entire cell using nanoparticles. The intricate frequency dependence of Brillouin scattering and of resonances for a very thin cell is analyzed using a semi-analytical model leading to the challenging measurement of the 3D-morphology of the immersed cell at thicknesses down to [Formula: see text] of the optical wavelength.
... For the thinner parts of the cell, typically the cytoplasm surrounding the nucleus, thicknesses can be assessed measuring the acoustic resonance frequencies of the thin cell layer. This 3D mapping of cell morphology has been compared with measurement performed by AFM on a same fixed macrophage cell in air [122]. Fig. 7 shows the Brillouin frequency map and thickness map of a macrophage cell. ...
In this article we first present the foundations of ultrafast photoacoustics, a technique where the acoustic wavelength in play can be considerably shorter than the optical wavelength. The physics primarily involved in the conversion of short light pulses into high frequency sound is described. The mechanical disturbances following the relaxation of hot electrons in metals and other processes leading to the breaking of the mechanical balance are presented, and the generation of bulk shear-waves, of surface and interface waves and of guided waves is discussed. Then, efforts to overcome the limitations imposed by optical diffraction are described. Next, the principles behind the detection of the so generated coherent acoustic phonons with short light pulses are introduced for both opaque and transparent materials. The striking instrumental advances, in the detection of acoustic displacements, ultrafast acquisition, frequency and space resolution are discussed. Then secondly, we introduce picosecond opto-acoustics as a remote and label-free novel modality with an excellent capacity for quantitative evaluation and imaging of the cell's mechanical properties, currently with micron in-plane and sub-optical in depth resolution. We present the methods for time domain Brillouin spectroscopy in cells and for cell ultrasonography. The current applications of this unconventional means of addressing biological questions are presented. This microscopy of the nanoscale intra-cell mechanics, based on the optical monitoring of coherent phonons, is currently emerging as a breakthrough method offering new insights into the supra-molecular structural changes that accompany cell response to a myriad of biological events.
... Although the image displayed in Fig. 4e and 4f are sectioned every 100 nm, the depth resolution is limited to ~400 nm from the spatial width of the wavelet used in the frequency analysis (see Materials and Methods). Note that the Brillouin frequency and the acoustic resonance frequency are hardly distinguishable for the sample thickness where the Brillouin frequency and acoustic resonance frequency or harmonics have close values [41], e.g., in the thin cytoplasm of the dehydrated HeLa cells. Therefore, we did not define the peak frequency as Brillouin frequency for the dehydrated HeLa cells sample. ...
Frequency- and time-domain Brillouin scattering spectroscopy are powerful tools to read out the mechanical properties of complex systems in material and life sciences. Indeed, coherent acoustic phonons in the time-domain method offer superior depth resolution and a stronger signal than incoherent acoustic phonons in the frequency-domain method. However, it requires scanning of delay time between laser pulses for pumping and probing coherent acoustic phonons. Here, we present Brillouin scattering spectroscopy that spans the time and frequency domains to allow the multichannel detection of Brillouin scattering light from coherent acoustic phonons. Our technique traces the time-evolve Brillouin oscillations at the instantaneous frequency of a chromatic-dispersed laser pulse. The spectroscopic heterodyning of Brillouin scattering light in the frequency domain allows a single-frame readout of gigahertz-frequency oscillations with a spectrometer. As a proof of concept, we imaged heterogeneous thin films and biological cells over a wide bandwidth with nanometer depth resolution.
... Scanning the BO properties (amplitude, frequency, lifetime, and phase) allows bulk elasticity in biological cells to be mapped [3]. In-plane or in-depth investigations [4][5][6][7][8] can reveal nucleus or cytoskeleton elasticity and thickness of the cell. * emmanuel.peronne@ensta-paris.fr ...
... It is known that the acoustic signal, measured in transmission geometry, can be used to catch the inner-cell structures [4][5][6][7][8][9][10][11][12][13][14]. As illustrated in our previous work [7,15] the nucleus can be resolved, for example, by sorting the cell signal according to the cross-correlation map. ...
Ultrafast acoustic imaging experiments are a powerful tool to investigate, at the nanometer scale, cell mechanical properties such as stiffness, viscosity, and adhesion, properties that play some roles in the life and death of cells. However, due to cell complex structures, the ultrafast acoustic signal analysis is quite intricate and depends on multiple parameters. Complex data analysis with poorly known parameters can be handled by a data clustering method as already shown in particle physics and biology. In this work, ultrafast acoustic data analysis is tackled by a spectral clustering method followed by a hierarchical agglomerating method. Coupled to conventional microscopy performed on the very same cell, the clustered data can be assigned to inner-cell features such as the nucleus, the cytoplasm, and the cytoskeleton. The signal dependency on the cell thickness and stiffness is highlighted. Moreover, thanks to the improvement of the signal-to-noise ratio, the nature of the adhesion is also assessed through observation and characterization of a polymerlike layer as thin as a few nanometers.
... The underlying physics of the PARS mechanism suggests that the time evolution of PARS signals may be dictated by material properties such as density, heat capacity, and speed of sound [5,9]. Previously, methods such as femtosecond ultrasonics [10] and Brillouin scattering [11] have facilitated optical measurement of similar material properties, enabling functionality such as identification of cancerous cells via acoustic properties [10]. ...
... The underlying physics of the PARS mechanism suggests that the time evolution of PARS signals may be dictated by material properties such as density, heat capacity, and speed of sound [5,9]. Previously, methods such as femtosecond ultrasonics [10] and Brillouin scattering [11] have facilitated optical measurement of similar material properties, enabling functionality such as identification of cancerous cells via acoustic properties [10]. ...
