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International audience; Photoacoustic imaging (PAI) is an emerging noninvasive and inexpensive bioimaging modality, which has stimulated a wide interest in recent years for cancer theranostics. Hybrid nano-and microgels have been designed as protective carriers to incorporate PAI contrast agents due to their biocompatibility and biodegradability, high loading capacity, versatile surface chemistry, and stimuliresponsive properties. These key features offer unprecedented opportunities for the design of innovative multifunctional cancer theranostic agents. This review focuses on the recent development of photoacoustic active nano-and microgels for biomedical applications in cancer theranostics and cancer hallmark imaging. The various chemical natures of gels and PAI contrast agents and the elaboration methods of hybrid matrices are summarized and discussed. Their applications in multimodal cancer theranostics and cancer hallmark imaging are presented, highlighting their merits and potential in treatment and diagnosis.

Diversity of the design and alignment of illumination and ultrasonic transducers empower the fine scalability and versatility of optoacoustic imaging. In this study, we implement an innovative high-resolution optoacoustic mesoscopy for imaging the vasculature and tissue oxygenation within subcutaneous and orthotopic cancerous implants of mice in vivo through acquisition of tomographic projections over 180° at a central frequency of 24 MHz. High-resolution volumetric imaging was combined with multispectral functional measurements to resolve the exquisite inner structure and vascularization of the entire tumor mass using endogenous and exogenous optoacoustic contrast. Evidence is presented for constitutive hypoxemia within the carcinogenic tissue through analysis of the hemoglobin absorption spectra and distribution. Morphometric readouts obtained with optoacoustic mesoscopy have been verified with high-resolution ultramicroscopic studies. The findings described herein greatly extend the applications of optoacoustic mesoscopy toward structural and multispectral functional measurements of the vascularization and hemodynamics within solid tumors in vivo and are of major relevance to basic and preclinical oncological studies in small animal models.

In this work, we experimentally investigate thermal-based nonlinear photoacoustic generation as a mean to discriminate between different types of absorbing particles. The photoacoustic generation from solutions of dye molecules and gold nanospheres (same optical densities) was detected using a high frequency ultrasound transducer (20MHz). Photoacoustic emission was observed with gold nanospheres at low fluence for an equilibrium temperature around 4°C, where the linear photoacoustic effect in water vanishes, highlighting the nonlinear emission from the solution of nanospheres. The photoacoustic amplitude was also studied as a function of the equilibrium temperature from 2°C to 20°C. While the photoacoustic amplitude from the dye molecules vanished around 4°C, the photoacoustic amplitude from the gold nanospheres remained significant over the whole temperature range. Our preliminary results suggest that in the context of high frequency photoacoustic imaging, nanoparticles may be discriminated from molecular absorbers based on nanoscale temperature rises.

In security applications, several methods are used for detection of hidden objects. Metal detectors and palpation are the most common methods. Non-contact acoustic waves imaging of elastic heterogeneities in superficial body tissues could be an interesting alternative.

Gas nuclei exist naturally in living bodies. Their activation initiates cavitation activity, and is possible using short ultrasonic excitations of high amplitude. However, little is known about the nuclei population in vivo, and therefore about the rarefaction pressure required to form bubbles in tissue. A novel method dedicated to in vivo investigations was used here that combines passive and active cavitation detection with a multi-element linear ultrasound probe (4–7 MHz). Experiments were performed in vivo on the brain of trepanated sheep. Bubble nucleation was induced using a focused single-element transducer (central frequency 660 kHz, f -number = 1) driven by a high power (up to 5 kW) electric burst of two cycles. Successive passive recording and ultrafast active imaging were shown to allow detection of a single nucleation event in brain tissue in vivo. Experiments carried out on eight sheep allowed statistical studies of the bubble nucleation process. The nucleation probability was evaluated as a function of the peak negative pressure. No nucleation event could be detected with a peak negative pressure weaker than −12.7 MPa, i.e. one order of magnitude higher than the recommendations based on the mechanical index. Below this threshold, bubble nucleation in vivo in brain tissues is a random phenomenon. (Some figures in this article are in colour only in the electronic version)

