17th European Molecular Imaging Meeting
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Ultrasound & Optoacoustic Technologies

Session chair: Carmel M. Moran (Edinburgh, UK); Xose Luis Dean Ben (Zurich, Switzerland)
 
Shortcut: PS 10
Date: Wednesday, 16 March, 2022, 5:30 p.m. - 7:00 p.m.
Session type: Parallel Session

Contents

Click at talk title to open the abstract

5:30 p.m. PS 10-01

Introductory Lecture

Xose Luis Dean Ben

Zurich, Switzerland

5:50 p.m. PS 10-02

Fast optoacoustic mesoscopy of microvascular endothelial dysfunction in cardiovascular risk and disease

Hailong He1, 2, Angelos Karlas1, 4, 5, Nikolina-Alexia Fasoula1, 2, Michael Kallmayer4, Juan Aguirre1, 2, Hans-Henning Eckstein4, 5, Vasilis Ntziachristos1, 2, 3

1 Technical University of Munich, Chair of Biological Imaging, Munich, Baden-Württemberg, Germany
2 Helmholtz Zentrum München, Institute of Biological and Medical Imaging, Munich, Germany
3 Munich Institute of Robotics and Machine Intelligence (MIRMI), Technical University of Munich, Germany, Munich, Germany
4 Technical University of Munich, Clinic of Vascular and Endovascular Surgery, Klinikum Rechts der Isar, Munich, Germany
5 DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany

Introduction

Endothelial dysfunction (ED) is a systemic condition that affects both the macro- and microvasculature and precedes cardiovascular disease (CVD), including coronary artery disease and heart failure. Microvascular ED precedes ED in larger arteries and is an early marker of CVD. While precise assessment of microvascular ED could thus be used for the early detection and risk stratification of CVD, detailed interrogation of skin microvascular ED is limited by available technology.

Methods

Herein, we applied a novel approach for the non-invasive assessment of skin microvascular ED by developing fast plane raster-scan optoacoustic mesoscopy (FP-RSOM) to visualize and quantify skin microvasculature perfusion changes during post-occlusive hyperemia (PORH) tests. We combined three-dimensional RSOM imaging with fast dynamic FP-RSOM measurements (1 frame / second) in human skin in-vivo, which allowed for the first time to fully visualize the cutaneous microvascular response and further quantify changes in individual vessel diameter, total blood volume and vessel density during the PORH process. We computed biomarkers to quantify skin endothelial function within different skin layers as a function of skin depth, which conventional approaches cannot achieve.

Results/Discussion

We determined several biomarkers, such as the maximum volume change (MVC), hyperemia ratio (HR) and the time to peak (TP), to enable the quantification of the endothelial function of skin microvasculature by quantifying the vessel density changes at various skin depths. Then, we benchmarked our FP-RSOM-based method by comparing the microvascular ED between age-matched healthy volunteers and smokers. We showcase for the first time the markedly different impairments to the endothelial function of upper and deeper dermal microvessels in the smoking group. We validated the FP-RSOM measurements with macrovascular ultrasound-based measurements of the radial artery diameter, as well as microvascular Laser Doppler flowmetry and tissue spectrometry (O2C©) measurements of skin blood flow, Hb and SO2. FP-RSOM provided higher sensitivity in detecting the effects of smoking and CVD on microvascular ED compared to LDF and tissue spectrometry.

Conclusions

Our results demonstrate the great potential of FP-RSOM for elucidating the morphology, functional state and reactivity of skin microvasculature via the definition of novel FP-RSOM-based biomarkers, with implications for investigating new aspects of endothelial function and its impairment in cardiovascular risk and disease groups.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 871763 (WINTHER) and No 687866 (INNODERM), from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme under grant agreement No 694968 (PREMSOT) and from Helmholtz Zentrum München through Physician Scientists for Groundbreaking Projects, in part by the Helmholtz Association of German Research Center, through the Initiative and Networking Fund, i3 (ExNet-0022-Phase2-3). We thank Dr. Sergey Sulima for his attentive reading and improvements of the manuscript and the staff at the Clinic of Vascular and Endovascular Surgery, Klinikum rechts der Isar at TUM for assisting with the patient studies.

