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High-intensity focused ultrasound (HIFU) is a promising non-invasive therapeutic technique in clinical applications. Challenges in stimulation or ablation HIFU therapy are to accurately target the treatment spot, flexibly deliver or fast-move focus points in the treatment region, and monitor therapy progress in real-time. In this paper, we develop an array-based HIFU system integrated with real-time ultrasound (US) and photoacoustic (PA) imaging. The array-based HIFU transducer can be dynamically focused in a lateral range of ∼16 mm and an axial range of ∼40 mm via electronically adjusting the excitation phase map. To monitor the HIFU therapy progress in real-time, sequential HIFU transmission, PA imaging, PA thermometry, and US imaging are implemented to display the dual-modal images and record the local temperature changes. Co-registered dual-modal images show structural and functional information and thus can guide the HIFU therapy for precise positioning and dosage control. Besides therapy, the multi-element HIFU transducer can also be used to acquire US images to precisely align the imaging coordinates with the HIFU coordinates. Phantom experiments validate the precise and dynamic steering capability of HIFU ablation. We also show that dual-modal imaging can guide HIFU in the designated region and monitor the temperature in biological tissue in real-time.

Ultrasonography is a major medical imaging technique that has been broadly applied in many disease diagnoses. However, due to strong aberration and scattering in the human skull, high-resolution transcranial ultrasonic imaging remains a grand challenge. Here, we explore the rotational-invariant property of ultrasonic speckle and develop high-resolution speckle-scanning ultrasonography to image sub-millimeter-sized features through thick bones. We experimentally validate the rotational invariance of ultrasonic speckle. Based on this property, we scan a random ultrasonic speckle pattern across an object sandwiched between two thick bones so that the object features can be encoded to the ultrasonic waves. After receiving the transmitted ultrasonic waves, we reconstruct the image of the object using an iterative phase retrieval algorithm. We successfully demonstrate imaging of hole and tube features sized as fine as several hundreds of microns between two 0.5 ~ 1-cm-thick bones. With 2.5-MHz excitation and the third-harmonic detection, we measure the spatial resolution as 352 µm. Rotational-invariant speckle-scanning ultrasonography offers a new approach to image through thick bones and paves an avenue towards high-resolution ultrasonic imaging of the human brain.

We present a new spatial compounding method to improve the contrast of ultrasonic images for non-delayed sequential beamforming (NDSB). Sequential beamforming adopts more than one beamformer to reconstruct B-mode images which has the advantage of simple front-end electronics and fast data transfer rate. Via field pattern analysis, we propose a compounding method where two more sub-images can be reconstructed along with the NDSB sub-image. These sub-images can be seen as being produced with different transmit origins, thus, their summation enhances image contrast. Image quality was analyzed in terms of spatial resolution, contrast ratio (CR), and contrast-to-noise ratio (CNR). The proposed compounding method improves the lateral resolution up to 41%. In vitro results confirm a 13.0-dB CR and 4.0-dB CNR improvement. In vivo results reveal 10.9-dB and 6.0-dB improvement in CR and CNR for cross-section jugular vein and 8.0-dB and 4.5-dB improvement in CR and CNR for the longitudinal-section carotid artery.

Hemostasis is vital to save lives, reducing risks of organ failure and hemorrhagic shock. Exploring novel hemostatic materials and precise monitoring of the hemostatic status is of great importance for efficient hemostasis. We present the development of chitosan–graphene oxide-based hemostatic composite and multi-scale photoacoustic evaluation of the hemostatic performance. The hemostatic sponge can quickly and efficiently absorb the blood with its porous cavity and specific surficial property. We inspect the hemostatic performance via an in vitro blood absorption test and in vivo mouse bleeding injury experiments. Results show that the synthesized hemostatic sponge can not only absorb plasma in blood fast with its interior porous structure but also stimulate the interfacial reaction with erythrocytes and platelets. The superiority of multi-scale photoacoustic imaging for guiding, monitoring, and evaluating the hemostatic stages of sponges is demonstrated with high spatial resolution and great sensitivity at depths. Photoacoustic evaluation of a chitosan–graphene oxide-based hemostatic sponge has the potential to be transferred toward the clinical assessment of wound healing.

