Optimizing multi-user indoor sound communications with acoustic reconfigurable metasurfaces.

Sound in indoor spaces forms a complex wavefield due to multiple scattering encountered by the sound. Indoor acoustic communication involving multiple sources and receivers thus inevitably suffers from cross-talks. Here, we demonstrate the isolation of acoustic communication channels in a room by wavefield shaping using acoustic reconfigurable metasurfaces (ARMs) controlled by optimization protocols based on communication theories. The ARMs have 200 electrically switchable units, each selectively offering 0 or π phase shifts in the reflected waves. The sound field is reshaped for maximal Shannon capacity and minimal cross-talk simultaneously. We demonstrate diverse acoustic functionalities over a spectrum much larger than the coherence bandwidth of the room, including multi-channel, multi-spectral channel isolations, and frequency-multiplexed acoustic communication. Our work shows that wavefield shaping in complex media can offer new strategies for future acoustic engineering.

Three-dimensional ultrasound matrix imaging.

Matrix imaging paves the way towards a next revolution in wave physics. Based on the response matrix recorded between a set of sensors, it enables an optimized compensation of aberration phenomena and multiple scattering events that usually drastically hinder the focusing process in heterogeneous media. Although it gave rise to spectacular results in optical microscopy or seismic imaging, the success of matrix imaging has been so far relatively limited with ultrasonic waves because wave control is generally only performed with a linear array of transducers. In this paper, we extend ultrasound matrix imaging to a 3D geometry. Switching from a 1D to a 2D probe enables a much sharper estimation of the transmission matrix that links each transducer and each medium voxel. Here, we first present an experimental proof of concept on a tissue-mimicking phantom through ex-vivo tissues and then, show the potential of 3D matrix imaging for transcranial applications.

Analysis of Non-Contact Multichannel Recording of Cardiac Vibration: Visual Seismocardiogram.

Seismocardiography (SCG) is the recent research focus for cardiac monitoring and diagnosis. Contact based single channel accelerometer recordings suffer from limitations due to sensor placements and propagation delay. This work uses the airborne ultrasound device named Surface Motion Camera (SMC) for non-contact multichannel recording of the chest surface vibrations and proposes visualization techniques (vSCG) to enable simultaneous evaluation of both time and spatial variations of the vibrations. Recordings are performed on 10 healthy volunteers. The time propagation of vertical scans and 2D vibration contour maps at specific cardiac events are shown. These allow for a reproducible way for in-depth analysis of cardio mechanical activities, as compared to single channel SCG.

Damping-Driven Time Reversal for Waves.

Damping is usually associated with irreversibility. Here, we present a counterintuitive concept to achieve time reversal of waves propagating in a lossless medium using a transitory dissipation pulse. Applying a sudden and strong damping in a limited time generates a time-reversed wave. In the limit of a high damping shock, this amounts to "freezing" the initial wave by maintaining the wave amplitude while canceling its time derivative. The initial wave then splits in two counterpropagating waves with half of its amplitude and time evolutions in opposite directions. We implement this damping-based time reversal using phonon waves propagating in a lattice of interacting magnets placed on an air cushion. We show with computer simulations that this concept also applies to broadband time reversal in complex disordered systems.

Ultrasound Matrix Imaging-Part I: The Focused Reflection Matrix, the F-Factor and the Role of Multiple Scattering.

