Lihong Wang earned his Ph.D. degree at Rice University, Houston, Texas under the tutelage of Robert Curl, Richard Smalley, and Frank Tittel. He is Bren Professor of Medical Engineering and Electrical Engineering at California Institute of Technology. His book entitled “Biomedical Optics: Principles and Imaging,” one of the first textbooks in the field, won the 2010 Joseph W. Goodman Book Writing Award. He also edited the first book on photoacoustic tomography. He has published 495 peer-reviewed articles in journals including Nature and Science and delivered 500 keynote, plenary, or invited talks. His Google Scholar h-index and citations have reached 122 and 61,000, respectively. His laboratory was the first to report functional photoacoustic tomography, 3D photoacoustic microscopy, photoacoustic endoscopy, photoacoustic reporter gene imaging, the photoacoustic Doppler effect, the universal photoacoustic reconstruction algorithm, microwave-induced thermoacoustic tomography, ultrasound-modulated optical tomography, time-reversed ultrasonically encoded (TRUE) optical focusing, nonlinear photoacoustic wavefront shaping (PAWS), compressed ultrafast photography (10 trillion frames/s, world’s fastest camera), Mueller-matrix optical coherence tomography, and optical coherence computed tomography. He was the Editor-in-Chief of the Journal of Biomedical Optics. He received the NIH’s FIRST, NSF’s CAREER, NIH Director’s Pioneer, and NIH Director’s Transformative Research awards. He also received the OSA C.E.K. Mees Medal, IEEE Technical Achievement Award, IEEE Biomedical Engineering Award, SPIE Britton Chance Biomedical Optics Award, Senior Prize of the International Photoacoustic and Photothermal Association, and OSA Michael S. Feld Biophotonics Award. He is a Fellow of the AIMBE, Electromagnetics Academy, IEEE, OSA, and SPIE. An honorary doctorate was conferred on him by Lund University, Sweden. He was inducted into the National Academy of Engineering.
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Digital optical phase conjugation (DOPC) enables many optical applications by permitting focusing of light through scattering media. However, DOPC systems require precise alignment of all optical components, particularly of the spatial light modulator (SLM) and camera, in order to accurately record the wavefront and perform playback through the use of time-reversal symmetry. We present a digital compensation technique to optimize the alignment of the SLM in five degrees of freedom, permitting focusing through thick scattering media with a thickness of 5 mm and transport scattering coefficient of 2.5 mm − 1 while simultaneously improving focal quality, as quantified by the peak-to-background ratio, by several orders of magnitude over an unoptimized alignment.
Optical-resolution photoacoustic microscopy (OR-PAM) has demonstrated fast, label-free volumetric imaging of optical-absorption contrast within the quasiballistic regime of photon scattering. However, the limited numerical aperture of the ultrasonic transducer restricts the detectability of the photoacoustic waves, thus resulting in incomplete reconstructed features. To tackle the limited-view problem, we added an oblique illumination beam to the original coaxial optical-acoustic scheme to provide a complementary detection view. The virtual augmentation of the detection view was validated through numerical simulations and tissue-phantom experiments. More importantly, the combination of top and oblique illumination successfully imaged a mouse brain in vivo down to 1 mm in depth, showing detailed brain vasculature. Of special note, it clearly revealed the diving vessels that were long missing in images from original OR-PAM.
We have developed a single-breath-hold photoacoustic computed tomography (SBH-PACT) system to detect tumors and reveal detailed angiographic information about human breasts. SBH-PACT provides high spatial and temporal resolutions with a deep in vivo penetration depth of over 4 cm. A volumetric breast image can be acquired by scanning the breast within a single breath hold (~15 sec). We imaged a healthy female volunteer and seven breast cancer patients. SBH-PACT clearly identified all tumors by revealing higher blood vessel densities and lower compliance associated with the tumors
Conventional photoacoustic computed tomography (PACT) images the spatial distribution of optical absorption, which is approximated as an isotropic optical property. The optical absorption of many biological tissues, however, is anisotropic. This anisotropy, known as dichroism or diattenuation, encodes rich information about molecular conformation and structural alignment. Here we report a novel imaging method called dichroism-sensitive PACT (DS-PACT). Using a lock-detection strategy, our method can measure the amplitude of tissue’s dichroism and the orientation of the optic axis of uniaxial dichroic tissue, even at a depth of 3.25 transport mean free paths. We experimentally demonstrated DS-PACT by imaging plastic polarizers and ex vivo bovine tendons deep inside scattering media. Our method extends the functionality of PACT to include a new capability, imaging tissue absorption anisotropy.
Accurate estimation of the initial pressure distribution in photoacoustic computed tomography (PACT) requires some knowledge of the sound speed distribution. However, the sound speed distribution is typically unknown. Further, the initial pressure and sound speed distributions cannot both, in general, be stably recovered from PACT measurements alone. In this work, a joint reconstruction method for the initial pressure distribution and a low-dimensional parameterized model of the sound speed distribution is proposed. By employing a priori information about the structure of the sound speed distribution, both the initial pressure and sound speed can be accurately recovered. The joint reconstruction problem is solved by use of a proximal optimization method that allows constraints and non-smooth regularization functions for initial pressure distribution. The gradients of the cost function with respect to the initial pressure and sound speed distributions are calculated by use of an adjoint state method that has the same per-iteration computational cost as calculating the gradient with respect to the initial pressure distribution alone. This approach is quantitatively evaluated through 2D computer-simulation studies for a small animal imaging model. The impact of the choice of the parameterized sound speed model is investigated. Even when the assumed parameterized sound speed model is inconsistent with the true sound speed distribution, the estimated initial pressure distribution is more accurate than that obtained by assuming a constant sound speed. The utility of the proposed approach is also demonstrated through application to experimental in vivo measurements of a mouse.
In cancer research, regions of increasingly lowered oxygenation in tissue (hypoxia), which are due to tumor development, are considered to play an important role in activating various signaling pathways that facilitate further development of the cancer. However, devising a minimally invasive method to monitor tissue oxygenation has remained a challenge. Photoacoustic microscopy has been posed as a solution in a variety of preclinical research studies. Here, using optical-resolution photoacoustic microscopy (OR-PAM), for the first time, we non-invasively measured oxygenation and vascularization in vivo caused by multiple myeloma (MM) progression. Mice injected with MM cells tagged with green fluorescent protein were monitored with a fluorescence microscope for tumor progression over the course of 28 days. OR-PAM evaluated the oxygen saturation (sO2) and the blood vessel density in the cerebral bone marrow, where MM cells home. At 28 days after the injection of MM cells, the total sO2 had dropped by 50% in the developing tumor regions, while in the non-tumor developing regions it had dropped by 20% compared with the value at one day after MM injection. The blood vessel density had dropped by 35% in the tumor developing regions, while in the non-tumor developing regions it had dropped by 8% compared with the value at one day after MM injection. In summary, non-invasive measurement by OR-PAM correlated the development of hypoxia with to MM progression. It revealed decreased vascularization surrounding the tumor areas, which we feel can be ascribed to the rapid tumor progression.
Wavefront shaping techniques are being actively developed to achieve optical focusing through and inside opaque scattering media. These techniques promise to revolutionize biophotonics by enabling deep-tissue non-invasive optical imaging, optogenetics, optical tweezing, and light-based therapy. Among the existing wavefront shaping techniques, optical time-reversal-based techniques determine the optimum wavefront globally based on the principle of time reversal, without the need to perform time-consuming iterations to optimize each mode in sequence. In all previous optical time-reversal-based wavefront shaping experiments, Nyquist sampling criterion was followed so that the scattered light field was well-sampled during wavefront measurement and wavefront reconstruction. In this work, we overturn this conventional practice by demonstrating that a high-quality optical focus can still be achieved even when the scattered light field is under-sampled. Even more strikingly, we show both theoretically and experimentally that the focus achieved by the under-sampling scheme can be one order of magnitude brighter than that achieved by the well-sampling schemes used in previous works, where 3×3 to 5×5 pixels sampled one speckle grain on average. Moreover, since neighboring pixels were uncorrelated in feedback-based wavefront shaping, introducing the concept of sub-Nyquist sampling in time-reversal-based wavefront shaping makes the optimal phase maps obtained using these two different methods consistent. We anticipate that this newly explored under-sampling scheme will transform the understanding of optical time reversal and boost the performance of optical imaging, manipulation, and communication through opaque scattering media.
Ultraviolet photoacoustic microscopy (UV-PAM) is a promising intraoperative tool for surgical margin assessment (SMA), one that can provide label-free histology-like images with high resolution. In this study, using a microlens array and a one-dimensional (1-D) array ultrasonic transducer, we developed a high-throughput multifocal UV-PAM (MF-UV-PAM). Our new system achieved a 1.6 ± 0.2 μm lateral resolution and produced images 40 times faster than the previously developed point-by-point scanning UV-PAM. MF-UV-PAM provided a readily comprehensible photoacoustic image of a mouse brain slice with specific absorption contrast in ∼16 min, highlighting cell nuclei. Individual cell nuclei could be clearly resolved, showing its practical potential for intraoperative SMA.
Photoacoustic tomography (PAT), combining optical and ultrasonic waves via the photoacoustic effect, provides in vivo functional, metabolic, molecular, and histologic imaging. PAT has the unique strength of high-resolution imaging across the length scales of organelles, cells, tissues, and organs with consistent contrast. PAT has the potential to empower multiscale biology research and accelerate translation from microscopic laboratory discoveries to macroscopic clinical practice. PAT may also hold the key to the earliest detection of cancer by in vivo label-free quantification of hypermetabolism, the quintessential hallmark of cancer. Broad applications include imaging of the breast, brain, skin, esophagus, colon, vascular system, and lymphatic system in both humans and animals.
In biological applications, optical focusing is limited by the diffusion of light, which prevents focusing at depths greater than ~1 mm in soft tissue. Wavefront shaping aims to extend the focusing depth by compensating for phase distortions induced by scattering. This allows for focusing light through biological tissue beyond the optical diffusion limit through constructive interference. However, due to random motion, scattering of light in tissue is deterministic only within a brief speckle correlation time. In in vivo tissue this speckle correlation time is on the order of milliseconds, thus it is vital to optimize the wavefront within the correlation time.
The speed of wavefront shaping has typically been limited by the time required to measure and display the optimal phase pattern due to the low speeds of cameras, data transfer and processing, and spatial light modulators (SLM). While methods of binary-phase modulation requiring only two images for phase measurement have recently been reported, the majority of studies require a minimum of four frames for full-phase measurement. Here, we present a full-phase digital optical phase conjugation method based on off-axis holography for single-shot optical focusing through scattering media. By using off-axis holography in conjunction with graphics processing unit (GPU) based processing; we take advantage of single-shot full-phase measurement while using parallel computation to quickly reconstruct the phase map. Using this system, we are able to focus light through scattering media with a system latency of approximately 10 milliseconds, on the order of the in vivo speckle correlation time.
Photoacoustic tomography (PAT), combining optical and ultrasonic waves via the photoacoustic effect, provides in vivo functional, metabolic, molecular, and histologic imaging. PAT has the unique strength of high-resolution imaging across the length scales of organelles, cells, tissues, and organs with consistent contrast. PAT has the potential to empower multiscale biology research and accelerate translation from microscopic laboratory discoveries to macroscopic clinical practice. PAT can image the entire brain of a rat or mouse with optical contrast in vivo. Broad applications include imaging of the breast, brain, skin, esophagus, colon, vascular system, and lymphatic system in both humans and animals.
Focusing light deep inside and through thick biological tissue is critical to many applications. However, optical scattering prevents light from being focused through thick biological tissue, which restricts biophotonics to a limited depth of about 1 mm. To break this optical diffusion limit, digital optical phase conjugation (DOPC) based wavefront shaping techniques are being actively developed. Previous DOPC systems employed spatial light modulators that modulated either the phase or the amplitude of the conjugate light field. Here, we achieve optical focusing through scattering media by using polarization modulation based generalized DOPC. First, we describe an algorithm to extract the polarization map from the measured scattered field. Then, we validate the algorithm through numerical simulations, and find the focusing contrast achieved by polarization modulation is similar to that achieved by phase modulation, and is higher than those achieved by binary-phase and binary-amplitude modulations. Finally, we build a system using an inexpensive twisted nematic liquid crystal based spatial light modulator, and experimentally demonstrate light focusing through 3-mm thick chicken breast tissue. Since the polarization modulation based SLMs are widely used in displays and are having more and more pixel counts with the prevalence of 4K displays, these SLMs are inexpensive and valuable devices for wavefront shaping. Thus, we anticipate that polarization modulation based SLMs will gain their prevalence in the field of wavefront shaping.
The development of photoacoustic computed tomography (PACT) for neuroimaging in humans will fill an important void left by available imaging techniques. However, due to the presence of the skull, accurate image reconstruction in transcranial PACT remains challenging. Variations in the shear and longitudinal wave speed distributions due to the skull can induce strong aberrations in the measured photoacoustic wavefields. To mitigate these artifacts, image reconstruction methods in transcranial PACT require knowledge of these acoustic properties. However, such information may be difficult to obtain in practice. To circumvent this, we developed a joint reconstruction (JR) method for transcranial PACT where the longitudinal and shear speed distributions are reconstructed concurrently with the sought-after initial pressure distribution.
The joint estimation of the initial pressure, longitudinal speed, and shear speed distributions from PACT data alone is unstable. To overcome this instability, we propose to incorporate prior information about the acoustic properties of the skull. Specifically, a low-dimensional parameterized acoustic representation of the skull is established with the aid of adjunct CT data. The use of a low-dimensional representation of the acoustic skull parameters effectively overcomes the instability of the JR problem and allows stable reconstruction of the acoustic skull parameters and the initial pressure distribution concurrently.
To validate the proposed method, we conducted 3D numerical studies based on realistic human skull models derived from adjunct CT data. The efficacy of the proposed JR method was demonstrated through accurate reconstruction of the initial pressure, longitudinal speed, and shear speed distributions from PACT measurement data alone.
In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming for large specimens. Thus, 20-60% of patients undergoing BCS still require second surgeries due to positive surgical margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional histology and measuring features related to the histological landscape without the need for labels. We demonstrate that our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast tissue.