In nature, animals or plants often use soft organs to move and hunt. Research works on bioinspired materials and devices have attracted more and more interest as which show the potential for future intelligent robots. As key components of soft robots, biomimetic soft actuators are adapted to greater requirements for convenient, accurate, and programmable controlling robots. Here, a class of materials and processing routes of ultrathin actuators are reported for bioinspired piecewise controllable soft robots, where the actuators associate with thermal-responsible soft silicone thin film with thickness as thin as 45 µm and electrically driven by well mechanical designed metallic thin film electrodes. Multiple electrodes in the robots in charge of individual segments control allow the soft robots exhibiting similar functionalities of animals or plants (for example, imitating the tongue of a reptile, such as chameleon to hunt moving preys, and mimicking vines to tightly wind around objects). These bionic results in the soft robots demonstrate their advantages in precise and flexible operation, which provides a good reference for the future research of intelligent soft actuators and robots. acceptedVersion

We report wide-field polygon-scanning functional OR-PAM that for the first time achieves 1-MHz A-line rate of oxygen saturation in vivo. We address two technical challenges. The first is a 1-MHz dual-wavelength pulsed laser that has sufficient pulse energy and ultrafast wavelength switching. The second is a polygon-scanning imaging probe that has a fast scanning speed, a large field of view, and great sensitivity. The OR-PAM system offers a B-scan rate of 477.5 Hz in a 12-mm range and a volumetric imaging rate of ∼1 Hz over a 12 × 5 mm2 scanning area. We image microvasculature and blood oxygen saturation in a 12 × 12 mm2 scanning area in 5 s. Dynamic imaging of oxygen saturation in the mouse ear is demonstrated to monitor fast response to epinephrine injection. The new wide-field fast functional imaging ability broadens the biomedical application of OR-PAM.

Type 1 diabetes (T1D) is an incurable disease that affects 1. 25 million Americans. Diabetic patients typically rely on subcutaneous insulin infusions to regulate their glucose levels. A major contributor to their blood glucose levels is the amount of sugar intake, which cannot be easily tracked. While ultrasound imaging has been used to investigate the relationship between food characteristics and tongue movement, the technique utilized a bulky transducer array that cannot be translated into daily monitoring. Capitalizing on advanced electronics and data processing technologies, we developed a portable system that utilizes only a single ceramic disk to quantify the tongue movement in response to various levels of sweetness. After acquiring 32 subject datasets, we found a significant correlation between food sweetness and tongue movement. Our system can potentially be miniaturized into a wearable device for monitoring sugar intake, which will ultimately help T1D patients to better monitor and control their blood glucose levels and balance their diets accordingly.

Three-dimensional (3D) optical imaging of whole biological organs with microscopic resolution has remained a challenge. Most versions of such imaging techniques require special preparation of the tissue specimen. Here we demonstrate microtomy-assisted photoacoustic microscopy (mPAM) of mouse brains and other organs, which automatically acquires serial distortion-free and registration-free images with endogenous absorption contrasts. Without tissue staining or clearing, mPAM generates micrometer-resolution 3D images of paraffin- or agarose-embedded whole organs with high fidelity, achieved by label-free simultaneous sensing of DNA/RNA, hemoglobins, and lipids. mPAM provides histology-like imaging of cell nuclei, blood vessels, axons, and other anatomical structures, enabling the application of histopathological interpretation at the organelle level to analyze a whole organ. Its deep tissue imaging capability leads to less sectioning, resulting in negligible sectioning artifact. mPAM offers a new way to better understand complex biological organs., The state-of-the-art three-dimensional biomedical imaging often requires specific tissue preparation that may alter the physical properties of the specimen causing loss of information. Here Wong et al. develop a microtomy-assisted photoacoustic microscopy that allows imaging of biological samples without labelling agents and with reduced sectioning.

Imaging of small animals has played an indispensable role in preclinical research by providing high-dimensional physiological, pathological and phenotypic insights with clinical relevance. Yet, pure optical imaging suffers from either shallow penetration (up to ~1–2 mm) or a poor depth-to-resolution ratio (~3), and non-optical techniques for whole-body imaging of small animals lack either spatiotemporal resolution or functional contrast. Here, we demonstrate that stand-alone single-impulse panoramic photoacoustic computed tomography (SIP-PACT) mitigates these limitations by combining high spatiotemporal resolution (125 μm in-plane resolution, 50 μs per frame data acquisition and 50 Hz frame rate), deep penetration (48 mm cross-sectional width in vivo), anatomical, dynamical and functional contrasts, and full-view fidelity. Using SIP-PACT, we imaged in vivo whole-body dynamics of small animals in real time and obtained clear sub-organ anatomical and functional details. We tracked unlabelled circulating melanoma cells and imaged the vasculature and functional connectivity of whole rat brains. SIP-PACT holds great potential for both preclinical imaging and clinical translation.