This is the first article in a series of two dealing with a matrix approach for aberration quantification and correction in ultrasound imaging. Advanced synthetic beamforming relies on a double focusing operation at transmission and reception on each point of the medium. Ultrasound matrix imaging (UMI) consists in decoupling the location of these transmitted and received focal spots. The response between those virtual transducers form the so-called focused reflection matrix that actually contains much more information than a confocal ultrasound image. In this paper, a time-frequency analysis of this matrix is performed, which highlights the single and multiple scattering contributions as well as the impact of aberrations in the monochromatic and broadband regimes. Interestingly, this analysis enables the measurement of the incoherent input-output point spread function at any pixel of this image. A fitting process enables the quantification of the single scattering, multiple scattering and noise components in the image. From the single scattering contribution, a focusing criterion is defined, and its evolution used to quantify the amount of aberration throughout the ultrasound image. In contrast to the state-of-the-art coherence factor, this new indicator is robust to multiple scattering and electronic noise, thereby providing a contrasted map of the focusing quality at a much better transverse resolution. After a validation of the proof-of-concept based on time-domain simulations, UMI is applied to the in-vivo study of a human calf. Beyond this specific example, UMI opens a new route for speed-of-sound and scattering quantification in ultrasound imaging.

Ultrasound Matrix Imaging-Part II: The Distortion Matrix for Aberration Correction Over Multiple Isoplanatic Patches.

This is the second article in a series of two which report on a matrix approach for ultrasound imaging in heterogeneous media. This article describes the quantification and correction of aberration, i.e. the distortion of an image caused by spatial variations in the medium speed-of-sound. Adaptive focusing can compensate for aberration, but is only effective over a restricted area called the isoplanatic patch. Here, we use an experimentally-recorded matrix of reflected acoustic signals to synthesize a set of virtual transducers. We then examine wave propagation between these virtual transducers and an arbitrary correction plane. Such wave-fronts consist of two components: (i) An ideal geometric wave-front linked to diffraction and the input focusing point, and; (ii) Phase distortions induced by the speed-of-sound variations. These distortions are stored in a so-called distortion matrix, the singular value decomposition of which gives access to an optimized focusing law at any point. We show that, by decoupling the aberrations undergone by the outgoing and incoming waves and applying an iterative strategy, compensation for even high-order and spatially-distributed aberrations can be achieved. After a numerical validation of the process, ultrasound matrix imaging (UMI) is applied to the in-vivo imaging of a gallbladder. A map of isoplanatic modes is retrieved and is shown to be strongly correlated with the arrangement of tissues constituting the medium. The corresponding focusing laws yield an ultrasound image with drastically improved contrast and transverse resolution. UMI thus provides a flexible and powerful route towards computational ultrasound.

Dynamic full-field optical coherence tomography allows live imaging of retinal pigment epithelium stress model.

Retinal degenerative diseases lead to the blindness of millions of people around the world. In case of age-related macular degeneration (AMD), the atrophy of retinal pigment epithelium (RPE) precedes neural dystrophy. But as crucial as understanding both healthy and pathological RPE cell physiology is for those diseases, no current technique allows subcellular in vivo or in vitro live observation of this critical cell layer. To fill this gap, we propose dynamic full-field OCT (D-FFOCT) as a candidate for live observation of in vitro RPE phenotype. In this way, we monitored primary porcine and human stem cell-derived RPE cells in stress model conditions by performing scratch assays. In this study, we quantified wound healing parameters on the stressed RPE, and observed different cell phenotypes, displayed by the D-FFOCT signal. In order to decipher the subcellular contributions to these dynamic profiles, we performed immunohistochemistry to identify which organelles generate the signal and found mitochondria to be the main contributor to D-FFOCT contrast. Altogether, D-FFOCT appears to be an innovative method to follow degenerative disease evolution and could be an appreciated method in the future for live patient diagnostics and to direct treatment choice.

Freeze-Dried Microfluidic Monodisperse Microbubbles as a New Generation of Ultrasound Contrast Agents.

We succeeded in freeze-drying monodisperse microbubbles without degrading their performance, that is, their monodispersity in size and echogenicity. We used microfluidic technology to generate cryoprotected highly monodisperse microbubbles (coefficient of variation [CV] <5%). By using a novel retrieval technique, we were able to freeze-dry the microbubbles and resuspend them without degradation, that is, keeping their size distribution narrow (CV <6%). Acoustic characterization performed in two geometries (a centimetric cell and a millichannel) revealed that the resuspended bubbles conserved the sharpness of the backscattered resonance peak, leading to CVs ranging between 5% and 10%, depending on the geometry. As currently observed with monodisperse bubbles, the peak amplitudes are one order of magnitude higher than those of commercial ultrasound contrast agents. Our work thus solves the question of storage and transportation of highly monodisperse bubbles. This work might open pathways toward novel clinical non-invasive measurements, such as local pressure, impossible to carry out with the existing commercial ultrasound contrast agents.