Disclosure

Vasilis Ntziachristos is an equity owner in and consultant for iThera Medical GmbH, Munich, Germany.

FP-RSOM in assessment of skin microvasculature endothelium function
a. Skin microvessel endothelium function by FP-RSOM, O2C and ultrasound (US) during PORH; O2C: Laser Doppler Flowmetry and White-Light Spectroscopy. b. The timeline of PORH assessment by FP-RSOM and O2C simultaneously. c. 3D RSOM scan a healthy volunteer, where the dermis vasculature (DV) can be grouped into microvessels in the subpapillary dermis (SD) layer and vessels in the reticular dermis (RD) layer. d,e. Corresponding MIP images of the epidermis (EP) and dermis (DR) layers of (c) in the coronal direction. f. Optoacoustic FP-RSOM scan. g. Reconstructed image. Scale bar: 500 µm.
RSOM imaging of skin microvasculature hyperaemia during the PORH.
10 RSOM images during the PORH and corresponding MIP images of the dermis layer in the coronal direction are shown in a-j. The white arrows in (h) indicates the new capillaries layer and dermal vessels induced during PORH. k. The diameter changes of three vessels (white arrow labels 1, 2 and 3 in a). l. Changes of the total blood volume in the subpapillary dermis (SD) layer, in the reticular dermis (RD) layer and the whole dermis vasculature (DV). m. O2C results including the blood flow, oxygen saturation (SO2) and partial blood volume (rHb).  Scale bar: 500 µm.
Keywords: Optoacoustic mesoscopy, endothelial function, skin imaging
6:00 p.m. PS 10-03

Optoacoustic Localization and Tracking of Microparticles In Vivo Enabled by a NIR-Absorbing Shell

Daniil Nozdriukhin1, 2, Sandeep Kumar Kalva1, 2, Weiye Li1, 2, Jim Zhao1, 2, Daniel Razansky1, 2, Xosé Luís Deán-Ben1, 2

1 University of Zurich, Institute of Pharmacology and Toxicology, Faculty of Medicine, Zürich, Switzerland
2 ETH Zürich, Institute for Biomedical Engineering, Department of Information Technology and Electrical Engineering, Zürich, Switzerland

Introduction

Light-absorbing microparticles can greatly enhance the performance of optoacoustic (OA) imaging by enabling new capabilities for characterizing microcirculation. Specifically, tracking of individual particles facilitates microvascular imaging and blood flow quantification. Herein, we demonstrate the feasibility of tracking intravenously injected 4 um microparticles with a gold-carbon absorbing shell in the mouse brain vasculature in vivo. Super-resolution imaging is further achieved with localization OA tomography (LOT) based on these particles [1, 2, 3].

Methods

Microparticles were fabricated using a layer-by-layer technique on silica microspheres with 4 µm diameter. 3 layers of polyelectrolytes were deposited to form a pillow for further deposition of two bilayers of multi-walled carbon nanotubes/gold nanoparticles with PEGylation in the end (Fig. 1). Particles are shown to provide a broad absorbance in the near-infrared (NIR) window (680-850 nm). OA imaging at 800 nm (100 frames per second) was performed on an athymic nude mouse with a 512-element spherical array (7 MHz central frequency, 140° angular coverage). The acquired sequence was processed with a singular value decomposition (SVD) filter facilitating the separation of moving particles from tissue background. Particle localization and tracking for LOT image formation were eventually done.

Results/Discussion

Microparticle synthesis was controlled by zeta-potential measurement and UV-Vis spectroscopy on each step of fabrication. The surface of the synthesized microspheres was further visualized with scanning electron microscopy (SEM). The OA properties of the particles were studied in agar phantoms with the aforementioned spherical array and with optical-resolution OA microscopy (Fig. 1). Microparticle flow was observed in the brain during 5 min after tail vein injection with a laser fluence at the tissue surface of 13 mJ/cm2. Image processing facilitated the isolation of particle motion and determination of their position in each time point together with tracking. This information provided the possibility to build a LOT image and estimate the blood flow velocity (Fig. 2).