In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.
Photoacoustic computed tomography (PACT) is a non-invasive imaging technique offering high contrast, high resolution, and deep penetration in biological tissues. We report a photoacoustic computed tomography (PACT) system equipped with a high frequency linear array for anatomical and functional imaging of the mouse whole brain. The linear array was rotationally scanned in the coronal plane to achieve the full-view coverage. We investigated spontaneous neural activities in the deep brain by monitoring the hemodynamics and observed strong interhemispherical correlations between contralateral regions, both in the cortical layer and in the deep regions.
Normal development of the visual system in infants relies on clear images being projected onto the retina, which can be disrupted by lens opacity caused by congenital cataract. This disruption, if uncorrected in early life, results in amblyopia (permanently decreased vision even after removal of the cataract). Doctors are able to prevent amblyopia by removing the cataract during the first several weeks of life, but this surgery risks a host of complications, which can be equally visually disabling. Here, we investigated the feasibility of focusing light noninvasively through highly scattering cataractous lenses to stimulate the retina, thereby preventing amblyopia. This approach would allow the cataractous lens removal surgery to be delayed and hence greatly reduce the risk of complications from early surgery. Employing a wavefront shaping technique named time-reversed ultrasonically encoded optical focusing in reflection mode, we focused 532-nm light through a highly scattering ex vivo adult human cataractous lens. This work demonstrates a potential clinical application of wavefront shaping techniques.
Based on the photoacoustic (PA) effect, PA tomography directly measures specific optical absorption, i.e., absorbed optical energy per unit volume. We recently developed a full-ring ultrasonic transducer array-based photoacoustic computed tomography (PACT) system for small-animal whole-body imaging. The system has a full-view detection angle and high in-plane resolution (∼100 μm). However, due to the bandpass frequency response of the piezoelectric transducer elements and the limited elevational detection coverage of the full-ring transducer array, the reconstructed images present bipolar (i.e., both positive and negative) pixel values, which cause ambiguities in image interpretation for physicians and biologists. We propose a multiview Hilbert transformation method to recover the unipolar initial pressure for full-ring PACT. The effectiveness of the proposed algorithm was first validated by numerical simulations and then demonstrated with ex vivo mouse brain structural imaging and in vivo mouse whole-body imaging.
Optical phase conjugation based wavefront shaping techniques are being actively developed to focus light through or inside scattering media such as biological tissue, and they promise to revolutionize optical imaging, manipulation, and therapy. The speed of digital optical phase conjugation (DOPC) has been limited by the low speeds of cameras and spatial light modulators (SLMs), preventing DOPC from being applied to thick living tissue. Recently, a fast DOPC system was developed based on a single-shot wavefront measurement method, a field programmable gate array (FPGA) for data processing, and a digital micromirror device (DMD) for fast modulation. However, this system has the following limitations. First, the reported single-shot wavefront measurement method does not work when our goal is to focus light inside, instead of through, scattering media. Second, the DMD performed binary amplitude modulation, which resulted in a lower focusing contrast compared with that of phase modulations. Third, the optical fluence threshold causing DMDs to malfunction under pulsed laser illumination is lower than that of liquid crystal based SLMs, and the system alignment is significantly complicated by the oblique reflection angle of the DMD. Here, we developed a simple but high-speed DOPC system using a ferroelectric liquid crystal based SLM (512 × 512 pixels), and focused light through three diffusers within 4.7 ms. Using focused-ultrasound-guided DOPC along with a double exposure scheme, we focused light inside a scattering medium containing two diffusers within 7.7 ms, thus achieving the fastest digital time-reversed ultrasonically encoded (TRUE) optical focusing to date.
Photoacoustic microscopy (PAM) has been extensively applied in biomedical study because of its ability to visualize tissue morphology and physiology in vivo in three dimensions (3D). However, conventional PAM suffers from a rapidly decreasing resolution away from the focal plane because of the limited depth of focus of an objective lens, which deteriorates the volumetric imaging quality inevitably. Here, we propose a novel method to synthesize an ultra-long light needle to extend a microscope’s depth of focus beyond its physical limitations with wavefront engineering method. Furthermore, it enables an improved lateral resolution that exceeds the diffraction limit of the objective lens. The virtual light needle can be flexibly synthesized anywhere throughout the imaging volume without mechanical scanning. Benefiting from these advantages, we developed a synthetic light needle photoacoustic microscopy (SLN-PAM) to achieve an extended depth of field (DOF), sub-diffraction and motionless volumetric imaging. The DOF of our SLN-PAM system is up to 1800 µm, more than 30-fold improvement over that gained by conventional PAM. Our system also achieves the lateral resolution of 1.8 µm (characterized at 532 nm and 0.1 NA objective), about 50% higher than the Rayleigh diffraction limit. Its superior imaging performance was demonstrated by 3D imaging of both non-biological and biological samples. This extended DOF, sub-diffraction and motionless 3D PAM will open up new opportunities for potential biomedical applications.
The brain has been likened to a great stretch of unknown territory consisting of a number of unexplored continents. Small animal brain imaging plays an important role charting that territory. By using 1064 nm illumination from the side, we imaged the full coronal depth of rat brains in vivo. The experiment was performed using a real-time full-ring-array photoacoustic computed tomography (PACT) imaging system, which achieved an imaging depth of 11 mm and a 100 μm radial resolution.
Because of the fast imaging speed of the full-ring-array PACT system, no animal motion artifact was induced. The frame rate of the system was limited by the laser repetition rate (50 Hz). In addition to anatomical imaging of the blood vessels in the brain, we continuously monitored correlations between the two brain hemispheres in one of the coronal planes. The resting states in the coronal plane were measured before and after stroke ligation surgery at a neck artery.
Metastasis is responsible for as many as 90% of cancer-related deaths, and the deadliest skin cancer, melanoma, has a high propensity for metastasis. Since hematogenous spread of circulating tumor cells (CTCs) is cancer’s main route of metastasis, detecting and destroying CTCs can impede metastasis and improve patients’ prognoses. Extensive studies employing exogenous agents to detect tumor-specific biomarkers and guide therapeutics to CTCs have achieved promising results, but biosafety remains a critical concern. Taking another approach, physical detection and destruction of CTCs is a safer way to evaluate and reduce metastasis risks. Melanoma cells strongly express melanosomes, providing a striking absorption contrast with the blood background in the red to near-infrared spectrum. Exploiting this intrinsic optical absorption contrast of circulating melanoma cells, we coupled dual-wavelength photoacoustic flow cytography with a nanosecond-pulsed laser killing mechanism that specifically targets melanoma CTCs. We have successfully achieved in vivo label-free imaging of rare single CTCs and CTC clusters in mice. Further, the photoacoustic signal from a CTC immediately hardware-triggers a lethal pinpoint laser irradiation that lyses it on the spot in a thermally confined manner. Our technology can facilitate early inhibition of metastasis by clearing circulating tumor cells from vasculature.
Imaging of small animals, especially rodents provides physiological, pathological, and phenotypical insights into the most relevant milieu—an intact, living system. Currently, non-optical small-animal wholebody imaging approaches lack either spatiotemporal resolution or functional contrasts, whereas pure optical imaging suffers from either shallow penetration (up to ~1 mm) or a poor resolution-to-depth ratio (~1/3). Here, we present a standalone system that breaks all the above limitations. Our system features high spatiotemporal resolution and deep penetration, and can capture anatomical and functional contrasts. We imaged mouse wholebody dynamics in real time with clear sub-organ anatomical and functional details.
Photoacoustic computed tomography (PACT) is an emerging computed imaging modality that exploits optical contrast and ultrasonic detection principles to form images of the photoacoustically induced initial pressure distribution within tissue. The PACT reconstruction problem corresponds to a time-domain inverse source problem, where the initial pressure distribution is recovered from the measurements recorded on an aperture outside the support of the source. A major challenge in transcranial PACT brain imaging is to compensate for aberrations in the measured data due to the propagation of the photoacoustic wavefields through the skull. To properly account for these effects, a wave equation-based inversion method should be employed that can model the heterogeneous elastic properties of the medium. In this study, an iterative image reconstruction method for 3D transcranial PACT is developed based on the elastic wave equation. To accomplish this, a forward model based on a finite-difference time-domain discretization of the elastic wave equation is established. Subsequently, gradient-based methods are employed for computing penalized least squares estimates of the initial source distribution that produced the measured photoacoustic data. The developed reconstruction algorithm is validated and investigated through computer-simulation studies.
Photoacoustic computed tomography (PACT) is an emerging computed imaging modality that exploits optical contrast and ultrasonic detection principles to form images of the absorbed optical energy density within tissue. If the object possesses spatially variant acoustic properties that are unaccounted for by the reconstruction method, the estimated image can contain distortions. While reconstruction methods have recently been developed to compensate for this effect, they generally require the object’s acoustic properties to be known a priori. To circumvent the need for detailed information regarding an object’s acoustic properties, we previously proposed a half-time reconstruction method for PACT. A half-time reconstruction method estimates the PACT image from a data set that has been temporally truncated to exclude the data components that have been strongly aberrated. However, this method can be improved upon when the approximate sizes and locations of isolated heterogeneous structures, such as bones or gas pockets, are known. To address this, we investigate PACT reconstruction methods that are based on a variable data truncation (VDT) approach. The VDT approach represents a generalization of the half-time approach, in which the degree of temporal truncation for each measurement is determined by the distance between the corresponding ultrasonic transducer location and the nearest known bone or gas void location. Computer-simulated and experimental data are employed to demonstrate the effectiveness of the approach in mitigating artifacts due to acoustic heterogeneities.
We report photoacoustic microscopy (PAM) of arteriovenous (AV) shunts in early stage tumors in vivo, and develop a pattern recognition framework for computerized tumor detection. Here, using a high-resolution photoacoustic microscope, we implement a new blood oxygenation (sO2)-based disease marker induced by the AV shunt effect in tumor angiogenesis. We discovered a striking biological phenomenon: There can be two dramatically different sO2 values in bloodstreams flowing side-by-side in a single vessel. By tracing abnormal sO2 values in the blood vessels, we can identify a tumor region at an early stage. To further automate tumor detection based on our findings, we adopt widely used pattern recognition methods and develop an efficient computerized classification framework. The test result shows over 80% averaged detection accuracy with false positive contributing 18.52% of error test samples on a 50 PAM image dataset.
Circulating tumor cell (CTC) clusters arise from multicellular grouping in the primary tumor and elevate the metastatic potential by 23 to 50 fold compared to single CTCs. High throughout detection and quantification of CTC clusters is critical for understanding the tumor metastasis process and improving cancer therapy. In this work, we report a linear-array-based photoacoustic tomography (LA-PAT) system capable of label-free high-throughput CTC cluster detection and quantification in vivo. LA-PAT detects CTC clusters and quantifies the number of cells in them based on the contrast-to-noise ratios (CNRs) of photoacoustic signals. The feasibility of LA-PAT was first demonstrated by imaging CTC clusters ex vivo. LA-PAT detected CTC clusters in the blood-filled microtubes and computed the number of cells in the clusters. The size distribution of the CTC clusters measured by LA-PAT agreed well with that obtained by optical microscopy. We demonstrated the ability of LA-PAT to detect and quantify CTC clusters in vivo by imaging injected CTC clusters in rat tail veins. LA-PAT detected CTC clusters immediately after injection as well as when they were circulating in the rat bloodstreams. Similarly, the numbers of cells in the clusters were computed based on the CNRs of the photoacoustic signals. The data showed that larger CTC clusters disappear faster than the smaller ones. The results prove the potential of LA-PAT as a promising tool for both preclinical tumor metastasis studies and clinical cancer therapy evaluation.
Elastography can noninvasively map the elasticity distribution of biological tissue, which is often altered in pathological states. In this work, we report quantitative photoacoustic elastography (QPAE), capable of measuring Young’s modulus of human tissue in vivo. By combining photoacoustic elastography with a stress sensor having known stress-strain behavior, QPAE can simultaneously measure strain and stress, from which Young’s modulus is calculated. We first applied QPAE to quantify the Young’s modulus of tissue-mimicking agar phantoms with different concentrations. The measured values fitted well with both the empirical expectations based on the agar concentrations and those measured in independent standard compression tests. We then demonstrated the feasibility of QPAE by measuring the Young’s modulus of human skeletal muscle in vivo. The data showed a linear relationship between muscle stiffness and loading. The results proved that QPAE can noninvasively quantify the absolute elasticity of biological tissue, thus enabling longitudinal imaging of tissue elasticity. QPAE can be exploited for both preclinical biomechanics studies and clinical applications.
We have enhanced photoacoustic computed tomography with dry acoustic coupling that eliminates water immersion anxiety and wrinkling of the animal and facilitates incorporating complementary modalities and procedures. The dry acoustic coupler is made of a tubular elastic membrane enclosed by a closed transparent water tank. The tubular membrane ensures water-free contact with the animal, and the closed water tank allows pressurization for animal stabilization. The dry coupler was tested using a whole-body small-animal ring-shaped photoacoustic computed tomography system. Dry coupling was found to provide image quality comparable to that of conventional water coupling.
We present single-shot real-time video recording of light scattering dynamics by second-generation compressed ultrafast photography (G2-CUP). Using G2-CUP at 100 billion frames per second, in a single camera exposure, we experimentally captured the evolution of the light intensity distribution in an engineered thin scattering plate assembly. G2-CUP, which implements a new reconstruction paradigm and a more efficient hardware design than its predecessors, markedly improves the reconstructed image quality. The ultrafast imaging reveals the instantaneous light scattering pattern as a photonic Mach cone. We envision that our technology will find a diverse range of applications in biomedical imaging, materials science, and physics.
Optical phase conjugation (OPC) based wavefront shaping techniques focus light through or within scattering media, which is critically important for deep-tissue optical imaging, manipulation, and therapy. However, to date, the sample thicknesses used in wavefront shaping experiments have been limited to only a few millimeters or several transport mean free paths. Here, by using a long-coherence-length laser and an optimized digital OPC system that efficiently delivers light power, we focused 532 nm light through tissue-mimicking phantoms up to 9.6 cm thick, as well as through ex vivo chicken breast tissue up to 2.5 cm thick.