Optical phase modulation by natural eye movements: application to time-domain FF-OCT image retrieval.

Eye movements are commonly seen as an obstacle to high-resolution ophthalmic imaging. In this context we study the natural axial movements of the in vivo human eye and show that they can be used to modulate the optical phase and retrieve tomographic images via time-domain full-field optical coherence tomography (TD-FF-OCT). This approach opens a path to a simplified ophthalmic TD-FF-OCT device, operating without the usual piezo motor-camera synchronization. The device demonstrates in vivo human corneal images under the different image retrieval schemes (2-phase and 4-phase) and different exposure times (3.5 ms, 10 ms, 20 ms). Data on eye movements, acquired with a spectral-domain OCT with axial eye tracking (180 B-scans/s), are used to study the influence of ocular motion on the probability of capturing high-signal tomographic images without phase washout. The optimal combinations of camera acquisition speed and amplitude of piezo modulation are proposed and discussed.

Fourier transform acousto-optic imaging with off-axis holographic detection.

Acousto-optic (AO) imaging is an in-depth optical imaging technique of highly scattering media. One challenging end-application for this technique is to perform imaging of living biological tissues. Indeed, because it relies on coherent illumination, AO imaging is sensitive to speckle decorrelation occurring on the millisecond time scale. Camera-based detections are well suited for in vivo imaging provided their integration time is lower than those decorrelation time scales. We present Fourier transform acousto-optic imaging combined with off-axis holography, which relies on plane waves and long-duration pulses. We demonstrate, for the first time to the best of our knowledge, a two-dimensional imaging system fully compatible with in vivo imaging prerequisites. The method is validated experimentally by performing in-depth imaging inside a multiple scattering sample.

Manifestation of aberrations in full-field optical coherence tomography.

We report on a theoretical model for image formation in full-field optical coherence tomography (FFOCT). Because the spatial incoherence of the illumination acts as a virtual confocal pinhole in FFOCT, its imaging performance is equivalent to a scanning time-gated coherent confocal microscope. In agreement with optical experiments enabling a precise control of aberrations, FFOCT is shown to have nearly twice the resolution of standard imaging at moderate aberration level. Beyond a rigorous study on the sensitivity of FFOCT with respect to aberrations, this theoretical model paves the way towards an optimized design of adaptive optics and computational tools for high-resolution and deep imaging of biological tissues.

Integrated process development: The key to improve Fab production in E. coli.

Bioprocess development and optimization is a challenging, costly, and time-consuming effort. In this multidisciplinary task, upstream processing (USP) and downstream processing (DSP) are conventionally considered distinct disciplines. This consideration fosters "one-way" optimization disregarding interdependencies between unit operations; thus, the full potential of the process chain cannot be achieved. Therefore, it is necessary to fully integrate USP and DSP process development to provide balanced biotechnological production processes. The aim of the present study was to investigate how different host/secretory signal/antigen binding fragment (Fab) combinations in E. coli expression systems influence USP, primary recovery performance and the final product quality. We ran identical fed-batch cultivations with 16 different expression clones to study growth and product formation kinetics, as well as centrifugation efficiency, viscosity, extracellular DNA, and endotoxin content, important parameters in DSP. We observed a severe influence on cell growth, product titer, extracellular product, and cell lysis, accompanied by a significant impact on the analyzed parameters of DSP performance. Our results provide the basis for future research on integrated process development considering interdependencies between USP and DSP; however, individual products need to be considered specifically. These interdependencies need to be understood for rational decision-making and efficient process development in research and industry.

High-throughput microbioreactor provides a capable tool for early stage bioprocess development.