Conclusions

We successfully assembled 4 µm core-shell microparticles providing sufficient light absorption for OA in vivo detection in the presence of the strong background absorption of blood in the mouse brain vasculature. Processing of the acquired image sequence was performed with an SVD filter and subsequent tracking for LOT image formation.

Acknowledgement

X. L. D. B. acknowledges support from the Werner und Hedy Berger-Janser Stiftung (Application No 08/2019) and the Helmut Horten Stiftung (Project Deep Skin). D. R. acknowledges support from the European Research Council under grant agreement ERC-CoG_2015_682379.

Disclosure

 I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Dean-Ben, XL, Razansky, D 2018, ‘Localization optoacoustic tomography’, Light: Science & Applications, 7(4), 18004-18004
[2] Dean-Ben, XL, Robin, J, Ni, R, Razansky, D 2020, ‘Noninvasive three-dimensional optoacoustic localization microangiography of deep tissues’, arXiv preprint, arXiv:2007.00372
 
[3] Nozdriukhin, D, Kalva, SK, Li, W, Yashchenok, A, Gorin, D, Razansky, D, Dean-Ben, XL 2021, ‘Rapid Volumetric Optoacoustic Tracking of Individual Microparticles In Vivo Enabled by a NIR-Absorbing Gold-Carbon Shell’, ACS Appl. Mater. Interfaces, 13(41), 48423–48432
 
Microparticles and their characterization

Figure 1. (a) SEM image of synthesized microparticles (b) UV-VIS and Optoacoustic spectra of particles and substances from which they were assembled, (c) Optoacoustic Microscopy image of the microparticles

Optoacoustic tomography

Fig. 2. (a) MIP image of the particles in the mouse brain (b) SVD-filtered image of the particles (blue) in the brain vessels (red), (c) LOT image of the same region based on the whole dataset. Scale bar – 1 mm.

Keywords: Localization optoacoustic tomography, Microparticles tracking, NIR
6:10 p.m. PS 10-04

Multi-Parametric Characterization of Fatty Liver Disease with Transmission-Reflection Optoacoustic Ultrasound (TROPUS)

Berkan Lafci1, 2, Anna Hadjihambi3, 4, Christos Konstantinou3, 4, Joaquin L. Herraiz5, 6, Neal C. Burton7, Xosé Luís Deán-Ben1, 2, Daniel Razansky1, 2

1 University of Zurich, Institute of Pharmacology and Toxicology and Institute for Biomedical Engineering, Faculty of Medicine, Zurich, Switzerland
2 ETH Zurich, Institute for Biomedical Engineering, Department of Information Technology and Electrical Engineering, Zurich, Switzerland
3 Foundation for Liver Research, The Roger Williams Institute of Hepatology, London, United Kingdom
4 University of Lausanne, Department of Biomedical Sciences, Lausanne, Switzerland
5 Complutense University of Madrid, Nuclear Physics Group and IPARCOS, Madrid, Spain
6 Hospital Clínico San Carlos (IdISSC), Health Research Institute, Madrid, Spain
7 Thera Medical GmbH, Munich, Germany

Introduction

Non-alcoholic fatty liver disease (NAFLD) refers to the early stage of liver fibrosis resulting from accumulation of lipid in liver tissues. Early detection and treatment of NAFLD is paramount in preventing long-term liver damage. We evaluated the diverse contrasts available with the hybrid transmission-reflection optoacoustic ultrasound (TROPUS), namely, optoacoustic, ultrasound reflectivity, and speed of sound for detection and multi-parametric assessment of NAFLD in mice. The results indicate that the proposed approach is suitable for assessing the NAFLD development in preclinical models.