Circulating tumor cell (CTC) clusters, arising from multicellular groupings in a primary tumor, greatly elevate the metastatic potential of cancer compared with single CTCs. High-throughput detection and quantification of CTC clusters are important for understanding the tumor metastatic process and improving cancer therapy. Here, we applied a linear-array-based photoacoustic tomography (LA-PAT) system and improved the image reconstruction for label-free high-throughput CTC cluster detection and quantification in vivo. The feasibility was first demonstrated by imaging CTC cluster ex vivo. The relationship between the contrast-to-noise ratios (CNRs) and the number of cells in melanoma tumor cell clusters was investigated and verified. Melanoma CTC clusters with a minimum of four cells could be detected, and the number of cells could be computed from the CNR. Finally, we demonstrated imaging of injected melanoma CTC clusters in rats in vivo. Similarly, the number of cells in the melanoma CTC clusters could be quantified. The data showed that larger CTC clusters had faster clearance rates in the bloodstream, which agreed with the literature. The results demonstrated the capability of LA-PAT to detect and quantify melanoma CTC clusters in vivo and showed its potential for tumor metastasis study and cancer therapy.
Optical-resolution photoacoustic microscopy (OR-PAM) offers label-free in vivo imaging 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.
While lasers have been commonly used as illumination sources in photoacoustic (PA) imaging, their high purchase and maintenance costs, as well as their bulkiness, have hindered the rapid clinical dissemination of PA imaging. With this in mind, we explore an alternative illumination source for PA tomography—a xenon flash lamp with high pulse energy and a microsecond pulse width. We demonstrate that, by using a single xenon flash lamp, we can image both a black latex cord placed in chicken breast tissue at a depth of up to 3.5 cm ex vivo and an entire mouse body in vivo. Our findings indicate that the xenon flash lamp, producing optical illumination that is safe for humans, can be potentially applied to human tissue imaging.
One of the prime limiting factors of optical imaging in biological applications is the diffusion of light by tissue, which prevents focusing at depths greater than the optical diffusion limit (typically ∼1 mm). To overcome this challenge, wavefront shaping techniques that use a spatial light modulator (SLM) to correct the phase of the incident wavefront have recently been developed. These techniques are able to focus light through scattering media beyond the optical diffusion limit. However, the low speeds of typically used liquid crystal SLMs limit the focusing speed. Here, we present a method using a digital micromirror device (DMD) and an electro-optic modulator (EOM) to measure the scattering-induced aberrations, and using a liquid crystal SLM to apply the correction to the illuminating wavefront. By combining phase modulation from an EOM with the DMD’s ability to provide selective illumination, we exploit the DMD’s higher refresh rate for phase measurement. We achieved focusing through scattering media in less than 8 ms, which is sufficiently short for certain in vivo applications, as it is comparable to the speckle correlation time of living tissue.
Optical phase conjugation (OPC)-based wavefront shaping techniques focus light through or within scattering media, which is critically important for deep-tissue optical imaging, manipulation, and therapy. However, to date, the sample thickness in OPC experiments has been limited to only a few millimeters. Here, by using a laser with a long coherence length and an optimized digital OPC system that can safely deliver more light power, we focused 532-nm light through tissue-mimicking phantoms up to 9.6 cm thick, as well as through ex vivo chicken breast tissue up to 2.5 cm thick. Our results demonstrate that OPC can be achieved even when photons have experienced on average 1000 scattering events. The demonstrated penetration of nearly 10 cm (∼100 transport mean free paths) has never been achieved before by any optical focusing technique, and it shows the promise of OPC for deep-tissue noninvasive optical imaging, manipulation, and therapy.
Capitalizing on endogenous hemoglobin contrast, photoacoustic-computed tomography (PACT), a deep-tissue high-resolution imaging modality, has drawn increasing interest in neuroimaging. However, most existing studies are limited to functional imaging on the cortical surface and the deep brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can potentially provide high-resolution label-free imaging of structures in the entire mouse brain. With 100-μm in-plane resolution, we can clearly identify major structures of the brain, which complements magnetic resonance microscopy for imaging small-animal brain structures. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome distribution and that the presence of lipid sharpens the image contrast; brain histology results provide further validation. The feasibility of imaging the structure of the brain in vivo is also discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.
Photoacoustic computed tomography (PACT) has emerged as a unique and promising technology for multiscale biomedical imaging. To fully realize its potential for various preclinical and clinical applications, development of systems with high imaging speed, reasonable cost, and manageable data flow are needed. Sparse-sampling PACT with advanced reconstruction algorithms, such as compressed-sensing reconstruction, has shown potential as a solution to this challenge. However, most such algorithms require iterative reconstruction and thus intense computation, which may lead to excessively long image reconstruction times. Here, we developed a principal component analysis (PCA)-based PACT (PCA-PACT) that can rapidly reconstruct high-quality, three-dimensional (3-D) PACT images with sparsely sampled data without requiring an iterative process. In vivo images of the vasculature of a human hand were obtained, thus validating the PCA-PACT method. The results showed that, compared with the back-projection (BP) method, PCA-PACT required ∼50% fewer measurements and ∼40% less time for image reconstruction, and the imaging quality was almost the same as that for BP with full sampling. In addition, compared with compressed sensing-based PACT, PCA-PACT had approximately sevenfold faster imaging speed with higher imaging accuracy. This work suggests a promising approach for low-cost, 3-D, rapid PACT for various biomedical applications.
We report quantitative photoacoustic elastography (QPAE) capable of measuring Young’s modulus of biological tissue in vivo in humans. By combining conventional PAE with a stress sensor having known stress–strain behavior, QPAE can simultaneously measure strain and stress, from which Young’s modulus is calculated. We first demonstrate the feasibility of QPAE in agar phantoms with different concentrations. The measured Young’s modulus values fit well with both the empirical expectation based on the agar concentrations and those measured in an independent standard compression test. Next, QPAE was applied to quantify the Young’s modulus of skeletal muscle in vivo in humans, showing a linear relationship between muscle stiffness and loading. The results demonstrated the capability of QPAE to assess the absolute elasticity of biological tissue noninvasively in vivo in humans, indicating its potential for tissue biomechanics studies and clinical applications.
Grueneisen relaxation photoacoustic microscopy (GR-PAM) can achieve optically defined axial resolution, but it has been limited to ex vivo demonstrations so far. Here, we present the first in vivo image of a mouse brain acquired with GR-PAM. To induce the GR effect, an intensity-modulated continuous-wave laser was employed to heat absorbing objects. In phantom experiments, an axial resolution of 12.5 μm was achieved, which is sixfold better than the value achieved by conventional optical-resolution PAM. This axial-resolution improvement was further demonstrated by imaging a mouse brain in vivo, where significantly narrower axial profiles of blood vessels were observed. The in vivo demonstration of GR-PAM shows the potential of this modality for label-free and high-resolution anatomical and functional imaging of biological tissues.
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.
Optical focusing plays a central role in biomedical optical imaging, manipulation, and therapy. However, in scattering media, direct optical focusing becomes infeasible beyond ~10 mean free paths. To break this limit, time-reversed ultrasonically encoded (TRUE) optical focusing phase-conjugates ultrasonically tagged diffuse light back to the ultrasonic focus, thus forming a focus deep inside scattering media. In previous works, the speed of wavefront measurement was limited by the low frame rate of the camera used to record the four images required for phase-shifting holography. Moreover, most of the bits of a pixel value were used to represent an informationless background caused by the large amount of untagged light, increasing the amount of data to transfer and necessitating the use of costly high-resolution analog-to-digital converters (ADCs). Here, we developed a digital TRUE focusing system based on a lock-in camera (300×300 pixels), in which each pixel performs analog lock-in detection on chip. Since only the information of the signal, not that of the background, is digitized, the lock-in camera reduces the amount of data to transfer, and enables the use of cheap low-resolution ADCs. Using this lock-in camera, we were able to measure the wavefront of ultrasonically tagged light in less than 0.3 ms, and to achieve TRUE focusing in between two ground glass diffusers. Even when the signal-to-background ratio dropped to 6.32×10^-4, a phase sensitivity as low as 0.51 rad could still be realized, which is more than enough for digital optical phase conjugation.
Focusing light deep inside scattering media plays a key role in such biomedical applications as high resolution optical imaging, control, and therapy. In recent years, wavefront shaping technologies have come a long way in controlling light propagation in complex media. A prominent example is time-reversed ultrasonically encoded (TRUE) focusing, which allows noninvasive introduction of “guide stars” inside biological tissue to guide light focusing. By measuring the optical wavefront emanating from an ultrasound focus created at the target location, TRUE determines the desired wavefront non-iteratively, and achieves focusing at the target position via a subsequent optical time reversal. Compared to digital counterparts that employ slow electronic spatial light modulators and cameras, analog TRUE focusing relies on nonlinear photorefractive crystals that inherently accommodate more spatial modes and eliminate the troublesome alignment and data transfer required by digital approaches. However, analog TRUE focusing suffers from its small gain, defined as the energy or power ratio between the focusing and probing beams in the focal volume. Here, by implementing a modified analog TRUE focusing scheme that squeezes the duration of the time-reversed photon packet below the carrier-recombination-limited hologram decay time of the crystal, we demonstrated a photon flux amplification much greater than unity at a preset focal voxel in between two scattering layers. Although the energy gain was still below unity, the unprecedented power gain will nevertheless benefit new biomedical applications.
Neural scientists can benefit greatly from imaging tools that can penetrate thick brain tissue. Compared with traditional optical microscopy methods, photoacoustic imaging can beat the optical diffusion limit and achieve such deep tissue imaging with high spatial resolution. In this study, we used an optical-resolution photoacoustic microscope to image the odor-evoked neuronal activities in a drosophila model. Drosophila brain neurons stably express GCaMP5G, a calcium-sensitive fluorescent protein whose optical absorption coefficient changes with calcium influx during action potentials. We recorded an ~20% odor-evoked fractional photoacoustic signal increase at all depths of the drosophila brain in vivo, with and without removal of the brain cuticle, at a recording rate of 1 kHz. Our results were confirmed by concurrent fluorescent recordings. Furthermore, by performing fast 2D scanning, we imaged the antenna lobe region, which is of particular interest in neuroscience, at a volumetric rate of ~1 Hz with a sub-neuron resolution of 3 m. Unlike optical imaging, which requires surgical removal of the scattering brain cuticle, our photoacoustic system can image through the cuticle and measure neuronal signals of the whole drosophila brain without invasive surgery, enabling minimal disturbance to the animal’s behaviors. In conclusion, we have demonstrated photoacoustic imaging of calcium signals in drosophila brains for the first time. Utilizing the deep imaging capability of photoacoustic tomography, our methods could potentially be extended to in vivo imaging of neuronal activities from deep brains in other animal models.
Optical imaging of brain voltage signals is significantly limited in depth due to optical scattering and the absorptive property of brain tissue. Photoacoustic (PA) imaging promises to break this hard limit by utilizing both ballistic and diffused photons. To demonstrate the feasibility of PA, we used an in vivo mouse model. The brain cortex tissue was stained with dipicrylamine dye, electrically stimulated, and imaged with a customized dual-isosbestic-wavelength PA microscope (DIW-PAM). DIW-PAM separates voltage-induced PA signals from blood-induced PA signals and thereby allows recording the voltage response of mouse cortex tissue without interference from hemoglobin responses. The resting state PA voltage response signal exhibited a noise-like signal in the frequency domain. Upon 3 Hz electrical stimulation, the PA voltage response signal showed frequency peaks of 3.2 Hz and 6.3 Hz (Fig. 1). Although dipicrylamine dye is not fast enough for recording neuron action potentials, it served well for the purpose of this feasibility study. In conclusion, we successfully demonstrated in vivo photoacoustic imaging of mouse brain voltage signals for the first time. If a fast voltage-sensitive dye is available, using photoacoustic computed tomography (PACT) instead of PA microscopy could allow acquiring full-field PA action potential images at a speed limited only by the laser pulse repetition rate.
Optical-resolution photoacoustic microscopy (OR-PAM) can achieve submicron lateral resolution by tightly focusing the excitation light, while the axial resolution is still limited by the frequency bandwidth of the ultrasonic transducer. The Grueneisen relaxation effect, in which the Grueneisen parameter changes within the thermal relaxation time following a laser impulse heating, can provide excellent axial resolution due to its optical sectioning property. Based on this effect, Grueneisen relaxation photoacoustic microscopy (GR-PAM) was developed and demonstrated ex vivo. Here, we present for the first time in vivo imaging of mouse brains with improved axial resolution based on GR-PAM. An intensity-modulated continuous-wave (CW) 532 nm laser thermally heated the in-focus absorber. Another 532 nm pulsed laser, which is aligned confocally with the CW laser, generated the photoacoustic (PA) signal from the absorber. The difference between the amplitudes of the photoacoustic signals with and without heating was used for image reconstruction. The achieved axial resolution is ~12.5 µm, which is fivefold better than the acoustically determined value for a 20 MHz-bandwidth ultrasound transducer. The system was demonstrated by imaging a blood-filled tube ex vivo and blood vessels of mouse brains in vivo. The blood-filled tube diameter obtained from the PA image by GR-PAM is 105 µm, which is much closer to its actual diameter (100 µm) than the value from conventional OR-PAM (160 µm). This axial resolution improvement was further validated in imaging mouse brains in vivo, and yielded significantly narrower axial profiles of the vessels. This in vivo demonstration of imaging by GR-PAM might inspire more applications in PA biomedical imaging and sensing.
Microwave-based thermoacoustic tomography (TAT), based on the measurement of ultrasonic waves induced by microwave pulses, can reveal tissue dielectric properties that may be closely related to the physiological and pathological status of the tissues. Using microwaves as the excitation source improved imaging depth because of their deep penetration into biological tissues.
We demonstrate, for the first time, in vivo microwave-based thermoacoustic imaging in rats. The transducer is rotated around the rat in a full circle, providing a full two-dimensional view. Instead of a flat ultrasonic transducer, we used a virtual line detector based on a cylindrically focused transducer. A 3 GHz microwave source with 0.6 µs pulse width and an electromagnetically shielded transducer with 2.25 MHz central frequency provided clear cross-sectional images of the rat’s body. The high imaging contrast, based on the tissue’s rate of absorption, and the ultrasonically defined spatial resolution combine to reveal the spine, kidney, muscle, and other deeply seated anatomical features in the rat’s abdominal cavity. This non-invasive and non-ionizing imaging modality achieved an imaging depth beyond 6 cm in the rat’s tissue.