Tremendous advancements in cell and protein engineering methodologies and bioinformatics have led to a vast increase in bacterial production clones and recombinant protein variants to be screened and evaluated. Consequently, an urgent need exists for efficient high-throughput (HTP) screening approaches to improve the efficiency in early process development as a basis to speed-up all subsequent steps in the course of process design and engineering. In this study, we selected the BioLector micro-bioreactor (µ-bioreactor) system as an HTP cultivation platform to screen E. coli expression clones producing representative protein candidates for biopharmaceutical applications. We evaluated the extent to which generated clones and condition screening results were transferable and comparable to results from fully controlled bioreactor systems operated in fed-batch mode at moderate or high cell densities. Direct comparison of 22 different production clones showed great transferability. We observed the same growth and expression characteristics, and identical clone rankings except one host-Fab-leader combination. This outcome demonstrates the explanatory power of HTP µ-bioreactor data and the suitability of this platform as a screening tool in upstream development of microbial systems. Fast, reliable, and transferable screening data significantly reduce experiments in fully controlled bioreactor systems and accelerate process development at lower cost.

Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts.

Antibody fragments (Fab) are often produced by recombinant methods in Escherichia coli as no glycosylation is needed. Besides the correctly expressed Fab molecule, a multitude of host cell impurities and product related impurities are present in the crude sample. The identification and characterization of the product-related impurities, such as modified Fab-molecules or free light chain, are of utmost importance. The objective of this work was to design a purification strategy to isolate and characterize Fab and related impurities. A three-dimensional chromatography method was established, consisting of two affinity steps (Protein G and Protein L) and subsequent cation exchange chromatography, followed by mass spectrometry analysis of the purified samples. The procedure was automated by collecting the eluted target species in loops and directly loading the samples onto the high-resolution cation exchange chromatography column. As an example, four different Fab molecules are characterized. All four samples contained mainly the correct Fab, while only one showed extensive N-terminal pyroglutamate formation of the Fab. In another case, we found a light chain variant with uncleaved amino acids from the lead molecule, which was not used for the formation of whole Fab as only correct Fab was found in that sample. Impurities with lower molecular weights, which were bound on the Protein L column, were observed in all samples, and identified as fragments of the light chain. In conclusion, we have devised a platform for characterizing Fab and Fab-related impurities, which significantly facilitated strain selection and optimization of cultivation conditions.

Coherence gate shaping for wide field high-resolution in vivo retinal imaging with full-field OCT.

Allying high-resolution with a large field-of-view (FOV) is of great importance in the fields of biology and medicine, but it is particularly challenging when imaging non-flat living samples such as the human retina. Indeed, high-resolution is normally achieved with adaptive optics (AO) and scanning methods, which considerably reduce the useful FOV and increase the system complexity. An alternative technique is time-domain full-field optical coherence tomography (FF-OCT), which has already shown its potential for in-vivo high-resolution retinal imaging. Here, we introduce coherence gate shaping for FF-OCT, to optically shape the coherence gate geometry to match the sample curvature, thus achieving a larger FOV than previously possible. Using this instrument, we obtained high-resolution images of living human photoreceptors close to the foveal center without AO and with a 1 mm × 1 mm FOV in a single shot. This novel advance enables the extraction of photoreceptor-based biomarkers with ease and spatiotemporal monitoring of individual photoreceptors. We compare our findings with AO-assisted ophthalmoscopes, highlighting the potential of FF-OCT, as a compact system, to become a routine clinical imaging technique.

Dynamic full-field optical coherence tomography: 3D live-imaging of retinal organoids.

Optical coherence tomography offers astounding opportunities to image the complex structure of living tissue but lacks functional information. We present dynamic full-field optical coherence tomography as a technique to noninvasively image living human induced pluripotent stem cell-derived retinal organoids. Coloured images with an endogenous contrast linked to organelle motility are generated, with submicrometre spatial resolution and millisecond temporal resolution, creating a way to identify specific cell types in living tissue via their function.