Methods

The hybrid TROPUS [1] imaging system consists of four components, namely, a circular transducer array (512 elements, 5 MHz), a nanosecond pumped laser source, a data acquisition system and a workstation for data processing. The laser wavelength was tuned between 700 and 1000 nm with step size of 20 nm for multispectral optoacoustic (OA) acquisition. OA images were reconstructed using back-projection algorithm [2]. Ultrasound (US) data acquisition was performed using synthetic transmit aperture technique [3,4]. Single transmission reflection ultrasound computed tomography (RUCT) images were beamformed by delay and sum algorithm and then compounded to create final image [5]. Speed of sound (SoS) images were reconstructed from transmitted US waves using full wave inversion method [6].

Results/Discussion

Exemplary in vivo TROPUS images from the liver region of a control mouse are shown in Fig. 1. In total, 11 (4 NAFLD and 7 control) in vivo mice were imaged using TROPUS. The livers were segmented using RUCT images, which clearly show the anatomical structures (Fig. 1b). The quantitative in vivo analysis on fat accumulation was further done by comparing lipid component of OA images and changes in SoS values. The detection capabilities of the system were also tested on ex vivo excised livers by comparing the lipid content in NAFLD and control livers. Specifically, ex vivo images were acquired from 6 (3 NAFLD and 3 control) excised livers. Then, histology analysis was performed to validate NAFLD and fibrosis development. Accumulation of lipid in NAFLD livers shows increase in lipid component of OA signals for ex vivo and in vivo images (Fig. 2a). However, SoS values of NAFLD livers decreases with lipid accumulation since lipid has lower SoS compared to healthy liver tissues (Fig. 2b).

Conclusions

We demonstrated the capabilities of TROPUS imaging for detection of NAFLD. Ex vivo and in vivo results show that TROPUS can be used for assessment of NAFLD development. Specifically, liver regions were segmented using RUCT. The lipid content was quantified by OA images. SoS images provided quantitative readings of lipid accumulation. The results indicate that the proposed approach is suitable for assessing the NAFLD development in mice models.

Acknowledgement

This work was supported by Swiss Data Science Center grant C19-04.

We would like to thank Luc Pellerin from UNIL for arranging the in vivo animal licenses.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Lafci, B., Merčep, E., Herraiz, J.L., Deán-Ben, X.L. and Razansky, D., 2020. Noninvasive multiparametric characterization of mammary tumors with transmission-reflection optoacoustic ultrasound. Neoplasia, 22(12), pp.770-777.
[2] Ozbek, A., Deán-Ben, X.L. and Razansky, D., 2013, May. Realtime parallel back-projection algorithm for three-dimensional optoacoustic imaging devices. In European conference on biomedical optics (p. 88000I). Optical Society of America.
[3] Merčep, E., Herraiz, J. L., Deán-Ben, X. L., & Razansky, D., 2019. Transmission–reflection optoacoustic ultrasound (TROPUS) computed tomography of small animals. Light: Science & Applications, 8(1), 1-12.
[4] Lafci, B., Merčep, E., Herraiz, J.L., Deán-Ben, X.L. and Razansky, D., 2021, March. Transmission-reflection optoacoustic ultrasound (TROPUS) imaging of mammary tumors. Photons Plus Ultrasound: Imaging and Sensing 2021 (Vol. 11642, p. 116422L). International Society for Optics and Photonics.
[5] Lafci, B., Robin, J., Dean-Ben, X. L., & Razansky, D. (2021). pyruct (Version 1.0.3) [Computer software]. https://doi.org/10.5281/zenodo.5599811
[6] Pérez-Liva, M., Herraiz, J.L., Udías, J.M., Miller, E., Cox, B.T. and Treeby, B.E., 2017. Time domain reconstruction of sound speed and attenuation in ultrasound computed tomography using full wave inversion. The Journal of the Acoustical Society of America, 141(3), pp.1595-1604.
In vivo Results
In vivo images of a control mouse using TROPUS imaging system. (a) Optoacoustic (OA) image of liver cross-section. (b) Reflection ultrasound computed tomography (RUCT) image of liver cross-section. (c) Speed of sound (SoS) image of liver cross-section.
Quantitative Results
Quantitative results from optoacoustic (OA) and speed of sound (SoS) images. (a) Averaged lipid signal intensity of ex vivo and in vivo OA signals from liver tissues. (b) SoS values inside the liver regions of non-alcoholic fatty liver disease (NAFLD) and control mice.
Keywords: Optoacoustics, Reflection Ultrasound Computed Tomography, Speed of Sound Imaging, Non-Alcoholic Fatty Liver Disease (NAFLD)
6:20 p.m. PS 10-05