Cancer diagnosis based on information about tissue properties from microwave band TAT can potentially be more accurate than has previously been achievable.
Photoacoustic tomography (PAT) exploits optical contrast and ultrasonic detection principles to form images of absorbed optical energy density within tissue. Based on the photoacoustic effect, PAT directly and quantitatively measures specific optical absorption. A full-ring ultrasonic transducer array based photoacoustic computed tomography (PACT) system was recently developed for small animal whole-body imaging with a full-view detection angle and high in-plane resolution (100 µm). However, due to the band-pass frequency response of the piezoelectric transducer elements, the reconstructed images present bipolar (both positive and negative) pixel values, which is artificial and counterintuitive for physicians and biologists seeking to interpret the image. Moreover, bipolar pixel values hinder quantification of physiological parameters, such as oxygen saturation and blood flow speed. Unipolar images can be obtained by deconvolving the raw channel data with the transducer’s electrical impulse response and applying non-negativity during iteration, but this process requires complex transducer modeling and time-consuming computation.
Here, we present a multi-view Hilbert transformation method to recover the unipolar initial pressure for full-ring PACT. Multi-view Hilbert transformation along the acoustic wave propagation direction minimizes reconstruction artifacts during envelope extraction and maintains the signal-to-noise ratio of the reconstructed images. The in-plane isotropic spatial resolution of this method was quantified to 168 μm within a 20 × 20 mm2 field of view. The effectiveness of the proposed algorithm was first validated by numerical simulations and then demonstrated with ex-vivo mouse brain structural imaging and in-vivo mouse wholebody imaging.
Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT’s basic principles, major implementations, imaging contrasts, and recent applications.
Optical imaging of genetically encoded probes has revolutionized biomedical studies by providing valuable information about targeted biological processes. Here, we report a novel imaging technique, termed reversibly switchable photoacoustic tomography (RS-PAT), which exhibits large penetration depth, high detection sensitivity, and super-resolution. RS-PAT combines advanced photoacoustic imaging techniques with, for the first time, a nonfluorescent photoswitchable bacterial phytochrome. This bacterial phytochrome is the most near-infrared shifted genetically encoded probe reported so far. Moreover, this bacterial phytochrome is reversibly photoconvertible between its far-red and near-infrared light absorption states. Taking maximum advantage of the powerful imaging capability of PAT and the unique photochemical properties of the phytochrome, RS-PAT has broken through both the optical diffusion limit for deep-tissue imaging and the optical diffraction limit for super-resolution photoacoustic microscopy. Specifically, with RS-PAT we have achieved an unprecedented detection sensitivity of ~2 μM, or as few as ~20 tumor cells, at a centimeter depth. Such high sensitivity is fully demonstrated in our study by monitoring tumor growth and metastasis at whole-body level with ~100 μm resolution. Moreover, our microscopic implementation of RS-PAT is capable of imaging mammalian cells with a sub-diffraction lateral resolution of ~140 nm and axial resolution of ~400 nm, which are respectively ~2-fold and ~75-fold finer than those of our conventional photoacoustic microscopy. Overall, RS-PAT is a new and promising imaging technology for studying biological processes at different length scales.
We applied a linear-array-based photoacoustic probe to detect the tumor depth and volume of melanin-containing melanoma in nude mice in vivo. We demonstrated the ability of this linear-array-based system to measure both the depth and volume of melanoma through phantom, ex vivo, and in vivo experiments. The volume detection ability also enables us to accurately calculate the rate of growth of the tumor, which is important in quantifying tumor activity. Our results show that this system can be used for clinical melanoma diagnosis and treatment at the bedside.
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 may deteriorate image quality when the Bessel beam is directly employed to excite photoacoustic signals in ORPAM. Here, we present a nonlinear approach based on the Grueneisen relaxation effect to suppress the side-lobe artifacts in photoacoustic 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.
A major limiting factor of optical imaging in biological applications is the diffusion of light by tissue, preventing focusing at depths greater than ~1 mm in the body. To overcome this issue, phase-based wavefront shaping alters the phase of sections of the incident wavefront to counteract aberrations in phase caused by scattering. This enables focusing through scattering media beyond the optical diffusion limit and increases signal compared to amplitude-based compensation. However, in previous studies, speed of optimization has typically been limited by the use of a liquid crystal spatial light modulator (SLM) for measurement and display. SLMs usually have refresh rates of less than 100 Hz and require much longer than the speckle correlation time of tissue in vivo, usually on the order of milliseconds, to determine the optimal wavefront. Here, we present a phase-based iterative wavefront shaping method based on an onaxis digital micromirror device (DMD) in conjunction with an electro-optic modulator (EOM) for measurement and a fast SLM for display. By combining phase modulation from an EOM with the modal selection of the DMD, we take advantage of DMDs higher refresh rate, approximately 23 kHz, for iterative phase measurement. The slower SLM requires one update for display following the rapid determination of the optimal wavefront via the DMD, allowing for high-speed wavefront shaping. Using this system, we are able to focus through scattering media using 64 modes in under 8 milliseconds, on the order of the speckle correlation time for tissue in vivo.
We propose a saline-injection-based method to quantify blood flow velocity in vivo with acoustic-resolution photoacoustic tomography. By monitoring the saline-blood-interface propagating in the blood vessel, we can resolve the flow velocity. In phantom experiments, a root-mean-squared error of prediction of 0.29 mm/s was achieved. By injecting saline into a mouse tail vein covered with 1 mm chicken tissue, we showed that the flow velocity in the tail vein could be measured at depth, which is especially pertinent to monitoring blood flow velocity in patients undergoing intravenous infusion.
Using a handheld photoacoustic probe, we proposed a cuffing-based method to quantify blood flow speed in humans. By cuffing and releasing the blood vessel, we can measure the blood flow speed downstream. In phantom experiments, we demonstrated that the minimum and maximum measurable flow speeds were 0.035 mm/s and 42 mm/s, respectively. In human experiments, flow speeds were measured in three different blood vessels: a radial artery in the right forearm, a radial artery in the index finger of the right hand, and a radial vein in the right forearm.
Photoacoustic computed tomography (PACT) is an emerging computed imaging modality that exploits optical contrast and ultrasonic detection principles to form images of the absorbed optical energy density within tissue. If the object possesses spatially variant acoustic properties that are unaccounted for by the reconstruction algorithm, the estimated image can contain distortions. While reconstruction algorithms have recently been developed for compensating for this effect, they generally require the objects acoustic properties to be known a priori. To circumvent the need for detailed information regarding an objects acoustic properties, we have previously proposed a half-time reconstruction method for PACT. A half-time reconstruction method estimates the PACT image from a data set that has been temporally truncated to exclude the data components that have been strongly aberrated. In this approach, the degree of temporal truncation is the same for all measurements. However, this strategy can be improved upon it when the approximate sizes and locations of strongly heterogeneous structures such as gas voids or bones are known. In this work, we investigate PACT reconstruction algorithms that are based on a variable temporal data truncation (VTDT) approach that represents a generalization of the half-time reconstruction approach. In the VTDT approach, the degree of temporal truncation for each measurement is determined by the distance between the corresponding transducer location and the nearest known bone or gas void location. Reconstructed images from a numerical phantom is employed to demonstrate the feasibility and effectiveness of the approach.
We applied compressed ultrafast photography (CUP), a computational imaging technique, to acquire three-dimensional (3D) images. The approach unites image encryption, compression, and acquisition in a single measurement, thereby allowing efficient and secure data transmission. By leveraging the time-of-flight (ToF) information of pulsed light reflected by the object, we can reconstruct a volumetric image (150 mm×150 mm×1050 mm, x × y × z) from a single camera snapshot. Furthermore, we demonstrated high-speed 3D videography of a moving object at 75 frames per second using the ToF-CUP camera.
Quantification of vascular elasticity can help detect thrombosis and prevent life-threatening conditions such as acute myocardial infarction or stroke. Here, we propose vascular elastic photoacoustic tomography (VE-PAT) to measure vascular elasticity in humans. VE-PAT was developed by incorporating a linear-array-based photoacoustic computed tomography system with a customized compression stage. By measuring the deformation of blood vessels under uniaxial loading, VE-PAT was able to quantify the vascular compliance. We first demonstrated the feasibility of VE-PAT in blood vessel phantoms. In large vessel phantoms, VE-PAT detected a decrease in vascular compliance due to simulated thrombosis, which was validated by a standard compression test. In small blood vessel phantoms embedded 3 mm deep in gelatin, VE-PAT detected elasticity changes at depths that are difficult to image using other elasticity imaging techniques. We then applied VE-PAT to assess vascular compliance in a human subject and detected a decrease in vascular compliance when an occlusion occurred downstream from the measurement point, demonstrating the potential of VE-PAT in clinical applications such as detection of deep venous thrombosis.
Most image reconstruction methods in photoacoustic computed tomography (PACT) assume that the acoustic properties of the object and the surrounding medium are homogeneous. This can lead to strong artifacts in the reconstructed images when there are significant variations in sound speed or density. Air voids represent a particular challenge due to the severity of the differences between the acoustic properties of air and water. In whole-body small animal imaging, the presence of air voids in the lungs, stomach, and gastrointestinal system can limit image quality over large regions of the object. Iterative reconstruction methods based on the photoacoustic wave equation can account for these acoustic variations, leading to improved resolution, improved contrast, and a reduction in the number of imaging artifacts. However, the strong acoustic heterogeneities can lead to instability or errors in the numerical wave solver. Here, the impact of air voids on PACT image reconstruction is investigated, and procedures for their compensation are proposed. The contributions of sound speed and density variations to the numerical stability of the wave solver are considered, and a novel approach for mitigating the impact of air voids while reducing the computational burden of image reconstruction is identified. These results are verified by application to an experimental phantom.
The single-shot compressed ultrafast photography (CUP) camera is the fastest receive-only camera in the world. In this work, we introduce an external CCD camera and a space- and intensity-constrained (SIC) reconstruction algorithm to improve the image quality of CUP. The CCD camera takes a time-unsheared image of the dynamic scene. Unlike the previously used unconstrained algorithm, the proposed algorithm incorporates both spatial and intensity constraints, based on the additional prior information provided by the external CCD camera. First, a spatial mask is extracted from the time-unsheared image to define the zone of action. Second, an intensity threshold constraint is determined based on the similarity between the temporally projected image of the reconstructed datacube and the time-unsheared image taken by the external CCD. Both simulation and experimental studies showed that the SIC reconstruction improves the spatial resolution, contrast, and general quality of the reconstructed image.
Angiogenesis in a tumor region creates arteriovenous (AV) shunts that cause an abnormal venous blood oxygen saturation (sO2) distribution. Here, we applied optical-resolution photoacoustic microscopy to study the AV shunting in vivo. First, we built a phantom to image sO2 distribution in a vessel containing converged flows from two upstream blood vessels with different sO2 values. The phantom experiment showed that the blood from the two upstream vessels maintained a clear sO2 boundary for hundreds of seconds, which is consistent with our theoretical analysis using a diffusion model. Next, we xenotransplanted O-786 tumor cells in mouse ears and observed abnormal sO2 distribution in the downstream vein from the AV shunts in vivo. Finally, we identified the tumor location by tracing the sO2 distribution. Our study suggests that abnormal sO2 distribution induced by the AV shunts in the vessel network may be used as a new functional benchmark for early tumor detection.
Characterization of blood vessel elastic properties can help in detecting thrombosis and preventing life-threatening conditions such as acute myocardial infarction or stroke. Vascular elastic photoacoustic tomography (VE-PAT) is proposed to measure blood vessel compliance in humans. Implemented on a linear-array-based photoacoustic computed tomography system, VE-PAT can quantify blood vessel compliance changes due to simulated thrombosis and occlusion. The feasibility of the VE-PAT system was first demonstrated by measuring the strains under uniaxial loading in perfused blood vessel phantoms and quantifying their compliance changes due to the simulated thrombosis. The VE-PAT system detected a decrease in the compliances of blood vessel phantoms with simulated thrombosis, which was validated by a standard compression test. The VE-PAT system was then applied to assess blood vessel compliance in a human subject. Experimental results showed a decrease in compliance when an occlusion occurred downstream from the measurement point in the blood vessels, demonstrating VE-PAT’s potential for clinical thrombosis detection.
Myoglobin is an essential oxygen-binding hemoprotein in skeletal and cardiac muscles that buffers intracellular oxygen (O2) concentration in response to hypoxia or elevated muscle activities. We present a method that uses photoacoustic computed tomography to measure the distribution of myoglobin in tissue and the oxygen saturation of myoglobin (sO2-Mb). From photoacoustic measurements of mice in different oxygenation states, we performed calibration-free quantification of the sO2-Mb change in the backbone muscle in vivo.
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.
We propose a saline injection-based method to quantify blood flow velocity in vivo with acoustic-resolution photoacoustic tomography. By monitoring the saline–blood interface propagating in the blood vessel, the flow velocity can be resolved. We first demonstrated our method in phantom experiments, where a root mean square error of prediction of 0.29 mm/s was achieved. By injecting saline into a mouse tail vein covered with 1 mm chicken tissue, we showed that the flow velocity in the tail vein could be measured at depths, which is especially pertinent to monitoring blood flow velocity in patients undergoing intravenous infusion.
Due to their low cost, hand-held convenience, wide selection of bandwidths, and ultrasound imaging capability, linear ultrasonic transducer arrays have been widely studied for photoacoustic computed tomography (PACT). As linear-array PACT suffers from a limited view, full-view imaging requires either the transducer or the object to be rotated. So far, both the central frequencies and bandwidth of linear transducer arrays applied in full-view PACT are low, limiting the spatial resolutions of the reconstructed images. Here, we present a multiview high-frequency PACT imaging system implemented with a commercial 40-MHz central frequency linear transducer array. By rotating the object through multiple angles with respect to the linear transducer array, we acquired full-view photoacoustic pressure measurements. Further, to quantify the unipolar initial pressures and overcome the limitations of the single-view Hilbert transformation, we developed a multiview Hilbert transformation method. The in-plane spatial resolution of this full-view linear-array PACT was quantified to be isotropically 60 μm within a 10×10 mm2 field of view. The system was demonstrated by imaging both a leaf skeleton and a zebrafish in vivo.
