Quantitative, precise and multi-wavelength evaluation of the light-to-heat conversion efficiency for nanoparticular photothermal agents with calibrated photoacoustic spectroscopy.

Biomedical photothermal therapy with optical nanoparticles is based on the conversion of optical energy into heat through three steps: optical absorption, thermal conversion of the absorbed energy and heat transfer to the surrounding medium. The light-to-heat conversion efficiency (LHCE) has become one of the main metrics to quantitatively characterize the last two steps and evaluate the merit of nanoparticules for photothermal therapy. The estimation of the LHCE is mostly performed by monitoring the temperature evolution of a solution under laser irradiation. However, this estimation strongly depends on the experimental set-up and the heat balance model used. We demonstrate here, theoretically and experimentally, that the LHCE at multiple wavelengths can be efficiently and directly determined, without the use of models, by calibrated photoacoustic spectroscopy. The method was validated using already characterized colloidal suspensions of silver sulfide nanoparticles and maghemite nanoflowers and an uncertainty of 3 to 7% was estimated for the LHCE determination. Photoacoustic spectroscopy provides a new, precise and robust method of analysis of the photothermal capabilities of aqueous solutions of nanoagents.

Motion Rejection and Spectral Unmixing for Accurate Estimation of In Vivo Oxygen Saturation Using Multispectral Optoacoustic Tomography.

Multispectral optoacoustic tomography (MSOT) uniquely enables spatial mapping in high resolution of oxygen saturation (SO2), with potential applications in studying pathological complications and therapy efficacy. MSOT offers seamless integration with ultrasonography, by using a common ultrasound (US) detector array. However, MSOT relies on multiple successive acquisitions of optoacoustic (OA) images at different optical wavelengths and the low frame rate of OA imaging makes the MSOT acquisition sensitive to body/respiratory motion. Moreover, the estimation of SO2 is highly sensitive to noise, and artifacts related to the respiratory motion of the animal were identified as the primary source of noise in MSOT. In this work, we propose a two-step image processing method for SO2 estimation in deep tissues. First, to mitigate motion artifacts, we propose a method of selection of OA images acquired only during the respiratory pause of the animal, using ultrafast ultrasound (US) images acquired immediately after each OA acquisition (US image acquisition duration of 1.4 ms and a total delay of 7 ms). We show that gating is more effective using US images than OA images at different optical wavelengths. Second, we propose a novel method that can estimate directly the SO2 value of a pixel and at the same time evaluate the amount of noise present in that pixel. Hence, the method can efficiently eliminate the pixels dominated by noise from the final SO2 map. Our postprocessing method is shown to outperform conventional methods for SO2 estimation, and the method was validated by in vivo oxygen challenge experiments.

Volumetric and Simultaneous Photoacoustic and Ultrasound Imaging With a Conventional Linear Array in a Multiview Scanning Scheme.

Volumetric, multimodal imaging with precise spatial and temporal coregistration can provide valuable and complementary information for diagnosis and monitoring. Considerable research has sought to combine 3-D photoacoustic (PA) and ultrasound (US) imaging in clinically translatable configurations; however, technical compromises currently result in poor image quality either for PA or ultrasonic modes. This work aims to provide translatable, high-quality, simultaneously coregistered dual-mode PA/US 3-D tomography. Volumetric imaging based on a synthetic aperture approach was implemented by interlacing PA and US acquisitions during a rotate-translate scan with a 5-MHz linear array (12 angles and 30-mm translation to image a 21-mm diameter, 19 mm long cylindrical volume within 21 s). For coregistration, an original calibration method using a specifically designed thread phantom was developed to estimate six geometrical parameters and one temporal offset through global optimization of the reconstructed sharpness and superposition of calibration phantom structures. Phantom design and cost function metrics were selected based on analysis of a numerical phantom and resulted in a high estimation accuracy for the seven parameters. Experimental estimations validated the calibration repeatability. The estimated parameters were used for the bimodal reconstruction of additional phantoms with either identical or distinct spatial distributions of US and PA contrasts. The superposition distance of the two modes was within < 10% of the acoustic wavelength, and a wavelength-order uniform spatial resolution was obtained. This dual-mode PA/US tomography should contribute to more sensitive and robust detection and follow-up of biological changes or the monitoring of slower-kinetic phenomena in living systems such as the accumulation of nanoagents.

Synergic Thermo- and pH-Sensitive Hybrid Microgels Loaded with Fluorescent Dyes and Ultrasmall Gold Nanoparticles for Photoacoustic Imaging and Photothermal Therapy.