3D Nonlinear Sound-Sheet Imaging of Acoustic Biomolecules

Baptiste Heiles1, Olivia Weidlich1, 2, Dion Terwiel1, Juancito Van Leeuwen1, David Maresca1

1 Delft University of Technology, Department of Imaging Physics, Delft, Netherlands
2 Technical University of Denmark, Department of Health Technology, Lyngby, Denmark

Introduction

Recently, nonlinear ultrasound imaging of genetically encoded acoustic biomolecules known as gas vesicles (GVs) has enabled deep 2D scanning of gene expression [1], enzyme activity [2] or tumor hypoxia [3]- [6].  Here, we introduce 3D nonlinear sound-sheet imaging (NSSI), a new ultrasound imaging method that modulates pressure amplitude across wide imaging planes at kilohertz framerates. Using a row-column addressed (RCA) probe architecture, we successfully imaged engineered GVss in a 1.2 cm3 volume.

Methods

A custom 15 MHz 128+128 elements RCA probe was driven using a programmable high-framerate ultrasound scanner. Rows (or columns) were used to transmit cross-propagating plane waves from two contiguous half-apertures that  intersect along a 2D plane referred to as a sound-sheet (Fig 1a)..  We developed a dedicated delay-and-sum beamforming of the echoes backscattered from the sound-sheet plane [8] (Fig 1b). To modulate pressure amplitude , each half-aperture was fired individually and the received signals were substracted  to the echoes of the double aperture transmission (Fig 1c). A large tissue volume could then be captured by sweeping sound-sheets along the array (Fig 1d). A proof of concept in agar phantoms containing wild-type GVs and harmonic GVs is presented.

Results/Discussion

We performed NSSI with a 50 μm step at a pressure eliciting nonlinear GV scattering (>300 kPa) to reconstruct volumetric images (Fig 2.b). NSSI detected wells filled with normal and aggregated harmonic GVs (hGV/hGV+, fig.2a) with high specificity (Contrast to Noise Ratio =5.1/9.7dB respectively), whereas echoes arising from the wtGV/wtGV+ were extinguished. We display in fig  2.c a sound-sheet image across the GV phantom. All images were log-compressed and are displayed with a 70dB range. On the left hand side, the linear image shows all GV samples loaded in the phantom, exhibiting higher contrast than the non-scattering agar control. In the middle image, NSSI specifically captures nonlinear signal from nlGV wells. The right hand size image shows that higher pressure transmissions collapse GVs, suppressing all acoustic signals (fig 2.d).

Conclusions

We introduce NSSI, a 3D ultrasound imaging method capable of visualizing genetically encoded acoustic biomolecules with a CNR of 13dB over a 1.2cm3 volume. The implementation of NSSI on an RCA probe addressed current framerate and field of view limitations in GV imaging |7] by achieving kHz framerates in 2D and 2.5 to 300 Hz in 3D across the full probe aperture. NSSI will enable deep time-resolved biomolecular imaging of intact opaque organisms.

Acknowledgement

 This work was supported by the 4TU Precision Medicine Program (The Netherlands) and a Marie-Sklodowska Curie fellowship (European Commission).

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1]  A. Farhadi, F. Sigmund, G. G. Westmeyer, and M. G. Shapiro, “Genetically encodable materials for non-invasive biological imaging,” Nat. Mater., vol. 20, no. 5, pp. 585–592, May 2021, doi: 10.1038/s41563-020-00883-3.
 