Photoacoustic tomography is expected to impact biology and medicine broadly by providing multiscale in vivo functional and molecular imaging of structures ranging from subcellular organelles to organs, enabling a noninvasive look at subcutaneous tissue at a deep level.
Lihong Wang holds the Gene K. Beare Distinguished Professorship of Biomedical Engineering at Washington University in St. Louis, and is Editor-in-Chief of the Journal of Biomedical Optics.
Wang was awarded the 2015 Britton Chance Biomedical Optics Award for his pioneering technical contributions and visionary leadership in the development and application of photoacoustic tomography, photoacoustic microscopy, and photon transport modeling.
Due to the various causes of methemoglobinemia and its potential to be confused with other diseases, in vivo measurements of methemoglobin have significant applications in the clinic. Using photoacoustic microscopy (PAM), we quantified the average and the distributed percentage of methemoglobin both in vitro and in vivo. Based on the absorption spectra of methemoglobin, oxyhemoglobin, and deoxyhemoglobin, three wavelengths were chosen to differentiate methemoglobin from the others. The methemoglobin concentrations calculated from the photoacoustic signals agreed well with the preset concentrations. Then we imaged the methemoglobin percentage in microtubes that mimicked blood vessels. Average percentages calculated for five samples with different methemoglobin concentrations also agreed well with the preset values. Finally, we demonstrated the ability of PAM to detect methemoglobin in vivo in a mouse ear. Our results show that PAM can quantitatively image methemoglobin distribution in vivo.
We have successfully developed a fully-sheathed, flexible shaft-based, mechanical scanning photoacoustic endoscopy (PAE) system for imaging the human gastrointestinal tract via the instrument channel of a clinical video endoscope. The endoscopic system uses a single element ultrasonic transducer and flexible shaft-based proximal actuation mechanism, and it has a 2.5 m long and 3.2 mm diameter catheter section, which can be accommodated in the 3.7 mm diameter instrument channel of a clinical video endoscope. Here, we demonstrate the intra-instrument channel workability and in vivo imaging capability of the PAE system.
To achieve real-time photoacoustic tomography (PAT), massive transducer arrays and data acquisition (DAQ) electronics are needed to receive the PA signals simultaneously, which results in complex and high-cost ultrasound receiver systems. To address this issue, we have developed a new PA data acquisition approach using acoustic time delay. Optical fibers were used as parallel acoustic delay lines (PADLs) to create different time delays in multiple channels of PA signals. This makes the PA signals reach a single-element transducer at different times. As a result, they can be properly received by single-channel DAQ electronics. However, due to their small diameter and fragility, using optical fiber as acoustic delay lines poses a number of challenges in the design, construction and packaging of the PADLs, thereby limiting their performances and use in real imaging applications. In this paper, we report the development of new silicon PADLs, which are directly made from silicon wafers using advanced micromachining technologies. The silicon PADLs have very low acoustic attenuation and distortion. A linear array of 16 silicon PADLs were assembled into a handheld package with one common input port and one common output port. To demonstrate its real-time PAT capability, the silicon PADL array (with its output port interfaced with a single-element transducer) was used to receive 16 channels of PA signals simultaneously from a tissue-mimicking optical phantom sample. The reconstructed PA image matches well with the imaging target. Therefore, the silicon PADL array can provide a 16× reduction in the ultrasound DAQ channels for real-time PAT.
Intravital microscopy techniques have become increasingly important in biomedical research because they can provide unique microscopic views of various biological or disease developmental processes in situ. Here we present an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system that visualizes internal organs with a much finer resolution than conventional acoustic-resolution photoacoustic endoscopy systems. By combining gradient index (GRIN) lens-based optical focusing and ultrasonic ring transducer-based acoustic focusing, we achieved a transverse resolution as fine as ~10 μm at an optical working distance of 6.5 mm. The OR-PAEM system’s high-resolution intravital imaging capability is demonstrated through animal experiments.
Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.
The addition of photoacoustic endoscopy to conventional endoscopic ultrasound offers imaging capabilities that may improve diagnosis and clinical care of gastrointestinal tract diseases. In this study, using a 3.8-mm diameter dual-mode photoacoustic and ultrasonic endoscopic probe, we investigated photoacoustic and ultrasonic image features of rabbit esophagi. Specifically, we performed ex vivo imaging of intact rabbit esophagi and correlated the acquired images with histology. Without motion artifact-based limitations, we were able to utilize the full resolving power of the endoscopic device and acquire the first three-dimensional vasculature map of the esophagus and mediastinum, along with coregistered tissue density information. Here, we present the experimental results and discuss potential clinical applications of the technique.
Conventional photoacoustic computed tomography (PACT) image reconstruction methods assume that the object and surrounding medium are described by a constant speed-of-sound (SOS) value. In order to accurately recover fine structures, SOS heterogeneities should be quantified and compensated for during PACT reconstruction. To address this problem, several groups have proposed hybrid systems that combine PACT with ultrasound computed tomography (USCT). In such systems, a SOS map is reconstructed first via USCT. Consequently, this SOS map is employed to inform the PACT reconstruction method. Additionally, the SOS map can provide structural information regarding tissue, which is complementary to the functional information from the PACT image. We propose a paradigm shift in the way that images are reconstructed in hybrid PACT-USCT imaging. Inspired by our observation that information about the SOS distribution is encoded in PACT measurements, we propose to jointly reconstruct the absorbed optical energy density and SOS distributions from a combined set of USCT and PACT measurements, thereby reducing the two reconstruction problems into one. This innovative approach has several advantages over conventional approaches in which PACT and USCT images are reconstructed independently: (1) Variations in the SOS will automatically be accounted for, optimizing PACT image quality; (2) The reconstructed PACT and USCT images will possess minimal systematic artifacts because errors in the imaging models will be optimally balanced during the joint reconstruction; (3) Due to the exploitation of information regarding the SOS distribution in the full-view PACT data, our approach will permit high-resolution reconstruction of the SOS distribution from sparse array data.
Capitalizing on endogenous hemoglobin contrast, photoacoustic computed tomography (PACT), a deep-tissue highresolution imaging modality, has drawn increasing interest in neuro-imaging. However, most existing studies are limited to functional imaging on the cortical surface, and the deep-brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep-brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can provide high-resolution label-free structural imaging through the entire mouse brain. With 100 μm in-plane resolution, we can clearly identify major structures of the brain, and the image quality is comparable to that of magnetic resonance microscopy. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome and lipid. The feasibility of imaging the structure of the brain in vivo has also been discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.
Conventional photoacoustic computed tomography (PACT) image reconstruction methods assume that the object and surrounding medium are described by a constant speed-of-sound (SOS) value. In order to accurately recover fine structures, SOS heterogeneities should be quantified and compensated for during PACT reconstruction. To address this problem, several groups have proposed hybrid systems that combine PACT with ultrasound computed tomography (USCT). In such systems, a SOS map is reconstructed first via USCT. Consequently, this SOS map is employed to inform the PACT reconstruction method. Additionally, the SOS map can provide structural information regarding tissue, which is complementary to the functional information from the PACT image. We propose a paradigm shift in the way that images are reconstructed in hybrid PACT-USCT imaging. Inspired by our observation that information about the SOS distribution is encoded in PACT measurements, we propose to jointly reconstruct the absorbed optical energy density and SOS distributions from a combined set of USCT and PACT measurements, thereby reducing the two reconstruction problems into one. This innovative approach has several advantages over conventional approaches in which PACT and USCT images are reconstructed independently: (1) Variations in the SOS will automatically be accounted for, optimizing PACT image quality; (2) The reconstructed PACT and USCT images will possess minimal systematic artifacts because errors in the imaging models will be optimally balanced during the joint reconstruction; (3) Due to the exploitation of information regarding the SOS distribution in the full-view PACT data, our approach will permit high-resolution reconstruction of the SOS distribution from sparse array data.
Photoacoustic computed tomography (PACT) holds great promise for transcranial brain imaging. However, the strong reflection, scattering, attenuation, and mode-conversion of photoacoustic waves in the skull pose serious challenges to establishing the method. The lack of an appropriate model of solid media in conventional PACT imaging models, which are based on the canonical scalar wave equation, causes a significant model mismatch in the presence of the skull and thus results in deteriorated reconstructed images. The goal of this study was to develop an image reconstruction algorithm that accurately models the skull and thereby ameliorates the quality of reconstructed images. The propagation of photoacoustic waves through the skull was modeled by a viscoelastic stress tensor wave equation, which was subsequently discretized by use of a staggered grid fourth-order finite-difference time-domain (FDTD) method. The matched adjoint of the FDTD-based wave propagation operator was derived for implementing a back-projection operator. Systematic computer simulations were conducted to demonstrate the effectiveness of the back-projection operator for reconstructing images in a realistic three-dimensional PACT brain imaging system. The results suggest that the proposed algorithm can successfully reconstruct images from transcranially-measured pressure data and readily be translated to clinical PACT brain imaging applications.
Various causes can lead to methemoglobinemia, and it has the potential to be confused with other diseases. In vivo measurements of methemoglobin have significant applications in the clinics. We quantified the average and the distributed percentage of methemoglobin both in vitro and in vivo using photoacoustic microscopy (PAM). Based on the absorption spectra of methemoglobin, oxyhemoglobin, and deoxyhemoglobin, three wavelengths were chosen to differentiate methemoglobin from the others. We imaged the methemoglobin percentage in microtubes that mimicked blood vessels as a phantom experiment. The methemoglobin concentrations calculated from the photoacoustic signals were in accordance with the preset concentrations. We also demonstrated the ability of PAM to quantitatively image methemoglobin distribution in vivo in a mouse ear.
We developed a handheld photoacoustic microscope (PAM) to detect melanoma and determine tumor depth in nude mice in vivo. Compared to our previous PAM system for melanoma imaging, a new light delivery mechanism is introduced to improve light penetration. We show that melanomas with 4.1 mm and 3.3 mm thicknesses can be successfully detected in phantom and in vivo experiments, respectively. With its deep melanoma imaging ability and novel handheld design, this system is promising for clinical melanoma diagnosis, prognosis, and surgical planning for patients at the bedside.
Linear transducer arrays are readily available for ultrasonic detection in photoacoustic computed tomography. They offer low cost, hand-held convenience, and conventional ultrasonic imaging. However, the elevational resolution of linear transducer arrays, which is usually determined by the weak focus of the cylindrical acoustic lens, is about one order of magnitude worse than the in-plane axial and lateral spatial resolutions. Therefore, conventional linear scanning along the elevational direction cannot provide high-quality three-dimensional photoacoustic images due to the anisotropic spatial resolutions. Here we propose an innovative method to achieve isotropic resolutions for three-dimensional photoacoustic images through combined linear and rotational scanning. In each scan step, we first elevationally scan the linear transducer array, and then rotate the linear transducer array along its center in small steps, and scan again until 180 degrees have been covered. To reconstruct isotropic three-dimensional images from the multiple-directional scanning dataset, we use the standard inverse Radon transform originating from X-ray CT. We acquired a three-dimensional microsphere phantom image through the inverse Radon transform method and compared it with a single-elevational-scan three-dimensional image. The comparison shows that our method improves the elevational resolution by up to one order of magnitude, approaching the in-plane lateral-direction resolution. In vivo rat images were also acquired.
We used photoacoustic microscopy (PAM) to assist diagnoses and monitor the progress and treatment outcome of complex regional pain syndrome type 1 (CRPS-1). Blood vasculature and oxygen saturation (sO2) were imaged by PAM in eight adult patients with CRPS-1. Patients’ hands and cuticles were imaged both before and after stellate ganglion block (SGB) for comparison. For all patients, both the vascular structure and sO2 could be assessed by PAM. In addition, more vessels and stronger signals were observed after SGB.
We present spatially Fourier-encoded photoacoustic microscopy using a digital micromirror device (DMD). The spatial fluence distribution of laser pulses is Fourier-encoded by the DMD, and a series of such encoded photoacoustic (PA) measurements enables decoding of the spatial distribution of optical absorption. By imaging a chromium target, we demonstrated the throughput and Fellgett advantages, which increased the PA signal-to-noise ratio (SNR) compared to raster scanning. The system was used to image two biological targets, a monolayer of red blood cells, and melanoma cells. The enhanced SNR benefited PA images by increasing the image’s contrast-to-noise ratio and target identifiability.
Optical diffusion in scattering media prevents focusing beyond shallow depths, causing optical imaging and sensing to suffer from low optical intensities, resulting in low signal-to-noise ratios (SNR). Here, we demonstrate focusing using a fast binary-amplitude digital micromirror device to characterize the transmission modes of the scattering medium. We then identify and selectively illuminate the transmission modes which contribute constructively to the intensity at the optical focus. Applying this method to photoacoustic flowmetry, we increased the optical intensity at the focus six-fold, and showed that the corresponding increase in SNR allows particle flow to be measured.
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.
We demonstrate, by means of internal light delivery, photoacoustic imaging of the deep brain of rats in vivo. With fiber illumination via the oral cavity, we delivered light directly into the bottom of the brain, much more than can be delivered by external illumination. The study was performed using a photoacoustic computed tomography (PACT) system equipped with a 512-element full-ring transducer array, providing a full two-dimensional view aperture. Using internal illumination, the PACT system provided clear cross sectional photoacoustic images from the palate to the middle brain of live rats, revealing deep brain structures such as the hypothalamus, brain stem, and cerebral medulla.
We propose using noninvasive longitudinal optical-resolution photoacoustic microscopy (L-ORPAM) to quantify blood flow flux, oxygen saturation (sO2), and thereby the metabolic rate of oxygen (MRO2), for a renal tumor model in the same mouse over weeks to months. Experiments showed that the sO2 difference between the artery and vein decreased greatly due to the arteriovenous shunting effect during tumor growth. Moreover, hypermetabolism was exhibited by an increase in MRO2.