Photoacoustic imaging can achieve high-resolution three-dimensional (3-D) visualization of optical absorbers at penetration depths of ∼1 cm in biological tissues by detecting optically induced high ultrasound frequencies. Tomographic acquisition with ultrasound linear arrays offers an easy implementation of single-side access, parallelized, and high-frequency detection, but usually comes with an image quality impaired by the directionality of the detectors. Indeed, a simple translation of the array perpendicular to its median imaging plane is often used, but results both in a poor resolution in the translation direction and strong limited-view artifacts.To improve the spatial resolution and the visibility of complex structures while retaining a planar detection geometry, we introduce, in this paper, a rotate-translate scanning scheme and investigate the performance ofa scanner implemented at 15 MHz center frequency. The developed system achieved a quasi-isotropic uniform 3-D resolution of ∼170 μm over a cubic volume of side length 8.5 mm, i.e., an improvement in the resolution in the translation direction by almost one order of magnitude. Dual-wavelength imaging was also demonstrated with ultrafast wavelength shifting. The validity of our approach was shown in vitro. We discuss the ability to enable in vivo imaging for preclinical and clinical studies.

We have developed an epi-illumination raster-scan optoacoustic mesoscopy system (RSOM), the new system is capable of imaging model organisms, and vasculature. The newly developed system is based on a custom designed; spherically focused detector with a Characterization of the system shows an isotropic lateral resolution of 18 μm, and an axial resolution of 4 μm. The scan times are on the order of 8 minutes for a field of view of 10×10 mm2. The achieved resolution is slightly degraded up to a depth of 5 mm. After characterizing the system we showcase it's performance on a zebrafish ex vivo, and an excised mouse ear. Additionally, to improve the visibility of small structures we have reconstructed the high frequencies, and the low frequencies separately, and at the end overplayed the two reconstructions using different colors, this way the high frequencies are not masked by the low frequencies which have a higher signal to noise ratio.

International audience; We study the potential of photoacoustic guidance for light focusing through scattering samples via wavefront-shaping and iterative optimization. We experimentally demonstrate that the focusing efficiency on an extended absorber can be improved by iterative optimization of the high frequency components of the broadband photoacous-tic signal detected with a spherically focused transducer. We demonstrate more than 12-fold increase in the photoacoustic signal generated by a 30 μm wire using a narrow frequency band around 60 MHz. By monitoring the speckle pattern evolution during the optimization process with a CCD camera, we experimentally confirm that such optimization leads to a smaller optical focus than what would be obtained by optimizing lower frequencies of the photoacoustic feedback.

Optoacoutic mesoscopy aims to bridge the gap between optoacoustic microscopy and optoacoustic tomography. We have developed a setup for optoacoustic mesoscopy where we use a high frequency, high numerical aperture spherically focused ultrasound transducer, with a wide bandwidth of 25-125 MHz. The excitation is performed using a diode laser capable of >500 μJ/pulse, 1.8ns pulse width, 1.4 kHz pulse repetition rate, at 515 nm. The system is capable to penetrate more than 5 mm with a resolution of 7 μm axially and 30 μm transversally. Using high-speed stages and scanning the transducer in a quasi-continuous mode, a field of view of 2×2 mm2 is scanned in less than 2 minutes. The system is suitable for imaging biological samples that have a diameter of 1-5 mm; zebrafish, drosophila melanogaster, and thin biological samples such as the mouse ear and mouse extremities. We have used our mesoscopic setup to generate 3- dimensional images of genetically modified drosophila fly, and drosophila pupae expressing GFP from the wings, high resolution images were generated in both cases, in the fly we can see the wings, the legs, the eyes, and the shape of the body. In the pupae the outline of the pupae, the spiracles at both ends and a strong signal corresponding to the location of the future wings are observed.

We investigate experimentally the use of speckle illumination for photoacoustic imaging. In particular, we demonstrate that otherwise invisible features are revealed through high-frequency signals fluctuations from different speckle realizations.

International audience; We implement the photoacoustic transmission matrix approach on a two-dimensional photoacoustic imaging system, using a 15 MHz linear ultrasound array. Using a black leaf skeleton as a complex absorbing structure, we demonstrate that the photoacoustic transmission matrix approach allows to reveal structural features that are invisible in conventional photoacoustic images, as well as to selectively control light focusing on absorbing targets, leading to a local enhancement of the photoacoustic signal.