[2]  A. Lakshmanan et al., “Acoustic biosensors for ultrasound imaging of enzyme activity,” Nat Chem Biol, vol. 16, no. 9, pp. 988–996, Sep. 2020, doi: 10.1038/s41589-020-0591-0.
 
[3]  R. C. Hurt et al., “Genomically Mined Acoustic Reporter Genes Enable In Vivo Monitoring of Tumors and Tumor-Homing Bacteria,” Bioengineering, preprint, Apr. 2021. doi: 10.1101/2021.04.26.441537.
[4]  A. Lakshmanan et al., “Molecular Engineering of Acoustic Protein Nanostructures,” ACS Nano, vol. 10, no. 8, pp. 7314–7322, Aug. 2016, doi: 10.1021/acsnano.6b03364.
 
[5] D. Maresca et al., “Nonlinear ultrasound imaging of nanoscale acoustic biomolecules,” Appl. Phys. Lett., vol. 110, no. 7, p. 073704, Feb. 2017, doi: 10.1063/1.4976105.
[6] C. Rabut, D. Wu, B. Ling, Z. Jin, D. Malounda, and M. G. Shapiro, “Ultrafast amplitude modulation for molecular and hemodynamic ultrasound imaging,” Appl. Phys. Lett., vol. 118, no. 24, p. 244102, Jun. 2021, doi: 10.1063/5.0050807.
[7] D. Maresca, D. P. Sawyer, G. Renaud, A. Lee-Gosselin, and M. G. Shapiro, “Nonlinear X-Wave Ultrasound Imaging of Acoustic Biomolecules,” Phys. Rev. X, vol. 8, no. 4, p. 041002, Oct. 2018, doi: 10.1103/PhysRevX.8.041002.
[8] M. F. Rasmussen, T. L. Christiansen, E. V. Thomsen, and J. A. Jensen, “3-D imaging using row-column-addressed arrays with integrated apodization - part i: apodization design and line element beamforming,” IEEE Trans. Ultrason., Ferroelect., Freq. Contr., vol. 62, no. 5, pp. 947–958, May 2015, doi: 10.1109/TUFFC.2014.006531.
Figure 1. Transmit and receive sequence for generation of Sound Sheets with a RCA probe

a Two opposite plane waves (blue) generate a double amplitude sound-sheet (red) at their intersection

b The sound-sheet elicits scattering from a thin plane, restoring elevational focus in the orthogonal array (red). Two scatterers at different y-positions become distinguishable

c Amplitude modulation with substraction of the single amplitude to double amplitude signals is performed with sound-sheet to retain only non-linear signal

d To reconstruct a 10×10×12 mm3, the sound-sheets are scanned across the entire array
Figure 2. Sound-Sheet and Non-linear Sound Sheet Imaging of GVs

a Imaging and phantom setup. Wild-type GVs are on the top wells, harmonic GVs on the bottom. Dextran aggregated GVs are denoted with the plus sign and should elicit higher contrast.

b 3D renderings of linear and non-linear sound-sheet imaging scans. wtGVs disappear in the non-linear mode and so do artifacts such as bubbles present in between the agar and the absorbing layer

c Linear and NSSI obtained with transmissions at pressure eliciting harmonic content. Right, NSSI after high pressure transmit to collapse GVs
Keywords: ultrasound, contrast, 3D
6:30 p.m. PS 10-06

Boosting matrix sensitivity for 3D large field transcranial ultrasound localization microscopy using multi-lens diffracting layer

Hugues Favre1, Mathieu Pernot1, Mickael Tanter1, Clément Papadacci1

1 1Physics for Medecine Paris, ESPCI Paris, Inserm U1273, CNRS UMR 8063, France, Paris, France

Introduction

Mapping blood flows of the whole brain is crucial for early diagnosis of cerebral diseases such as aneurism. Ultrasound localization microscopy (ULM) was recently applied to map and quantify blood flows in 2D in brains of adult patients down to a micron-scale resolution [1]. 3D clinical ULM remains challenging due to the transcranial energy loss which significantly reduces the imaging sensitivity. Here, we developed a new matrix combining a limited number of element (elt) and large aperture, based on large diverging elts or multi-lens diffracting layer to increase sensitivity.