Photoacoustic remote sensing (PARS) microscopy is an emerging label-free optical absorption imaging modality. PARS operates by capturing nanosecond-scale optical fluctuations produced by photoacoustic pressures. These time-domain (TD) variations are usually projected by amplitude to determine optical absorption magnitude. However, valuable details on a target’s material properties (e.g., density, speed of sound) are contained within the TD signals. This work uses a novel, to the best of our knowledge, clustering method to learn TD features, based on signal shape, which relate to underlying material traits. A modified K-means method is used to cluster TD data, capturing representative signal features. These features are then used to form virtual colorizations which may highlight tissues based on their underlying material properties. Applied in fresh resected murine brain tissue, colorized visualizations highlight distinct regions of tissue. This may potentially facilitate differentiation of tissue constituents (e.g., myelinated and unmyelinated axons, cell nuclei) in a single acquisition.
... This spreading reveals an increase in the roughness of the nucleus surface. The B-POM has already proved its capability to measure greater nucleus roughness for abnormal cells than for healthy cells [36]. Nuclear herniations and blebbing are indeed cell phenotypes that have long been considered for some cancer diagnosis and for tumor grading [7,37]. ...
How DNA damage and repair processes affect the biomechanical properties of the nucleus interior remains unknown. Here, an opto-acoustic microscope based on time-domain Brillouin spectroscopy (TDBS) was used to investigate the induced regulation of intra-nuclear mechanics. With this ultrafast pump-probe technique, coherent acoustic phonons were tracked along their propagation in the intra-nucleus nanostructure and the complex stiffness moduli and thicknesses were measured with an optical resolution. Osteosarcoma cells were exposed to methyl methanesulfonate (MMS) and the presence of DNA damage was tested using immunodetection targeted against damage signaling proteins. TDBS revealed that the intra-nuclear storage modulus decreased significantly upon exposure to MMS, as a result of the chromatin decondensation and reorganization that favors molecular diffusion within the organelle. When the damaging agent was removed and cells incubated for 2 hours in the buffer solution before fixation the intra-nuclear reorganization led to an inverse evolution of the storage modulus, the nucleus stiffened. The same tendency was measured when DNA double-strand breaks were caused by cell exposure to ionizing radiation. TDBS microscopy also revealed changes in acoustic dissipation, another mechanical probe of the intra-nucleus organization at the nano-scale, and changes in nucleus thickness during exposure to MMS and after recovery.
... Picosecond laser ultrasound, in which acoustic waves are photoacoustically generated by shining pulsed laser light on an optically absorbing layer on a substrate that is in mechanical contact with the sample and which are optically detected by a probe laser beam, has been convincingly shown to overcome many of the above-mentioned limitations. [39][40][41][42][43][44][45][46][47][48] The high bandwidth provided by picosecond and femtosecond laser pulses makes it possible to generate acoustic wavelengths that are shorter than optical wavelengths. The generated ultrasound wavefront, which is traveling both through the sample and through the substrate, can be detected either (i) at the photoacoustic actuator layer, e.g., by detecting the ultrasound modulated optical reflection, or, (ii) in case the sample is sufficiently translucent for probe laser light, via Brillouin oscillations 49 caused by dynamic optical interference between a static part of reflected probe beam laser light (e.g., the fraction of the probe light that is reflected at the metal layer or at a cover window) and a part that is optically reflected at the traveling acoustic wavefront due to the accompanying refractive index changes. ...
This contribution intends to convince readers that by virtue of the rich physics involved, optical excitation, thermal diffusion, thermal expansion, and acoustic wave propagation, and of the optical nature of the involved excitation and detection, photoacoustic and photothermal methods offer a unique combination of features that makes them very attractive for exploitation in a wide area of scientific and technological fields that involve material property evaluation. A perspective is also given on the high potential of these methods for substantial advances beyond the state of the art in a diverse selection of scientific disciplines: biomedical diagnostics, cell and tissue mechanobiology, thin film and interface characterization, characterization of the microstructure of solids, and the physics of relaxation in glass-forming liquids.
... Following the advent of the scanning acoustic microscope in 1974 (ref. 20 ), the most pragmatic breakthroughs in high-resolution acoustics have been provided by optoacoustics, i.e., the optical detection of acoustic phenomena [14][15][16][21][22][23][24][25] . Among these techniques, picosecond ultrasonics (PU) 26 and Brillouin scattering 27 are of particular interest as they offer picosecond temporal resolution and direct read-out of viscoelastic properties (respectively) with optical lateral resolution. ...
We show for the first time that a single ultrasonic imaging fibre is capable of simultaneously accessing 3D spatial information and mechanical properties from microscopic objects. The novel measurement system consists of two ultrafast lasers that excite and detect high-frequency ultrasound from a nano-transducer that was fabricated onto the tip of a single-mode optical fibre. A signal processing technique was also developed to extract nanometric in-depth spatial measurements from GHz frequency acoustic waves, while still allowing Brillouin spectroscopy in the frequency domain. Label-free and non-contact imaging performance was demonstrated on various polymer microstructures. This singular device is equipped with optical lateral resolution, 2.5 μm, and a depth-profiling precision of 45 nm provided by acoustics. The endoscopic potential for this device is exhibited by extrapolating the single fibre to tens of thousands of fibres in an imaging bundle. Such a device catalyses future phonon endomicroscopy technology that brings the prospect of label-free in vivo histology within reach.