Distortion matrix concept for deep optical imaging in scattering media.

In optical imaging, light propagation is affected by the inhomogeneities of the medium. Sample-induced aberrations and multiple scattering can strongly degrade the image resolution and contrast. On the basis of a dynamic correction of the incident and/or reflected wavefronts, adaptive optics has been used to compensate for those aberrations. However, it only applies to spatially invariant aberrations or to thin aberrating layers. Here, we propose a global and noninvasive approach based on the distortion matrix concept. This matrix basically connects any focusing point of the image with the distorted part of its wavefront in reflection. A singular value decomposition of the distortion matrix allows to correct for high-order aberrations and forward multiple scattering over multiple isoplanatic modes. Proof-of-concept experiments are performed through biological tissues including a turbid cornea. We demonstrate a Strehl ratio enhancement up to 2500 and recover a diffraction-limited resolution until a depth of 10 scattering mean free paths.

Distortion matrix approach for ultrasound imaging of random scattering media.

Focusing waves inside inhomogeneous media is a fundamental problem for imaging. Spatial variations of wave velocity can strongly distort propagating wave fronts and degrade image quality. Adaptive focusing can compensate for such aberration but is only effective over a restricted field of view. Here, we introduce a full-field approach to wave imaging based on the concept of the distortion matrix. This operator essentially connects any focal point inside the medium with the distortion that a wave front, emitted from that point, experiences due to heterogeneities. A time-reversal analysis of the distortion matrix enables the estimation of the transmission matrix that links each sensor and image voxel. Phase aberrations can then be unscrambled for any point, providing a full-field image of the medium with diffraction-limited resolution. Importantly, this process is particularly efficient in random scattering media, where traditional approaches such as adaptive focusing fail. Here, we first present an experimental proof of concept on a tissue-mimicking phantom and then, apply the method to in vivo imaging of human soft tissues. While introduced here in the context of acoustics, this approach can also be extended to optical microscopy, radar, or seismic imaging.

Functional ultrasound imaging of deep visual cortex in awake nonhuman primates.

Deep regions of the brain are not easily accessible to investigation at the mesoscale level in awake animals or humans. We have recently developed a functional ultrasound (fUS) technique that enables imaging hemodynamic responses to visual tasks. Using fUS imaging on two awake nonhuman primates performing a passive fixation task, we constructed retinotopic maps at depth in the visual cortex (V1, V2, and V3) in the calcarine and lunate sulci. The maps could be acquired in a single-hour session with relatively few presentations of the stimuli. The spatial resolution of the technology is illustrated by mapping patterns similar to ocular dominance (OD) columns within superficial and deep layers of the primary visual cortex. These acquisitions using fUS suggested that OD selectivity is mostly present in layer IV but with extensions into layers II/III and V. This imaging technology provides a new mesoscale approach to the mapping of brain activity at high spatiotemporal resolution in awake subjects within the whole depth of the cortex.

Real-time non-contact cellular imaging and angiography of human cornea and limbus with common-path full-field/SD OCT.

In today's clinics, a cell-resolution view of the cornea can be achieved only with a confocal microscope (IVCM) in contact with the eye. Here, we present a common-path full-field/spectral-domain OCT microscope (FF/SD OCT), which enables cell-detail imaging of the entire ocular surface in humans (central and peripheral cornea, limbus, sclera, tear film) without contact and in real-time. Real-time performance is achieved through rapid axial eye tracking and simultaneous defocusing correction. Images contain cells and nerves, which can be quantified over a millimetric field-of-view, beyond the capability of IVCM and conventional OCT. In the limbus, palisades of Vogt, vessels, and blood flow can be resolved with high contrast without contrast agent injection. The fast imaging speed of 275 frames/s (0.6 billion pixels/s) allows direct monitoring of blood flow dynamics, enabling creation of high-resolution velocity maps. Tear flow velocity and evaporation time can be measured without fluorescein administration.