Methods

Via simulation using FieldII [2,3], matrix arrays (10 cm², 256 elts, 1MHz) with small (λ/2), large (4λ), and large curved elts (4λ, curvature radius 4.8mm) were simulated. Mono-elt and focused pressure fields were generated for the 3 configurations. Ultrafast N=9 transmits and dynamic receive focusing were implemented. Point Spread Functions (PSF) were assessed. A simulated 10cm3 vascular phantom, in transcranial context, with microbubbles was imaged using the three matrix arrays. A 3D ULM algorithm was used, and bubbles detection statistics were assessed. Finally, a prototype, composed of 16 elts (1MHz, 4λ diameter) with removable epoxy lenses was made to verify simulations. Mono-elt and focused pressure fields were measured via an interferometer [4]. Microbubbles in a tube were imaged.

Results/Discussion

At the same transmit pressure, the simulated maximum axial pressure at 10cm, is 40 times higher for the 4λ convex elt than the λ/2 elt, and angular aperture at -20dB is 2.4 higher for the 4λ convex elt (120°) than the 4λ flat elt (50°). Because of its high directivity, the 4λ flat elts matrix array lacks focusing and shows high secondary lobes in the PSF (-5dB), contrary to 4λ convex matrix (-18dB). The maximal amplitude of the PSF is 45dB higher using 4λ convex than λ/2 flat elts. So when imaging bubbles, λ/2 array shows limited SNR (<10dB) due to weak sensitivity contrary to 4λ convex array (<20dB). For the 4D vascular phantom, 7% of the bubbles are detected with the λ/2 elts, 10% with 4λ elts and 93% using the 4λ convex elts. Experimental pressure fields confirmed the simulated results between large elts without lens (50° angular aperture, 9mm focusing lateral width at half maximum (FLWHM)), and with lenses (120°, 2mm FLWHM). A density map of the tube was obtained only using lenses.

Conclusions

We proved via a simulation study, strengthen by experimental results obtained with a 16 elts prototype, the imaging capabilities of an ultrasensitive probe with 256 large diverging elts that can be performed using a multi-lens diffracting layer.  This new technology has a strong potential to enable 3D ULM over a large field of view through the bones and could be a future whole-organ imaging systems standard to visualize blood flows.

Acknowledgement

This work was supported by the AXA research found and Inserm research accelerator (Inserm ART).

Disclosure

 I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Demené C, Robin J, Dizeux A, Heiles B, Pernot M, Tanter M and Perren F 2021 Transcranial ultrafast ultrasound localization microscopy of brain vasculature in patients Nature Biomedical Engineering 5 219–28
[2] Jensen J A 1996 FIELD: A Program for Simulating Ultrasound Systems 10th Nordicbaltic Conference on Biomedical Imaging, Vol. 4, Supplement 1, Part 1:351–353 pp 351–3
[3] Jensen J A and Svendsen N B 1992 Calculation of pressure fields from arbitrarily shaped, apodized, and excited ultrasound transducers IEEE Trans. Ultrason., Ferroelect., Freq. Contr. 39 262–7
[4] Royer D, Dubois N and Fink M 1992 Optical probing of pulsed, focused ultrasonic fields using a heterodyne interferometer Appl. Phys. Lett. 61 153–5
3D large field imaging simulation of transcranial ULM
Experimental Prototype & focused pressure field
Keywords: ultrasound imaging, blood flow, super-resolution, 3D imaging, transcranial imaging
6:40 p.m. PS 10-07

Looking and listening at biomolecular vibrations: mid-infrared optoacoustic and optothermal sensing for bioanalysis and imaging

Miguel A. Pleitez1, 2, Tao Yuan1, 2, Francesca Gasparin1, 2, Vasilis Ntziachristos1, 2