Optical-resolution photoacoustic microscopy (OR-PAM) offers label-freeitalicin vivo/italicimaging with high spatial resolution by acoustically detecting optical absorption contrasts via the photoacoustic effect. We developed a compact handheld OR-PAM probe for fast photoacoustic imaging. Different from benchtop microscopes, the handheld probe provides flexibility in imaging various anatomical sites. Resembling a cup in size, the probe uses a two-axis water-immersible microelectromechanical system mirror to scan both the illuminating optical beam and resultant acoustic beam. The system performance was tested in vivo by imaging the capillary bed in a mouse ear and both the capillary bed and a mole on a human volunteer.

As a window on the microcirculation, human cuticle capillaries provide rich information about the microvasculature, such as its morphology, density, dimensions, or even blood flow speed. Many imaging technologies have been employed to image human cuticle microvasculature. However, almost none of these techniques can noninvasively observe the process of oxygen release from single red blood cells (RBCs), an observation which can be used to study healthy tissue functionalities or to diagnose, stage, or monitor diseases. For the first time, we adapted single-cell resolution photoacoustic (PA) microscopy (PA flowoxigraphy) to image cuticle capillaries and quantified multiple functional parameters. Our results show more oxygen release in the curved cuticle tip region than in other regions of a cuticle capillary loop, associated with a low of RBC flow speed in the tip region. Further analysis suggests that in addition to the RBC flow speed, other factors, such as the drop of the partial oxygen pressure in the tip region, drive RBCs to release more oxygen in the tip region.

The short focal depth of a Gaussian beam limits the volumetric imaging speed of optical resolution photoacoustic microscopy (OR-PAM). A Bessel beam, which is diffraction free, provides a long focal depth, but its side lobes deteriorate image quality when the Bessel beam is directly employed to excite photoacoustic (PA) signals in OR-PAM. We present a nonlinear approach based on the Grueneisen relaxation effect to suppress the side-lobe artifacts in PA imaging. This method extends the focal depth of OR-PAM and speeds up volumetric imaging. We experimentally demonstrated a 1-mm focal depth with a 7-μm lateral resolution and volumetrically imaged a carbon fiber and red blood cell samples.

Photoacoustic (PA) imaging of arbitrarily-shaped or oriented objects may miss important features because PA waves propagate normal to structure boundaries and may miss the acoustic detectors when the detection view has a limited angular range. To overcome this long-standing problem, we present an ultrasonic thermal encoding approach that is universally applicable. We exploit the temperature dependence of the Grueneisen parameter and encode a confined [[What does confined mean here?]] voxel using heat generated by a focused ultrasonic transducer. The PA amplitude from the encoded voxel is increased while those from the neighboring voxels are unchanged. Consequently, the amplitude-increased PA waves propagate in all directions due to the round cross-section of the encoded region and thus can be received at any viewing angle on the cross-sectional plane [[Please check throughout the manuscript for similar places.]]. We built a mathematical model for the thermally encoded PA tomography, performed a numerical simulation, and experimentally validated the ultrasonic thermal encoding efficiency. As a proof of concept, we demonstrate full-view in vivo vascular imaging and compare it to the original linear-array PA tomography system, showing dramatically enhanced imaging of arbitrarily oriented blood vessels. Since ultrasonic heating can be focused deeply, this method can be applied to deep tissue imaging and is promising for full-view imaging of other features of biomedical interest, such as tumor margins.

We present fast functional photoacoustic microscopy (PAM), which is capable of three-dimensional high-resolution high-speed imaging of the mouse brain, complementary to other imaging modalities. A single-wavelength pulse-width-based method was implemented to image blood oxygenation with capillary-level resolution and a one-dimensional imaging rate of 100 kHz. We applied PAM to image the vascular morphology, blood oxygenation, blood flow, and oxygen metabolism in the brain in both resting and stimulated states.

Combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for super-resolution photoacoustic imaging of both fluorescent and non-fluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. By scanning the excitation beam, we performed three-dimensional sub-diffraction imaging of varied fluorescent and non-fluorescent species. A lateral resolution of 80 nm and an axial resolution of 370 nm have been demonstrated. This technique has the potential to enable label-free super-resolution imaging, and can be transferred to other optical imaging modalities or combined with other super-resolution methods.