Smart microgels (µGels) made of polymeric particles doped with inorganic nanoparticles have emerged recently as promising multifunctional materials for nanomedicine applications. However, the synthesis of these hybrid materials is still a challenging task with the necessity to control several features, such as particle sizes and doping levels, in order to tailor their final properties in relation to the targeted application. We report herein an innovative modular strategy to achieve the rational design of well-defined and densely filled hybrid particles. It is based on the assembly of the different building blocks, i.e., µGels, dyes, and small gold nanoparticles (<4 nm), and the tuning of nanoparticle loading within the polymer matrix through successive incubation steps. The characterization of the final hybrid networks using UV-vis absorption, fluorescence, transmission electron microscopy, dynamic light scattering, and small-angle X-ray scattering revealed that they uniquely combine the properties of hydrogel particles, including high loading capacity and stimuli-responsive behavior, the photoluminescent properties of dyes (rhodamine 6G, methylene blue and cyanine 7.5), and the features of gold nanoparticle assembly. Interestingly, in response to pH and temperature stimuli, the smart hybrid µGels can shrink, leading to the aggregation of the gold nanoparticles trapped inside the polymer matrix. This stimuli-responsive behavior results in plasmon band broadening and red shift toward the near-infrared region (NIR), opening promising prospects in biomedical science. Particularly, the potential of these smart hybrid nanoplatforms for photoactivated hyperthermia, photoacoustic imaging, cellular internalization, intracellular imaging, and photothermal therapy was assessed, demonstrating well controlled multimodal opportunities for theranostics.

Calibrated Photoacoustic Spectrometer Based on a Conventional Imaging System for In Vitro Characterization of Contrast Agents.

Photoacoustic (PA) imaging systems are spreading in the biomedical community, and the development of new PA contrast agents is an active area of research. However, PA contrast agents are usually characterized with spectrophotometry or uncalibrated PA imaging systems, leading to partial assessment of their PA efficiency. To enable quantitative PA spectroscopy of contrast agents in vitro with conventional PA imaging systems, we have developed an adapted calibration method. Contrast agents in solution are injected in a dedicated non-scattering tube phantom imaged at different optical wavelengths. The calibration method uses a reference solution of cupric sulfate to simultaneously correct for the spectral energy distribution of excitation light at the tube location and perform a conversion of the tube amplitude in the image from arbitrary to spectroscopic units. The method does not require any precise alignment and provides quantitative PA spectra, even with non-uniform illumination and ultrasound sensitivity. It was implemented on a conventional imaging setup based on a tunable laser operating between 680 nm and 980 nm and a 5 MHz clinical ultrasound array. We demonstrated robust calibrated PA spectroscopy with sample volumes as low as 15 µL of known chromophores and commonly used contrast agents. The validated method will be an essential and accessible tool for the development of new and efficient PA contrast agents by improving their quantitative characterization.

Biocompatible and Photostable Photoacoustic Contrast Agents as Nanoparticles Based on Bodipy Scaffold and Polylactide Polymers: Synthesis, Formulation, and In Vivo Evaluation.

We have designed a new Bodipy scaffold for efficient in vivo photoacoustic (PA) imaging of nanoparticles commonly used as drug nanovectors. The new dye has an optimized absorption band in the near-infrared window in biological tissue and a low fluorescence quantum yield that leads to a good photoacoustic generation efficiency. After Bodipy-initiated ring-opening polymerization of lactide, the polylactide-Bodipy was formulated into PEGylated nanoparticles (NPs) by mixing with PLA-PEG at different concentrations. Formulated NPs around 100 nm exhibit excellent PA properties: an absorption band at 760 nm and a molar absorption coefficient in between that of molecular PA absorbers and gold NPs. Highly improved photostability compared to cyanine-labeled PLA NPs as well as innocuity in cultured macrophages were demonstrated. After intravenous injection in healthy animals, NPs were easily detected using a commercial PA imaging system and spectral unmixing, opening the way to their use as theranostic agents.

Anisotropic Superattenuation of Capillary Waves on Driven Glass Interfaces.

Metrological atomic force microscopy measurements are performed on the silica glass interfaces of photonic band-gap fibers and hollow capillaries. The freezing of attenuated out-of-equilibrium capillary waves during the drawing process is shown to result in a reduced surface roughness. The roughness attenuation with respect to the expected thermodynamical limit is determined to vary with the drawing stress following a power law. A striking anisotropic character of the height correlation is observed: glass surfaces thus retain a structural record of the direction of the flow to which the liquid was submitted.

Non-contact and through-clothing measurement of the heart rate using ultrasound vibrocardiography.

We present a novel non-contact system for monitoring the heart rate on human subjects with clothes. Our approach is based on vibrocardiography, and measures locally skin displacements. Vibrocardiography with a laser Doppler vibrometer already allows monitoring of this vital sign, but can only be used on bare skin and requires an expensive piece of equipment. We propose here to use an airborne pulse-Doppler ultrasound system operating in the 20-60 kHz range, and comprised of an emitter focusing the ultrasound pulses on skin and a microphone recording the reflected waves. Our implementation was validated in vitro and on two healthy human subjects, using simultaneously laser vibrocardiography and electrocardiography as references. Accurate measurements of the heart rate on clothed skin suggest that our non-contact ultrasonic method could be implemented both inside and outside the clinical environment, and therefore benefit both medical and safety applications.

Complementary Speckle Patterns: Deterministic Interchange of Intrinsic Vortices and Maxima through Scattering Media.