Using internal illumination with an optical fiber in the oral cavity, we demonstrate, for the first time, photoacoustic computed tomography (PACT) of the deep brain of rats in vivo. The experiment was performed on a full-ring-array PACT system, with the capability of providing high-speed cross-sectional imaging of the brain. Compared with external illumination through the cranial skull, internal illumination delivers more light to the base of the brain. Consequently, in vivo photoacoustic images clearly reveal deep brain structures such as the hypothalamus, brain stem, and cerebral medulla.
Despite its critical function in coordinating the egress of inflammatory and immune cells out of tissues and maintaining fluid balance, the causative role of lymphatic network dysfunction in pathological settings is still understudied. Engineered-animal models and better noninvasive high spatial-temporal resolution imaging techniques in both preclinical and clinical studies will help to improve our understanding of different lymphatic-related pathologic disorders. Our aim was to take advantage of our newly optimized noninvasive wide-field fast-scanning photoacoustic (PA) microcopy system to coordinately image the lymphatic vasculature and its flow dynamics, while maintaining high resolution and detection sensitivity. Here, by combining the optical-resolution PA microscopy with a fast-scanning water-immersible microelectromechanical system scanning mirror, we have imaged the lymph dynamics over a large field-of-view, with high spatial resolution and advanced detection sensitivity. Depending on the application, lymphatic vessels (LV) were spectrally or temporally differentiated from blood vessels. Validation experiments were performed on phantoms and in vivo to identify the LV. Lymphatic flow dynamics in nonpathological and pathological conditions were also visualized. These results indicate that our newly developed PA microscopy is a promising tool for lymphatic-related biological research.
Accurate quantification of microvasculature remains of interest in fundamental pathophysiological studies and clinical trials. Current photoacoustic microscopy can noninvasively quantify properties of the microvasculature, including vessel density and diameter, with a high spatial resolution. However, the depth range of focus (i.e., focal zone) of optical-resolution photoacoustic microscopy (OR-PAM) is often insufficient to encompass the depth variations of features of interest—such as blood vessels—due to uneven tissue surfaces. Thus, time-consuming image acquisitions at multiple different focal planes are required to maintain the region of interest in the focal zone. We have developed continuous three-dimensional motorized contour-scanning OR-PAM, which enables real-time adjustment of the focal plane to track the vessels’ profile. We have experimentally demonstrated that contour scanning improves the signal-to-noise ratio of conventional OR-PAM by as much as 41% and shortens the image acquisition time by 3.2 times. Moreover, contour-scanning OR-PAM more accurately quantifies vessel density and diameter, and has been applied to studying tumors with uneven surfaces.
Complex regional pain syndrome (CRPS) is a chronic pain syndrome that causes intractable pain, disability, and poor quality of life for patients. The etiology and pathophysiology of CRPS are still poorly understood. Due to a lack of proper diagnostic tools, the prognosis of CRPS is primarily based on clinical observation. The objective of this work is to evaluate a new imaging modality, photoacoustic microscopy (PAM), for assisting diagnoses and monitoring the progress and treatment outcome of CRPS. Blood vasculature and oxygen saturation (sO2) were imaged by PAM from eight adult patients with CRPS-1. Patients’ hands and cuticles were imaged both before and after stellate ganglion block (SGB) for comparison. For all patients, both vascular structure and sO2 could be assessed by PAM. In addition, more vessels and stronger signals were observed after SGB. The results show that PAM can help diagnose and monitor CRPS.
Confocal microscopy with optical sectioning has revolutionized biological studies by providing sharper images than conventional optical microscopy. Here, we introduce a fluorescence imaging method with enhanced resolution and imaging contrast, which can be implemented using a commercial confocal microscope setup. This approach, called the reversibly switchable photo-imprint microscopy (rsPIM), is based on the switching dynamics of reversibly switchable fluorophores. When the fluorophores are switched from the bright (ON) state to the dark (OFF) state, their switching rate carries the information about the local excitation light intensity. In rsPIM, a polynomial function is used to fit the fluorescence signal decay during the transition. The extracted high-order coefficient highlights the signal contribution from the center of the excitation volume, and thus sharpens the resolution in all dimensions. In particular, out-of-focus signals are greatly blocked for large targets, and thus the image contrast is considerably enhanced. Notably, since the fluorophores can be cycled between the ON and OFF states, the whole imaging process can be repeated. RsPIM imaging with enhanced image contrast was demonstrated in both fixed and live cells using a reversibly switchable synthetic dye and a genetically encoded red fluorescent protein. Since rsPIM does not require the modification of commercial microscope systems, it may provide a simple and cost-effective solution for subdiffraction imaging of live cells.
The development of the first miniaturized parallel acoustic delay line (PADL) probe for handheld photoacoustic tomography (PAT) is reported. Using fused-silica optical fibers with low acoustic attenuation, we constructed two arrays of eight PADLs. Precision laser micromachining was conducted to produce robust and accurate mechanical support and alignment structures for the PADLs, with minimal acoustic distortion and interchannel coupling. The 16 optical-fiber PADLs, each with a different time delay, were arranged to form one input port and two output ports. A handheld PADL probe was constructed using two single-element transducers and two data acquisition channels (equal to a channel reduction ratio of 8∶1). Photoacoustic (PA) images of a black-ink target embedded in an optically scattering phantom were successfully acquired. After traveling through the PADLs, the eight channels of differently time-delayed PA signals reached each single-element ultrasonic transducer in a designated nonoverlapping time series, allowing clear signal separation for PA image reconstruction. Our results show that the PADL technique and the handheld probe can potentially enable real-time PAT, while significantly reducing the complexity and cost of the ultrasound receiver system.
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 report a flexible shaft-based mechanical scanning photoacoustic endoscopy (PAE) system that can be potentially used for imaging the human gastrointestinal tract via the instrument channel of a clinical video endoscope. The development of such a catheter endoscope has been an important challenge to realize the technique’s benefits in clinical settings. We successfully implemented a prototype PAE system that has a 3.2-mm diameter and 2.5-m long catheter section. As the instrument’s flexible shaft and scanning tip are fully encapsulated in a plastic catheter, it easily fits within the 3.7-mm diameter instrument channel of a clinical video endoscope. Here, we demonstrate the intra-instrument channel workability and in vivo animal imaging capability of the PAE system.
Human brain mapping has become one of the most exciting contemporary research areas, with major breakthroughs expected in the coming decades. Modern brain imaging techniques have allowed neuroscientists to gather a wealth of anatomic and functional information about the brain. Among these techniques, by virtue of its rich optical absorption contrast, high spatial and temporal resolutions, and deep penetration, photoacoustic tomography (PAT) has attracted more and more attention, and is playing an increasingly important role in brain studies. In particular, PAT complements other brain imaging modalities by providing high-resolution functional and metabolic imaging. More importantly, PAT’s unique scalability enables scrutinizing the brain at both microscopic and macroscopic scales, using the same imaging contrast. In this review, we present the state-of-the-art PAT techniques for brain imaging, summarize representative neuroscience applications, outline the technical challenges in translating PAT to human brain imaging, and envision potential technological deliverables.
In order to monitor dynamic physiological events in near-real time, a variety of photoacoustic computed tomography (PACT) systems have been developed that can rapidly acquire data. Previously reported studies of dynamic PACT have employed conventional static methods to reconstruct a temporally ordered sequence of images on a frame-by-frame basis. Frame-by-frame image reconstruction (FBFIR) methods fail to exploit correlations between data frames and are known to be statistically and computationally suboptimal. In this study, a low-rank matrix estimation-based spatiotemporal image reconstruction (LRME-STIR) method is investigated for dynamic PACT applications. The LRME-STIR method is based on the observation that, in many PACT applications, the number of frames is much greater than the rank of the ideal noiseless data matrix. Using both computer-simulated and experimentally measured photoacoustic data, the performance of the LRME-STIR method is compared with that of conventional FBFIR method followed by image-domain filtering. The results demonstrate that the LRME-STIR method is not only computationally more efficient but also produces more accurate dynamic PACT images than a conventional FBFIR method followed by image-domain filtering.
We propose a calibration-free photoacoustic (PA) method for transverse flow measurements. In this method, a pulsed periodically structured (i.e., grating patterned) optical beam is used to illuminate flowing absorptive particles in an optically scattering medium. The PA signal amplitudes measured over consecutive laser pulses carry an imprint of the illumination structure. The modulation frequency of the imprint is proportional to the component of the flow speed projected onto the normal axis of the striped illumination pattern. This method can tolerate high particle density, and is insensitive to the particle size, thus calibration-free. Bovine blood and microsphere phantoms were used to validate the proposed method. Blood flow in a mouse ear was measured in vivo as well.
A decade of research has pushed photoacoustic computed tomography to the forefront of molecular-level imaging, notes SPIE Fellow Lihong Wang (Washington University, St. Louis) in his plenary talk, "Photoacoustic Tomography: Ultrasonically Beating Optical Diffusion and Diffraction."
Modern optical microscopy has resolution and diffraction limitations. But noninvasive functional photoacoustic computed tomography has overcome this limit, offering deep penetration with optical contrast and ultrasonic resolution of 1 cm depth or more -- up to 7 cm of penetration in some cases, such as evaluating sentinel lymph nodes for breast cancer staging. This opens up applications in whole body imaging, brain function, oxygen saturation, label-free cell analysis, and noninvasive cancer biopsies.
Intralipid is widely used as an optical scattering agent in tissue-mimicking phantoms. Accurate control when using Intralipid is critical to match the optical diffusivity of phantoms to the prescribed value. Currently, most protocols of Intralipid-based hydrogel phantom fabrication focus on factors such as Intralipid brand and concentration. In this note, for the first time to our knowledge, we explore the dependence of the optical reduced scattering coefficient (at 532 nm optical wavelength) on the temperature and the time of mixing Intralipid with gelatin-water solution. The studied samples contained 1% Intralipid and were measured with oblique-incidence reflectometry. It was found that the reduced scattering coefficient increased when the Intralipid-gelatin-water mixture began to solidify at room temperature. For phantoms that had already solidified completely, the diffusivity was shown to be significantly influenced by the temperature and the duration of the mixing course. The dependence of the measured diffusivity on the mixing conditions was confirmed by experimental observations. Moreover, the mechanism behind the dependence behavior is discussed.
We present a generic sub-diffraction-limited imaging method – photobleaching imprinting microscopy (PIM) – for biological fluorescence imaging. A lateral resolution of 110 nm was measured, more than a two-fold improvement over the optical diffraction limit. Unlike other super-resolution imaging techniques, PIM does not require complicated illumination modules or specific fluorescent dyes. PIM is expected to facilitate the conversion of super-resolution imaging into a routine lab tool, making it accessible to a much broader biological research community.
The invention of green fluorescent protein and other molecular fluorescent probes has promoted applications of confocal and two-photon fluorescence microscopy in biology and medicine. However, exogenous fluorescence contrast agents may affect cellular structure and function, and fluorescence microscopy cannot image nonfluorescent chromophores. We overcome this limitation by integrating optical-resolution photoacoustic microscopy into a modern Olympus IX81 confocal, two-photon, fluorescence microscope setup to provide complementary, label-free, optical absorption contrast. Automatically coregistered images can be generated from the same sample. Imaging applications in ophthalmology, developmental biology, and plant science are demonstrated. For the first time, in a familiar microscopic fluorescence imaging setting, this trimodality microscope provides a platform for future biological and medical discoveries.
Currently, laser fluence calibration is typically required for quantitative measurement of particle concentration in photoacoustic imaging. Here, we present a calibration-free method to quantify the absolute particle concentration by statistically analyzing photoacoustic signals. The proposed method is based on the fact that Brownian motion induces particle count fluctuation in the detection volume. If the count of particles in the detection volume is assumed to follow the Poisson distribution, its expected value can be calculated by the photoacoustic signal mean and variance. We first derived a theoretical model for photoacoustic signals. Then, we applied our method to quantitative measurement of different concentrations of various particles, including red blood cells. Finally, we performed in vivo experiments to demonstrate the potential of our method in biological applications. The experimental results agreed well with the predictions from the theoretical model suggesting that our method can be used for noninvasive measurement of absolute particle concentrations in deep tissue without fluence calibration.
Measuring intracellular temperature is critical to understanding many cellular functions but still remains challenging. Here we present a technique – fluorescence-assisted photoacoustic thermometry (FAPT) – for intracellular temperature mapping applications. To demonstrate FAPT, we monitored the intracellular temperature distribution of HeLa cells with sub-degree (0.7 °C) temperature resolution and sub-micron (0.23 μm) spatial resolution at a sampling rate of 1 kHz. Compared to traditional fluorescence-based methods, FAPT features the unique capability of transforming a regular fluorescence probe into a concentration- and excitation-independent temperature sensor, bringing a large collection of commercially available generic fluorescent probes into the realm of intracellular temperature sensing.
Photoacoustic computed tomography (PACT) provides structural and functional information when used in small animal brain imaging. Acoustic distortion caused by bone structures largely limits the deep brain image quality. In our work, we present ex vivo PACT images of freshly excised mouse brain, intending that can serve as a gold standard for future PACT in vivo studies on small animal brain imaging. Our results show that structures such as the striatum, hippocampus, ventricles, and cerebellum can be clearly di erentiated. An artery feature called the Circle of Willis, located at the bottom of the brain, can also be seen. These results indicate that if acoustic distortion can be accurately accounted for, PACT should be able to image the entire mouse brain with rich structural information.
Focusing light inside highly scattering media beyond the ballistic regime is a challenging task in biomedical optical imaging, manipulation, and therapy. This challenge can be overcome by time reversing ultrasonically encoded (TRUE) diffuse light to the ultrasonic focus inside a turbid medium. In TRUE optical focusing, a photorefractive crystal or polymer is used as the phase conjugate mirror for optical time reversal. Accordingly, a relatively long ultrasound burst, whose duration matches the response time of the photorefractive material, is used to encode the diffuse light. With this long ultrasound burst, the resolution of the TRUE focus along the acoustic axis is poor. In this work, we used two transducers, emitting two intersecting ultrasound beams at 3.4 MHz and 3.6 MHz respectively, to modulate the diffuse light within their intersection volume at the beat frequency. We show that light encoded at the beat frequency can be time-reversed and converge to the intersection volume. Experimentally, TRUE focusing with an acoustic axial resolution of ~1.1 mm was demonstrated inside turbid media, agreeing with the theoretical estimation.