The temperature-dependent property of the Grueneisen parameter has been employed in photoacoustic imaging mainly to measure tissue temperature. Here we explore this property using a different approach and develop Grueneisen relaxation photoacoustic microscopy (GR-PAM), a technique that images nonradiative absorption with confocal optical resolution. GR-PAM sequentially delivers two identical laser pulses with a microsecond-scale time delay. The first laser pulse generates a photoacoustic signal and thermally tags the in-focus absorbers. When the second laser pulse excites the tagged absorbers within the thermal relaxation time, a photoacoustic signal stronger than the first one is produced, owing to the temperature dependence of the Grueneisen parameter. GR-PAM detects the amplitude difference between the two colocated photoacoustic signals, confocally imaging the nonradiative absorption. We greatly improved axial resolution from 45 μm to 2.3 μm and, at the same time, slightly improved lateral resolution from 0.63 μm to 0.41 μm. In addition, the optical sectioning capability facilitates the measurement of the absolute absorption coefficient without fluence calibration.

Super-resolution microscopy techniques - capable of overcoming the diffraction limit of light - have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.

We present an optically encoded photoacoustic flow imaging method based on optical-resolution photoacoustic microscopy. An intensity-modulated continuous-wave laser photothermally encodes the flowing medium, and a pulsed laser generates photoacoustic waves to image the encoded heat pattern. Flow speeds can be calculated by cross-correlation. The method was validated in phantoms, at flow speeds ranging from 0.23 mm/s to 11 mm/s. Venous blood flow speed in a mouse ear was also measured.

Combined optical and mechanical scanning (COMS) in optical-resolution photoacoustic microscopy (OR-PAM) has provided five scanning modes with fast imaging speed and wide field of view (FOV). With two-dimensional (2D) galvanometer-based optical scanning, we have achieved a 2 KHz B-scan rate and 50 Hz volumetric-scan rate, which enables real-time tracking of cell activities in vivo . With optical-mechanical hybrid 2D scanning, we are able to image a wide FOV (10×8 mm 2 ) within 150 seconds, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechani cal-based contour scanning, we can maintain the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects w ith uneven surfaces, which is ideal for fast and quantitative studies of tumors and the brain. Keywords: Medical and biological imaging, Photoacoustic imaging, Scanning microscopy, Three-dimensional microscopy. 1. INTRODUCTION Optical-resolution photoacoustic microscopy (OR-PAM), based on the photoacoustic effect [1], detects optical absorption contrasts with greater sensitivity than all other optical imaging modalities [2]. The high detection efficiency of intrinsic optical absorbers (e.g., oxy-hemoglobin (HbO2) and deoxy-hemoglobin (HbR) [3, 4]) enables OR-PAM to provide label-free high-resolution [5, 6] functional imaging of fine structures [7]. OR-PAM has already demonstrated broad biomedical applications in the fields of neurology [8], ophthalmology [9-12], and vascular biology [13]. However, the imaging speed of second-generation (G2) OR-PAM is insufficient for tracking certain fast dynamic activities in vivo [14]. Here, we report a combination of optical and mechanical scanning OR -PAM (COMS-OR-PAM), which provides five scanning modes with fast imaging speed and a wide field of view (FOV).

Sub-diffraction optical microscopy allows the imaging of cellular and subcellular structures with resolution finer than the diffraction limit. Here, combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for sub-diffraction photoacoustic imaging of both fluorescent and non-fluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. The differential signal between the two excitations preserves the signal contribution mostly from the center of the excitation volume, and dramatically sharpens the lateral resolution. Moreover, due to the nonlinear nature of the signal, our method offers inherent optical sectioning capability, which is lacking in conventional photoacoustic microscopy. By scanning the excitation beam, we performed three-dimensional sub-diffraction imaging of varied fluorescent and non-fluorescent species. As any molecules have absorption, this technique has the potential to enable label-free sub-diffraction imaging, and can be transferred to other optical imaging modalities or combined with other sub-diffraction methods.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technique that directly images optical absorption in tissue at high spatial resolution. To date, the majority of OR-PAM systems are based on single focused optical excitation and ultrasonic detection, limiting the wide-field imaging speed. While one-dimensional multifocal OR-PAM (1D-MFOR-PAM) has been developed, the potential of microlens and transducer arrays has not been fully realized. Here, we present the development of two-dimensional multifocal optical-resolution photoacoustic computed microscopy (2D-MFOR-PACM), using a 2D microlens array and a full-ring ultrasonic transducer array. The 10 × 10 mm2 microlens array generates 1800 optical foci within the focal plane of the 512-element transducer array, and raster scanning the microlens array yields optical-resolution photoacoustic images. The system has improved the in-plane resolution of a full-ring transducer array from ≥100 µm to 29 µm and achieved an imaging time of 36 seconds over a 10 × 10 mm2 field of view. In comparison, the 1D-MFOR-PAM would take more than 4 minutes to image over the same field of view. The imaging capability of the system was demonstrated on phantoms and animals both ex vivo and in vivo.