Intensity maxima and zeros of speckle patterns obtained behind a diffuser are experimentally interchanged by applying a spiral phase delay of charge ±1 to the impinging coherent beam. This transform arises from the expectation that tightly focused beams, which have a planar wave front around the focus, are so changed into vortex beams and vice versa. The statistics of extrema locations and the intensity distribution of the so-generated "complementary" patterns are characterized by numerical simulations. It is demonstrated experimentally that the incoherent superposition of the three "complementary speckle patterns" yield a synthetic speckle grain size enlarged by a factor of sqrt[3]. A cyclic permutation of optical vortices and intensity maxima is unexpectedly observed and discussed.

Optical imaging of post-embryonic zebrafish using multi orientation raster scan optoacoustic mesoscopy.

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.

Nondestructive measurement of the roughness of the inner surface of hollow core-photonic bandgap fibers.

We present optical and atomic force microscopy measurements of the roughness of the core wall surface within a hollow core photonic bandgap fiber (HC-PBGF) over the [3×10-2 µm-1-30 µm-1] spatial frequency range. A recently developed immersion optical profilometry technique with picometer-scale sensitivity was used to measure the roughness of air-glass surfaces inside the fiber at unprecedentedly low spatial frequencies, which are known to have the highest impact on HC-PBGF scattering loss and, thus, determine their loss limit. Optical access to the inner surface of the core was obtained by the selective filling of the cladding holes with index matching liquid using techniques borrowed from micro-fluidics. Both measurement techniques reveal ultralow roughness levels exhibiting a 1/f spectral power density dependency characteristic of frozen surface capillary waves over a broad spatial frequency range. However, a deviation from this behavior at low spatial frequencies was observed for the first time, to the best of our knowledge.

High-Resolution Multispectral Optoacoustic Tomography of the Vascularization and Constitutive Hypoxemia of Cancerous Tumors.

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.

Optical mesoscopy without the scatter: broadband multispectral optoacoustic mesoscopy.

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.

Single-side access, isotropic resolution, and multispectral three-dimensional photoacoustic imaging with rotate-translate scanning of ultrasonic detector array.

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.

Influence of nanoscale temperature rises on photoacoustic generation: Discrimination between optical absorbers based on thermal nonlinearity at high frequency.

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 (20 MHz). 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.

Improving photoacoustic-guided optical focusing in scattering media by spectrally filtered detection.

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 photoacoustic 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.

Ultrawideband reflection-mode optoacoustic mesoscopy.

We developed a reflection-mode optoacoustic mesoscopy system, based on raster-scanning of a custom designed spherically focused ultrasound detector, enabling seamless epi-illumination of the volume imaged. We study the performance of acoustic-resolution mesoscopy operating at an ultrawideband bandwidth of 20-180 MHz. i.e., a frequency band spreading over virtually an order of magnitude. Using tomographic reconstruction we showcase previously unreported, to our knowledge, axial resolutions of 4 µm and transverse resolutions of 18 µm reaching depths of up to 5 mm. We further investigate the frequency-dependence of features seen on the images to understand the implications of ultrawideband measurements. We show the overall imaging performance and the frequency ranges that contribute to observable resolution improvements from phantoms and animals.

Light focusing and two-dimensional imaging through scattering media using the photoacoustic transmission matrix with an ultrasound array.

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.

24-MHz scanner for optoacoustic imaging of skin and burn.

Optoacoustic (photoacoustic) imaging uniquely visualizes optical contrast in high resolution and comes with very attractive characteristics for clinical imaging applications. In this paper, we showcase the performance of a scanner based on a 24 MHz center-frequency 128 element array, developed for applications in dermatology. We perform system characterization to examine the imaging performance achieved. We then showcase its imaging ability on healthy tissue and cancer. Finally, we image burns and human lesions in vivo and gain insights on the benefits and challenges of this approach as it is considered for diagnostic and treatment follow-up applications in dermatology and beyond.

Model-based optoacoustic image reconstruction of large three-dimensional tomographic datasets acquired with an array of directional detectors.

Image quality in 3-D optoacoustic (photoacoustic) tomography is greatly influenced by both the measurement system, in particular the number and spatial arrangement of ultrasound sensors, and the ability to account for the spatio-temporal response of the sensor element(s) in the reconstruction algorithm. Herein we present a reconstruction procedure based on the inversion of a time-domain forward model incorporating the spatial impulse response due to the shape of the transducer, which is subsequently applied in a tomographic system based on a translation-rotation scan of a linear detector array. The proposed method was also adapted to cope with the data-intensive requirements of high-resolution volumetric optoacoustic imaging. The processing of 2 · 10 (4) individual signals resulted in well-resolved images of both ~ 200 µm absorbers in phantoms and complex vascular structures in biological tissue. The results reported herein demonstrate that the introduced model-based methodology exhibits a better contrast and resolution than standard back-projection and model-based algorithms that assume point detectors. Moreover, the capability of handling large datasets anticipates that model-based methods incorporating the sensor properties can become standard practice in volumetric opto acoustic image formation.