We studied the phenomenon of photothermal bleaching — a gradual reduction of contrast agent particles during repeated scans in photoacoustic microscopy. The dependence of the photothermal bleaching rate on the excitation pulse energy was determined while the laser focal diameter was held constant. Our results showed that, the dependence of the photothermal bleaching rate on the excitation pulse energy differed before and after the absorbers were raised to their melting point by the deposited laser energy. Based on this finding, we suggested an optimal excitation pulse energy, which balances the photothermal bleaching rate and signal amplitude, for time-lapse imaging applications.
We have developed three-dimensional arbitrary trajectory (3-DAT) scanning, which can rapidly image vessels of interest over a large field of view (FOV) and maintain a high signal-to-noise ratio (SNR) along the depth direction. The concept of 3-DAT scanning was demonstrated by imaging a human hair within a FOV of 3.5 × 2.0 mm2. Further, we showed that hemoglobin oxygen saturation (sO2) and blood flow can be measured simultaneously. The frame rate was 67 times faster than a traditional two-dimensional raster scan. We also observed sO2 dynamics in response to a switch between systemic hyperoxia and hypoxia.
Wavefront distortion in scattering media can be compensated for using optical wavefront shaping. In this technique, a spatial light modulator (SLM) is used to apply a spatially distributed phase shift to the optical field. A genetic optimization algorithm was used to obtain the SLM pattern which best focuses light within the medium. The target volume is defined by using a focused ultrasound beam to encode light travelling within the acoustic focus. The ultrasonically-encoded light is measured and used as feedback to the algorithm, which then searches for the pattern which maximizes the encoded light intensity. We call this technique ultrasonically-encoded wavefront shaping (SEWS). Using SEWS, we focused light into a scattering medium consisting of ground glass diffuser and a gelatin phantom. The optical intensity at the target was increased by 11 times over the original intensity. These results were validated using fluorescent imaging at the ultrasonic focus.
We present a photoacoustic microscopy (PAM) technique with an optical sectioning capability. By combining crossoptical- beam illumination with nonlinear PAM, an axial resolution of 8.7 μm was measured, demonstrating a fourfold improvement over the acoustically determined value. Compared to methods relying on high-frequency ultrasound transducers to improve the axial resolution, our approach offers a greater working distance and a higher signal-to-noise ratio.
In current photoacoustic tomography (PAT), l-D or 2-D ultrasound arrays and multi-channel data acquisition (DAQ) electronics are used to detect the photoacoustic signals simultaneously for “real-time” image construction. However, as the number of transducer elements and DAQ channels increase, the construction and operation of the ultrasound receiving system will become complex and costly. This situation can be addressed by using parallel acoustic delay lines (PADLs) to create true time delays in multiple PA signal channels. The time-delayed PA signals will reach the ultrasound transducer at different times and therefore can be received by one single-element transducer without mixing with each other. In this paper, we report the development of the first miniaturized PADL probe suitable for handheld operations. Fusedsilica optical fibers with low acoustic attenuation were used to construct the 16 PADLs with specific time delays. The handheld probe structure was fabricated using precision laser-micromachining process to provide robust mechanical support and accurate alignment of the PADLs with minimal acoustic distortion and inter-channel coupling. The 16 optical-fiber PADLs were arranged to form one input port and two output ports. Photoacoustic imaging of a black-ink target embedded in an optically-scattering phantom was successfully conducted using the handheld PADL probe with two single-element transducers and two DAQ channels (equal to a channel reduction ratio of 8:1). Our results show that the PADL technique and the handheld probe could provide a promising solution for real-time PAT with significantly reduced complexity and cost of the ultrasound receiver system.
Photoacoustic computed tomography (PACT) holds great promise for transcranial brain imaging. However, the strong reflection, scattering and attenuation of acoustic waves in the skull present significant challenges to developing this method. We report on a systematic computer-simulation study of transcranial brain imaging using PACT. The goal of this study was to identify an effective imaging system design that can be translated for clinical use. The propagation of photoacoustic waves through a model skull was studied by use of an elastic finite-difference time-domain (FDTD) method. The acoustic radiation pattern from a photoacoustic source just beneath the skull was observed with a ring transducer array that was level with the source. The observed radiation pattern was found to contain stronger contributions from waves that were converted to shear waves in skull than longitudinal waves that did not undergo mode conversion. Images reconstructed from the pressure data that contain shear wave components possess better resolution than images reconstructed from the data that only contain the longitudinal wave signals. These observations revealed that the detection system should be designed to capture photoacoustic signals that travel through the skull in the form of shear waves as well as in the form of longitudinal waves. A preliminary investigation on the effect of the presence of absorption in the skull is also reported. This study provides an insight into the wave phenomena in transcranial PACT imaging, as well as a concrete detection design strategy that mitigates the degraded resolution of reconstructed images.
A novel method – photoacoustic recovery after photothermal bleaching (PRAP) – is proposed and implemented to study particle dynamics and medium properties at the micron scale via photoacoustic imaging. PRAP is an intuitive way to visualize as well as quantify dynamic processes in many kinds of media. We demonstrate PRAP first in a phantom study, and then in live cells. PRAP provides high signal-to-noise ratio imaging with minimal bleaching-induced artifacts during the recovery stage, ideal for monitoring the diffusive and kinetic phenomena inside a cell.
Photoacoustic computed tomography (PACT) is a hybrid technique that combines optical excitation and ultrasonic detection to provide high resolution images in deep tissues. In the image reconstruction, a constant speed of sound (SOS) is normally assumed. This assumption, however, is often not strictly satisfied in deep tissue imaging, due to acoustic heterogeneities within the object and between the object and coupling medium. If these heterogeneities are not accounted for, they will cause distortions and artifacts in the reconstructed images. In this paper, we incorporated ultrasonic computed tomography (USCT), which measures the SOS distribution within the object, into our full-ring array PACT system. Without the need for ultrasonic transmitting electronics, USCT was performed using the same laser beam as for PACT measurement. By scanning the laser beam on the array surface, we can sequentially fire different elements. As a first demonstration of the system, we studied the effect of acoustic heterogeneities on photoacoustic vascular imaging. We verified that constant SOS is a reasonable approximation when the SOS variation is small. When the variation is large, distortion will be observed in the periphery of the object, especially in the tangential direction.
Photoacoustic computed tomography (PACT) with a linear transducer array suffers from limited detection view. To increase the detection aperture, it is possible to circularly scan the linear transducer array around the object at the expense of imaging speed. Here we propose an alternative method to double or triple the detection view angle without sacrificing the imaging speed. By using a planar acoustic reflector which creates a virtual linear transducer array, the detection view angle is doubled. Similarly, by using two planar acoustic reflectors placed at 120 degrees to each other, we can form two virtual linear transducer arrays, and the detection view angle is tripled. This paper comparatively studies the two cases. We found that the planar acoustic reflectors greatly increase the detection aperture and thus significantly enhance the image quality of linear-array-base PACT systems.
Noninvasive and accurate blood flow measurement is critical for medical diagnoses. We proposed a cross-correlationbased method to quantitatively measure transverse flow velocity, using an optical-resolution photoacoustic microscope with a digital micromirror device (DMD). The DMD alternately delivers two spatially separated laser beams to the target. The slow-time photoacoustic signal profiles measured from the two beams are cross-correlated. The magnitude and sign of the time shift in the cross-correlation profile are used to simultaneously calculate the speed and direction of transverse flow. The proposed method was first demonstrated in an aqueous suspension of microspheres flowing in capillary tubing. Using 10-μm-microspheres, transverse flows in the range of 0.50–6.84 mm/s were measured with a root-mean-squared accuracy of 0.22 mm/s. Using three different sizes of microspheres (3, 6, and 10 μm in diameter), we proved experimentally that the flow measurements were independent of the particle size for flows in the velocity range of 0.55–6.49 mm/s. We also observed an expected parabolic distribution of flow velocity along the depth direction. Finally, we used this method to measure blood flow in a mouse ear in vivo.
The scanning mechanism is a major technical focus in optical-resolution photoacoustic microscopy. Flexible scanning access with fast scanning speed is desired to monitor biological and physiological dynamics with high temporal resolution. We developed random-access optical-resolution photoacoustic microscopy (RA-OR-PAM) using a digital micromirror device (DMD). Each micromirror on the DMD can be independently controlled, allowing imaging of regions of interest with arbitrary user-selected shapes without extraneous information. A global structural image is first acquired, and the regions of interest are selected. The laser beam then scans these regions exclusively, resulting in a faster frame rate than in a conventional raster scan. This system can rapidly scan arbitrarily shaped regions of interest with a lateral resolution of 3.6 μm within a 40×40 μm2 imaging area, a size comparable to the focal spot size of a 50 MHz ultrasound transducer. We demonstrated the random-access ability of RA-OR-PAM by imaging a monolayer of red blood cells. This system was then used to monitor blood flow in vivo within user-selected capillaries in a mouse ear. By imaging only the capillary of interest, the frame rate was increased by up to 13.3 times.
Proc. SPIE. 8943, Photons Plus Ultrasound: Imaging and Sensing 2014
KEYWORDS: Breast cancer, Tumors, Imaging systems, Ultrasonography, Biopsy, Acquisition tracking and pointing, Photoacoustic spectroscopy, In vivo imaging, Multichannel imaging systems, Lymphatic system
Sentinel lymph node biopsy (SLNB) has emerged as an accurate, less invasive alternative to axillary lymph node dissection, and it has rapidly become the standard of care for patients with clinically node-negative breast cancer. The sentinel lymph node (SLN) hypothesis states that the pathological status of the axilla can be accurately predicted by determining the status of the first (i.e., sentinel) lymph nodes that drain from the primary tumor. Physicians use radio-labeled sulfur colloid and/or methylene blue dye to identify the SLN, which is most likely to contain metastatic cancer cells. However, the surgical procedure causes morbidity and associated expenses. To overcome these limitations, we developed a dual-modality photoacoustic and ultrasound imaging system to noninvasively detect SLNs based on the accumulation of methylene blue dye. Ultimately, we aim to guide percutaneous needle biopsies and provide a minimally invasive method for axillary staging of breast cancer. The system consists of a tunable dye laser pumped by a Nd:YAG laser, a commercial ultrasound imaging system (Philips iU22), and a multichannel data acquisition system which displays co-registered photoacoustic and ultrasound images in real-time. Our clinical results demonstrate that real-time photoacoustic imaging can provide sensitive and specific detection of methylene blue dye in vivo. While preliminary studies have shown that in vivo detection of SLNs by using co-registered photoacoustic and ultrasound imaging is feasible, further investigation is needed to demonstrate robust SLN detection.
Time-reversed ultrasonically encoded (TRUE) optical focusing is an emerging technique that focuses light deep into scattering media by phase-conjugating ultrasonically encoded diffuse light. In previous work, the speed of TRUE focusing was limited to no faster than 1 Hz by the response time of the photorefractive phase conjugate mirror, or the data acquisition and streaming speed of the digital camera; photorefractive-crystal-based TRUE focusing was also limited to the visible spectral range. These time-consuming schemes prevent this technique from being applied in vivo, since living biological tissue has a speckle decorrelation time on the order of a millisecond. In this work, using a Tedoped Sn2P2S6 photorefractive crystal at a near-infrared wavelength of 793 nm, we achieved TRUE focusing inside dynamic scattering media having a speckle decorrelation time as short as 7.7 ms. As the achieved speed approaches the tissue decorrelation rate, this work is an important step forward toward in vivo applications of TRUE focusing in deep tissue imaging, photodynamic therapy, and optical manipulation.
The increasing use of mouse models for human brain disease studies, coupled with the fact that existing functional imaging modalities cannot be easily applied to mice, presents an emerging need for a new functional imaging modality. Utilizing acoustic-resolution photoacoustic microscopy (AR-PAM), we imaged spontaneous cerebral hemodynamic fluctuations and their associated functional connections in the mouse brain. The images were acquired noninvasively in B-scan mode with a fast frame rate, a large field of view, and a high spatial resolution. At a location relative to the bregma 0, correlations were investigated inter-hemispherically between bilaterally homologous regions, as well as intra-hemispherically within the same functional regions. The functional connectivity in different functional regions was studied. The locations of these regions agreed well with the Paxinos mouse brain atlas. The functional connectivity map obtained in this study can then be used in the investigation of brain disorders such as stroke, Alzheimer’s, schizophrenia, multiple sclerosis, autism, and epilepsy. Our experiments show that photoacoustic microscopy is capable to detect connectivities between different functional regions in B-scan mode, promising a powerful functional imaging modality for future brain research.
The axial resolution of photoacoustic microscopy (PAM) can be enhanced by reducing the speed of sound within the imaging region of interest. This principle was demonstrated on a previously-reported PAM system, which utilized a 125 MHz ultrasonic transducer for signal detection and the Wiener deconvolution for signal processing. With sound slowed by silicone oil immersion, we have achieved a finest axial resolution of 5.8 μm for PAM, as validated by phantom experiments. The axial resolution was also enhanced in vivo when mouse ears injected with silicone oil were imaged. After injection of silicone oil, the blood vessels were resolved more clearly. When tissue-compatible low-speed liquids become available, this approach may find applications in PAM as well as in other imaging modalities, such as photoacoustic computed tomography and ultrasound imaging.
Both the spatial resolution and maximum penetration depth of optical-resolution photoacoustic microscopy (ORPAM) deteriorate sharply with depth due to strong light scattering in tissue. To reduce tissue scattering, we propose to use glycerol as an optical clearing agent in OR-PAM. Our results show that the imaging performance of OR-PAM can be greatly enhanced by optical clearing both in vitro and in vivo.