1 Helmholtz Zentrum München, IBMI, Neuherberg, Baden-Württemberg, Germany
2 Technische Universität München, CBI, Münich, Germany

Introduction

Mid-infrared (mid-IR) excitation and optoacoustic/optothermal (OA/OT) sensing are excellent examples of perfectly complementary technologies. Mid-IR absorption excites molecular-specific vibrational-transitions that are deexcited in the form of heat, generating OA and OT signal which intensity primarily depends on efficient heat deposition. This chemical-bond specific combination is highly sensitive and we apply it for longitudinal assessment of biomolecular composition in living cells, tissues, and small animal without the need of exogenous labels; i.e., for lable-free metabolic imaging.

Methods

We present two different approaches for label-free metabolic imaging: 1) Mid-infraRed Optoacoustic Microscopy (MiROM) [1] and 2) Wide-field Optothermal Mid-infrared Microscopy (WOMiM) [2]. MiROM uses tightly focused optical excitation with coaxially focused ultrasound detection and Chemical-contrast imaging is obtained by raster scanning the sample along the focal plane; simultaneously acquiring OA signals produced at specific molecular vibrations excited by pulsed mid-IR radiation. WOMiM is a new wide-field chemical-contrast imaging method using pump-probe detection of OT signals by optical phase change due to mid-IR irradiation. Chemical-contrast imaging is obtained by taking the difference between Phase Contrast images of the sample with and without Mid-IR illumination.

Results/Discussion

Using intrinsic molecular contrast only, by MiROM, we are able to monitor lipid, protein, and carbohydrate dynamics down to the single-cell level with a lateral resolution of ~5 µm. For instance, in living adipocytes, we were able to observe the spatio-temporal distribution of carbohydrates used for triglyceride formation during adipogenesis. Intrinsic molecular contrast was obtained by excitation of symmetric CH2 vibration of lipids at 2857 cm-1 and from the amide II band of proteins at 1550 cm-1, mainly from NH bending and CN stretching. Carbohydrates were detected at excitations between 1085 and 1000 cm−1 (C–O stretching and C–O–H deformation). While with WOMIM, in Triglyceride drops, we achieved chemical-contrast for field-of-views up to 180 μm in diameter, at imaging speeds of 1 ms/frame. The maximum possible imaging speed of WOMiM was determined by the relaxation time of optothermal heat, measured to be 32.8 μs in water, corresponding to a frame rate of ~30 kHz.

Conclusions

The unique features resulting from the combination of mid-IR excitation with OA/OT sensing have lead to the development of positive-contrast label-free metabolic imaging of living-cells. This presentation will discuss the basic principles of mid-IR spectroscopy and detection as well as the most recent developments on mid-IR optoacoustic and optothermal sensing, focusing on label-free live-cell molecular microscopy for metabolic research.

Acknowledgement

The research leading to these results has received funding from the Deutsche Forschungsgemeinschaft (DFG), Germany [Gottfried Wilhelm Leibniz Prize 2013; NT 3/10-1], as well as from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme under grant agreement No 694968 (PREMSOT).

Disclosure

V.N. is an equity owner and consultant at iThera Medical GmbH, member of the Scientific Advisory Board at SurgVision BV / Bracco Sp.A, owner at Spear UG, founder and consultant at I3. V.N. and M.A.P. are founders of sThesis GmbH.

References
[1] Pleitez, M. A.; Khan, A. A.; Soldà, A.; Chmyrov, A.; Reber, J.; Gasparin, F.; Seeger, M. R.; Schätz, B.; Herzig, S.; Scheideler, M., Label-free metabolic imaging by mid-infrared optoacoustic microscopy in living cells, Nat. Biotechnol. 2019, 1-4.
[2] Yuan, T.; Pleitez, M. A.; Gasparin, F.; Ntziachristos, V., Wide-field mid-infrared hyperspectral imaging by snapshot phase contrast measurement of optothermal excitation, Anal. Chem. 2021, 93, 15323-15330.
Keywords: Optoacoustic, mid-infrared, optothermal, Chemical-microscopy