In high-frequency photoacoustic imaging with uniform illumination, homogeneous photo-absorbing structures may be invisible because of their large size or limited-view issues. Here we show that, by exploiting dynamic speckle illumination, it is possible to reveal features which are normally invisible with a photoacoustic system comprised of a 20MHz linear ultrasound array. We demonstrate imaging of a 5 mm diameter absorbing cylinder and a 30 micrometer black thread arranged in a complex shape. The hidden structures are directly retrieved from photoacoustic images recorded for different random speckle illuminations of the phantoms by assessing the variation in the value of each pixel over the illumination patterns., Comment: 4 pages, 3 figures, Accepted in Optics letters

International audience; Bubbles can form in biological tissues through ultrasonic activation of natural gas nuclei. The damaging aftereffects raise safety concerns. However, the population of nuclei is currently unknown, and bubble nucleation is stochastic and thus unpredictable. This study investigates the statistical behavior of bubble nucleation experimentally and introduces a model-based analysis to determine the distribution of nuclei in biological samples-two pig blood samples in vitro. Combined ultra-fast passive and active cavitation detection with a linear array was used to detect nucleation from pulsed ultrasound excitations at 660 kHz. Single nucleation events were detected at peak rarefaction pressures from -3.6 to -24 MPa, and the nucleation probability over the range 0 to 1 was estimated from more than 330 independent acquisitions per sample. Model fitting of the experimental probability revealed that the distribution of nuclei is most likely continuous, and nuclei are rare in comparison to blood cells.

Broadband optoacoustic waves generated by biological tissues excited with nanosecond laser pulses carry information corresponding to a wide range of geometrical scales. Typically, the frequency content present in the signals generated during optoacoustic imaging is much larger compared to the frequency band captured by common ultrasonic detectors, the latter typically acting as bandpass filters. To image optical absorption within structures ranging from entire organs to microvasculature in three dimensions, we implemented optoacoustic tomography with two ultrasound linear arrays featuring a center frequency of 6 and 24 MHz, respectively. In the present work, we show that complementary information on anatomical features could be retrieved and provide a better understanding on the localization of structures in the general anatomy by analyzing multi-bandwidth datasets acquired on a freshly excised kidney.

Whole-body optical imaging of post-embryonic stage model organisms is a challenging and long sought-after goal. It requires a combination of high-resolution performance and high-penetration depth. Optoacoustic (photoacoustic) mesoscopy holds great promise, as it penetrates deeper than optical and optoacoustic microscopy while providing high-spatial resolution. However, optoacoustic mesoscopic techniques only offer partial visibility of oriented structures, such as blood vessels, due to a limited angular detection aperture or the use of ultrasound frequencies that yield insufficient resolution. We introduce 360° multi orientation (multi-projection) raster scan optoacoustic mesoscopy (MORSOM) based on detecting an ultra-wide frequency bandwidth (up to 160 MHz) and weighted deconvolution to synthetically enlarge the angular aperture. We report unprecedented isotropic in-plane resolution at the 9–17 μm range and improved signal to noise ratio in phantoms and opaque 21-day-old Zebrafish. We find that MORSOM performance defines a new operational specification for optoacoustic mesoscopy of adult organisms, with possible applications in the developmental biology of adulthood and aging. An optoacoustic technique has been used to realize whole-body imaging of post-embryonic zebrafish by researchers in Germany and France. The whole-body imaging of biological specimens beyond the embryonic stage is highly desired for biological studies. While showing promise, raster-scan optoacoustic mesoscopy (RSOM) is limited by the low numerical aperture of the ultrasonic detectors used. Now, Murad Omar at the Technical University of Munich and co-workers have demonstrated a Multi Orientation RSOM technique that, similar to X-ray computed tomography, collects projections over 360 degrees. As it uses broad-field illumination and its resolution is limited only by ultrasound diffraction, the new method can achieve a high resolution and wide range of spatial frequencies. The team demonstrated it by obtaining high-resolution whole-body images of 21-day-old zebrafish, which cannot be obtained by optical microscopy or conventional optoacoustic mesoscopy.

International audience; The activation of natural gas nuclei to induce larger bubbles is possible using short ultrasonic excitations of high amplitude, and is required for ultrasound cavitation therapies. However, little is known about the distribution of nuclei in tissues. Therefore, the acoustic pressure level necessary to generate bubbles in a targeted zone and their exact location are currently difficult to predict. To monitor the initiation of cavitation activity, a novel all-ultrasound technique sensitive to single nucleation events is presented here. It is based on combined passive detection and ultrafast active imaging over a large volume using the same multi-element probe. Bubble nucleation was induced using a focused transducer (660 kHz, f-number = 1) driven by a high-power electric burst (up to 300 W) of one to two cycles. Detection was performed with a linear array (4 to 7 MHz) aligned with the single-element focal point. In vitro experiments in gelatin gel and muscular tissue are presented. The synchronized passive detection enabled radio-frequency data to be recorded, comprising high-frequency coherent wave fronts as signatures of the acoustic emissions linked to the activation of the nuclei. Active change detection images were obtained by subtracting echoes collected in the unnucleated medium. These indicated the appearance of stable cavitating regions. Because of the ultrafast frame rate, active detection occurred as quickly as 330 μs after the high-amplitude excitation and the dynamics of the induced regions were studied individually.