We report the development of photoacoustic flowmetry assisted by high-intensity focused ultrasound (HIFU). This novel method employs HIFU to generate a heating impulse in the flow medium, followed by photoacoustic monitoring of the thermal decay process. Photoacoustic flowmetry in a continuous medium remains a challenge in the optical diffusive regime. Here, both the HIFU heating and photoacoustic detection can focus at depths beyond the optical diffusion limit (~1 mm in soft tissue). This method can be applied to a continuous medium, i.e., a medium without discrete scatterers or absorbers resolvable by photoacoustic imaging. Flow speeds up to 41 mm⋅s^(-1) have been experimentally measured in a blood phantom covered by 1.5-mm-thick tissue.

Photoacoustic tomography (PAT) is a hybrid imaging technique that has broad preclinical and clinical applications. Based on the photoacoustic effect, PAT directly measures specific optical absorption, which is the product of the tissue-intrinsic optical absorption coefficient and the local optical fluence. Therefore, quantitative PAT, such as absolute oxygen saturation (sO_2) quantification, requires knowledge of the local optical fluence, which can only be estimated through invasive measurements or sophisticated modeling of light transportation. In this Letter, we circumvent this requirement by taking advantage of the dynamics in sO_2. The new method works when the sO_2 transition can be simultaneously monitored with multiple wavelengths. For each wavelength, the ratio of photoacoustic amplitudes measured at different sO_2 states is utilized. Using the ratio cancels the contribution from optical fluence and allows calibration-free quantification of absolute sO_2. The new method was validated through both phantom and in vivo experiments.

Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO 2 ) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (C Hb ), oxygen saturation (sO 2 ), sO 2 gradient ( ), flow speed (v f ), and oxygen release rate (rO 2 ), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO 2 , , v f , and rO 2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications.

Photoacoustic microscopy (PAM), whose spatial resolution and penetration depth are both scalable, has made great progress in recent years. According to their different lateral resolutions, PAM systems can be categorized into either optical-resolution (OR) PAM, with optical-diffraction-limited lateral resolution, or acoustic-resolution (AR) PAM, with acoustically limited resolution and a deeper maximum imaging depth. In this report, we present a combined OR and AR PAM system with resolutions of 2.2 μm and 40 μm, respectively. Sharing most components between the OR and AR implementations, the system achieves separated illumination for OR and AR imaging by an optical fiber bundle through different channels, and two discrete lasers are used to provide either high-power energy for AR imaging or highrepetition- rate pulses for OR imaging. The design enables automatically co-registered OR and AR photoacoustic imaging in one single system, which extends the usability of current photoacoustic systems and simplifies the imaging procedure.

By offering images with high spatial resolution and unique optical absorption contrast, optical-resolution photoacoustic microscopy (OR-PAM) has gained increasing attention in biomedical research. Recent developments in OR-PAM have improved its imaging speed, but have sacrificed either the detection sensitivity or field of view or both. We have developed a wide-field fast-scanning OR-PAM by using a water-immersible MEMS scanning mirror (MEMS-ORPAM). Made of silicon with a gold coating, the MEMS mirror plate can reflect both optical and acoustic beams. Because it uses an electromagnetic driving force, the whole MEMS scanning system can be submerged in water. In MEMS-ORPAM, the optical and acoustic beams are confocally configured and simultaneously steered, which ensures uniform detection sensitivity. A B-scan imaging speed as high as 400 Hz can be achieved over a 3 mm scanning range. A diffraction-limited lateral resolution of 2.4 μm in water and a maximum imaging depth of 1.1 mm in soft tissue have been experimentally determined. Using the system, we imaged the flow dynamics of both red blood cells and carbon particles in a mouse ear in vivo. By using Evans blue dye as the contrast agent, we also imaged the flow dynamics of lymphatic vessels in a mouse tail in vivo. The results show that MEMS-OR-PAM could be a powerful tool for studying highly dynamic and time-sensitive biological phenomena.