Brain treatment with High Intensity Focused Ultrasound (HIFU) can be achieved by multichannel arrays through the skull using time-reversal focusing. Such a method requires a reference signal either sent by a real source embedded in brain tissues or computed from a virtual source, using the acoustic properties of the skull deduced from CT images. This non-invasive computational method allows precise focusing, but is time consuming and suffers from unavoidable modeling and repositioning errors which reduce the accessible acoustic pressure at the focus in comparison with real experimental time-reversal using an implanted hydrophone. Ex vivo simulations with a half skull immersed in a water tank allow us to reach at low amplitude levels a pressure ratio of 83% of the reference pressure (real time-reversal) at 1 MHz. Using this method to transcranially focus a pulse signal in an agar gel (model for in vivo bubble formation), we induced a cavitation bubble that generated an ultrasonic wave received by the array. Selecting the 1 MHz component, the signal was time reversed and re-emitted, allowing 97%plusmn1.1% of pressure ratio to be restored. To target points in the vicinity of the geometrical focus, conventional electronic steering from the reference signal has been achieved. Skull aberrations severely degrade the accessible pressure while moving away from the focus ( ~90% at 11 mm in the focal plane). Nevertheless, inducing cavitation bubbles close to the limit of the primary accessible zone allowed us to acquire multiple references signal to increase the electronic steering area by 50%.

Optical mesoscopy extends the capabilities of biological visualization beyond the limited penetration depth achieved by microscopy. However, imaging of opaque organisms or tissues larger than a few hundred micrometers requires invasive tissue sectioning or chemical treatment of the specimen for clearing photon scattering, an invasive process that is regardless limited with depth. We developed previously unreported broadband optoacoustic mesoscopy as a tomographic modality to enable imaging of optical contrast through several millimeters of tissue, without the need for chemical treatment of tissues. We show that the unique combination of three-dimensional projections over a broad 500 kHz–40 MHz frequency range combined with multi-wavelength illumination is necessary to render broadband multispectral optoacoustic mesoscopy (2B-MSOM) superior to previous optical or optoacoustic mesoscopy implementations.

We developed a raster-scan acoustic resolution broadband optoacoustic mesoscopy system and investigated the imaging performance using ultrasonic frequencies up to 125 MHz. The developed system achieves 7 μm axial resolution and transverse resolution of 30 μm reaching depths of at least 5 mm. This unprecedented performance is achieved by operating at out-of-focus ultrasonic detection and tomographic reconstruction. We demonstrate the limits reached due to the width of the laser pulse employed and showcase the technique on drosophila fly and drosophila pupae ex vivo.

Optoacoustic (photoacoustic) mesoscopic and microscopic imaging is often implemented by linearly scanning a spherically focused ultrasound transducer. In this case, the resolution and sensitivity along the scan direction are limited by diffraction and therefore degrade rapidly for imaging depths away from the focal point. Partial restoration of the lost resolution can be achieved by using data-processing techniques, such as the virtual detector delay-and-sum method. However, these techniques are based on an approximate description of the detector properties, which limits the improvement in image quality they achieve. Herein we propose a reconstruction method based on an exact model of the optoacoustic generation and propagation that incorporates the spatial response of the sensor. The proposed method shows superior imaging performance over previously considered techniques.

We present optical and atomic force microscopy measurements of the roughness of the inner surfaces within glass capillary tubes and Hollow Core Photonic Bandgap Fibers over the [3×10−2 μηm-1–30 μm-1] spatial frequency range.

In optoacoustic imaging absorbing structures excited with short laser pulses generate broadband ultrasound waves, which tomographically detected outside the sample enable reconstruction of initial pressure distribution. As light scatters in biological tissues, the excitation has a three-dimensional (3D) pattern allocation. Accurate reconstruction of the 3D distribution of optical absorption requires a large solid angle of detection of the ultrasonic field. Moreover, the center frequency and bandwidth of a given detector define the range of structure sizes it is able to resolve. Therefore, detectors with different frequency bandwidths record different subsets of information. A volumetric optoacoustic system using linear ultrasound arrays with different central frequencies, 6MHz and 24MHz, is introduced. By employing a novel scanning geometry that takes advantage of the high sensitivity on the transversal dimension of these linear probes, high resolution optoacoustic signals are being recorded. Resolution performance and biological capabilities are demonstrated with a 20um crossed-suture phantom and an excised mouse liver lobe.