Resting-state functional connectivity (RSFC) imaging is an emerging neuroimaging approach that aims to identify spontaneous cerebral hemodynamic fluctuations and their associated functional connections. Clinical studies have demonstrated that RSFC is altered in brain disorders such as stroke, Alzheimer’s, autism, and epilepsy. However, conventional neuroimaging modalities cannot easily be applied to mice, the most widely used model species for human brain disease studies. For instance, functional magnetic resonance imaging (fMRI) of mice requires a very high magnetic field to obtain a sufficient signal-to-noise ratio and spatial resolution. Functional connectivity mapping with optical intrinsic signal imaging (fcOIS) is an alternative method. Due to the diffusion of light in tissue, the spatial resolution of fcOIS is limited, and experiments have been performed using an exposed skull preparation. In this study, we show for the first time, the use of photoacoustic computed tomography (PACT) to noninvasively image resting-state functional connectivity in the mouse brain, with a large field of view and a high spatial resolution. Bilateral correlations were observed in eight regions, as well as several subregions. These findings agreed well with the Paxinos mouse brain atlas. This study showed that PACT is a promising, non-invasive modality for small-animal functional brain imaging.
Many breast cancer patients receive neoadjuvant treatment to reduce tumor size and enable breast conserving therapy. Most imaging methods used to monitor response to neoadjuvant chemotherapy or hormone therapy depend on overall gross tumor morphology and size measurements, which may not be sensitive or specific, despite tumor response on a cellular level. A more sensitive and specific method of detecting response to therapy might allow earlier adjustments in treatment, and thus result in better outcomes while avoiding unnecessary morbidity. We developed an imaging system that combines spectral photoacoustic tomography and ultrasonography to predict breast neoadjuvant therapeutic response based on blood volume and blood oxygenation contrast. The system consists of a tunable dye laser pumped by a Nd:YAG laser, a commercial ultrasound imaging system (Philips iU22), and a multichannel data acquisition system which displays co-registered photoacoustic and ultrasound images in real time. Early studies demonstrate functional imaging capabilities, such as oxygen saturation and total concentration of hemoglobin, in addition to ultrasonography of tumor morphology. Further study is needed to determine if the co-registered photoacoustic tomography and ultrasonography system may provide an accurate tool to assess treatment efficacy by monitoring tumor response in vivo.
We propose time and frequency domain methods for homogenous flow measurement based on the photoacoustic Doppler effect. Excited by spatially modulated laser pulses, the flowing medium induces a Doppler frequency shift in the received photoacoustic signals. The frequency shift is proportional to the component of the flow speed projected onto the acoustic beam axis. These methods do not rely on particle heterogeneity in the medium. A red-ink phantom flowing in a tube immersed in water was used to validate the methods in both frequency and time domains.
To achieve localized light delivery beyond turbid layers, TRUE optical focusing has been previously implemented by both analog and digital devices. The digital scheme offers a higher energy gain than the analog version. In many biological applications, the reflection-mode configuration, which uses backscattered light from the sample, is more suitable than the transmission-mode configuration. Although reflection-mode analog TRUE focusing has been demonstrated, its digital implementation has not been explored. Here, we report a reflection-mode digital TRUE focusing to concentrate light through a turbid layer. Further, by simply moving the ultrasound focus, we show the system's dynamic focusing capability.
The metabolic rate and oxygen consumption of the brain is reflected in jugular venous oxygen saturation. In many clinical conditions, such as head trauma, stroke, and low cardiac output states, the brain is at risk for hypoxic-ischemic injury. The current gold standard for monitoring brain oxygenation is invasive and requires jugular vein catheterization under fluoroscopic guidance; and therefore it is rarely used. Photo-acoustic tomography in combination with ultrasound can be used to estimate oxygen saturation of the internal jugular vein in real-time. This noninvasive method will enable earlier detection and prevention of impending hypoxic brain injury. A wavelength-tunable dye laser pumped by a Nd:YAG laser delivers light through an optical fiber bundle, and a modified commercial ultrasound imaging system (Philips iU22) detects both the pulse-echo ultrasound (US) and photoacoustic (PA) signals. A custom-built multichannel data acquisition system renders co-registered ultrasound and photoacoustic images at 5 frames per second. After the jugular vein was localized in healthy volunteers, dualwavelength PA images were used to calculate the blood hemoglobin oxygen saturation from the internal jugular vein in vivo. The preliminary results raise confidence that this emerging technology can be used clinically as an accurate, noninvasive indicator of cerebral oxygenation.
Photoacoustic (PA) tomography imaging is an emerging, versatile, and noninvasive imaging modality, which combines the advantages of both optical imaging and ultrasound imaging. It opens up opportunities for noninvasive imaging of angiogenesis, a feature of skin pathologies including cancers and psoriasis. In this study, high-density copper oleate encapsulated within a phospholipid surfactant (CuNPs) generated a soft nanoparticle with PA contrast comparable to gold. Within the near-infrared window, the copper nanoparticles can provide a signal more than 7 times higher that of blood. ανβ3-targeted of CuNPs in a Matrigel mouse model demonstrated prominent PA contrast enhancement of the neovasculature compared to mice given nontargeted or competitively inhibited CuNPs. Incorporation of a sn-2 lipase-labile fumagillin prodrug into the CuNPs produced marked antiangiogenesis in the same model, demonstrating the theranostic potential of a PA agent for the first time in vivo. With a PA signal comparable to gold-based nanoparticles yet a lower cost and demonstrated drug delivery potential, ανβ3-targeted CuNPs hold great promise for the management of skin pathologies with neovascular features.
Controllable light delivery to the region of interest is essential to biomedical optical imaging methods like photoacoustic microscopy. It is, however, challenging beyond superficial depths in biological tissue (~1 mm beneath human skin) due to the strong scattering of light that scrambles the photon propagation paths. Recently, optical wavefront shaping has been proposed to modulate the incident light wavefront to compensate for the scattering-induced phase distortions, and consequentially, convey light optimally to a desired location behind or inside turbid media. To reach an optimum wavefront, a searching algorithm is usually required to optimize a feedback signal. In this work, we present our latest explorations, which use photoacoustic signals as the feedback to remotely and non-invasively guide the wavefront shaping process. Our method does not require direct optical access to the target region or the invasive embedding of fluorescence probes inside turbid media. Experimentally, we have demonstrated that diffuse light can be converged to the ultrasound focus by maximizing the amplitude of photoacoustic emissions from the intended absorbing site. Moreover, we show that wavefront-shaped light focusing can enhance existing optical imaging modalities like photoacoustic microscopy, in regard to signal-to-noise ratio, imaging depth, and potentially, resolution.
To focus light beyond one transport mean free path, time-reversed ultrasonically encoded (TRUE) optical focusing has previously been implemented by both analog and digital devices. By allowing wavefront recording with finer resolution and larger aperture, the analog scheme, which uses photorefractive materials as the phase-conjugate mirror, generates a more complete set of time-reversed optical modes than the digital scheme. Here, we report the direct visualization of localized fluorescence excitation inside a turbid medium by photorefractive time reversal. Further, we imaged fluorescent targets embedded in a turbid phantom whose thickness was four transport mean free paths.
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 mm2) within 150 seconds, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical-based contour scanning, we can maintain the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is ideal for fast and quantitative studies of tumors and the brain.
An important and interesting question in photoacoustic computed tomography (PACT) is whether the absorbed optical energy density distribution, A(r), and the speed of sound distribution, c(r), can both be accurately determined from the measured photoacoustic data alone. However, in many cases c(r) is unknown or cannot be accurately estimated. Therefore, it would be practically beneficial if A(r) and c(r) can be jointly reconstructed from the measurements. In this work, we propose a reconstruction approach to the joint reconstruction of both properties in PACT.
Currently, laser fluence calibration is typically required for quantitative measurement of particle concentration in photoacoustic microscopy. In this paper, we present another quantitative approach to measure absolute absorber concentrations by photoacoustic correlation spectroscopy. The proposed method is based on the fact that the Brownian motion induces particle count fluctuation in the detection volume. We first derived a theoretical model for photoacoustic signals and then applied our method to quantitative measurement of different concentrations of various particles. The experimental results agreed well with the predictions from the theoretical model, suggesting that our method can be used for absolute particle concentrations measurement.
In order to monitor dynamic physiological events in near-real time, a variety of photoacoustic computed tomography (PACT) systems have been developed that can rapidly acquire data. Previously reported studies of dynamic PACT have employed conventional static methods to reconstruct a temporally ordered sequence of images on a frame-by-frame basis. Frame-by-frame image reconstruction (FBFIR) methods fail to exploit correlations between data frames and are known to be statistically and computationally suboptimal. In this study, a low-rank matrix estimation-based spatio-temporal image reconstruction (LRME-STIR) method is investigated for dynamic PACT applications. The LRME-STIR method is based on the observation that, in many PACT applications, the number of frames is much greater than the rank of the ideal noiseless data matrix. Using computer-simulated photoacoustic data, the performance of the LRME-STIR method is compared with that of conventional FBFIR method. The results demonstrate that LRME-STIR method is not only computationally more efficient but also produces more accurate dynamic PACT images than a conventional FBFIR method.
Photoacoustic (PA) techniques can measure temperature in biological tissues because PA signal amplitude is sensitive to tissue temperature. So far, temperature-measuring PA techniques have focused on sensing of temperature changes at a single position. In this work, we photoacoustically measured spatial distribution of temperature in deep tissue. By monitoring the temperature at a single position using a thermocouple, the relationship between the PA signal amplitude and the actual temperature was determined. The relationship was then used to translate a PA image into a temperature map. This study showed that it is possible to calibrate the system for the temperature range of hyperthermia using single-point measurements over a smaller temperature range. Our experimental results showed a precision of −0.8±0.4°C (mean±standard error ) in temperature measurement, and a spatial resolution as fine as 1.0 mm. PA techniques can be potentially applied to monitor temperature distribution deep in tissue during hyperthermia treatment of cancer.
The Grüneisen parameter, a constitutive parameter in photoacoustics, is usually measured from isobaric thermal expansion, which may not be valid for a biological medium due to its heterogeneity. Here, we directly measured the Grüneisen parameter by applying photoacoustic spectroscopy. Laser pulses at wavelengths between 460 and 1800 nm were delivered to tissue samples, and photoacoustic signals were detected by flat water-immersion ultrasonic transducers. Least-squares fitting photoacoustic spectra to molar optical absorption spectra showed that the Grüneisen parameter was 0.81±0.05 (mean±SD ) for porcine subcutaneous fat tissue and 0.69±0.02 for porcine lipid at room temperature (22°C). The Grüneisen parameter of a red blood cell suspension was linearly related to hemoglobin concentration, and the parameter of bovine serum was 9% greater than that of water at room temperature.
Peripheral neuropathy is a common neurological problem that affects millions of people worldwide. Diagnosis and treatment of this condition are often hindered by the difficulties in making objective, noninvasive measurements of nerve fibers. Photoacoustic microscopy (PAM) has the ability to obtain high resolution, specific images of peripheral nerves without exogenous contrast. We demonstrated the first proof-of-concept imaging of peripheral nerves using PAM. As validated by both standard histology and photoacoustic spectroscopy, the origin of photoacoustic signals is myelin, the primary source of lipids in the nerves. An extracted sciatic nerve sandwiched between two layers of chicken tissue was imaged by PAM to mimic the in vivo case. Ordered fibrous structures inside the nerve, caused by the bundles of myelin-coated axons, could be observed clearly. With further technical improvements, PAM can potentially be applied to monitor and diagnose peripheral neuropathies.
Photoacoustic tomography (PAT) is an emerging technique that has a great potential for preclinical whole-body imaging. To date, most whole-body PAT systems require multiple laser shots to generate one cross-sectional image, yielding a frame rate of <1 Hz . Because a mouse breathes at up to 3 Hz, without proper gating mechanisms, acquired images are susceptible to motion artifacts. Here, we introduce, for the first time to our knowledge, retrospective respiratory gating for whole-body photoacoustic computed tomography. This new method involves simultaneous capturing of the animal’s respiratory waveform during photoacoustic data acquisition. The recorded photoacoustic signals are sorted and clustered according to the respiratory phase, and an image of the animal at each respiratory phase is reconstructed subsequently from the corresponding cluster. The new method was tested in a ring-shaped confocal photoacoustic computed tomography system with a hardware-limited frame rate of 0.625 Hz. After respiratory gating, we observed sharper vascular and anatomical images at different positions of the animal body. The entire breathing cycle can also be visualized at 20 frames/cycle .
The versatility and real-time imaging capability of commercial linear array transducers make them widely used in clinical ultrasound and photoacoustic imaging. However, they often suffer from limited detection view. For instance, acoustic waves traveling at a grazing angle to the transducer surface are difficult to detect. In this letter, we propose a simple and easy approach to ameliorate this problem by using a 45-deg acoustic reflector. The reflector forms a virtual array that is perpendicular to the physical array, thereby doubling the detection coverage. The improvement in image quality in photoacoustic tomography was demonstrated through a hair phantom, a leaf skeleton phantom, and an ex vivo mouse ear experiment.
Focusing light inside highly scattering media is a challenging task in biomedical optical imaging, manipulation, and therapy. A recent invention has overcome this challenge by time reversing ultrasonically encoded diffuse light to an ultrasound-modulated volume inside a turbid medium. In this technique, a photorefractive (PR) crystal or polymer can be used as the phase conjugate mirror for optical time reversal. Accordingly, a relatively long ultrasound burst, whose duration matches the PR response time of the PR material, is usually used to encode the diffuse light. This long burst results in poor focusing resolution along the acoustic axis. In this work, we propose to use two intersecting ultrasound beams, emitted from two ultrasonic transducers at different frequencies, to modulate the diffuse light at the beat frequency within the intersection volume. We show that the time reversal of the light encoded at the beat frequency can converge back to the intersection volume. Experimentally, an acoustic axial resolution of ∼1.1 mm was demonstrated inside turbid media, agreeing with theoretical estimation.
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.
We present an innovative method, photoacoustic recovery after photothermal bleaching (PRAP), for studying particle dynamics at micron scale via photoacoustic imaging. As an intuitive way to visualize and quantify dynamic processes, PRAP is demonstrated first in a simple phantom study and then in a more complex measurement involving live cells. Compared with the conventional fluorescence-based approach, PRAP provides high signal-to-noise ratio (SNR) imaging with minimal bleaching-induced artifacts during the recovery stage, ideal for monitoring the diffusive and kinetic processes inside a cell.
The fundamental limitations of photoacoustic microscopy for detecting optically absorbing molecules are investigated both theoretically and experimentally. We experimentally demonstrate noise-equivalent detection sensitivities of 160,000 methylene blue molecules (270 zeptomol or 2.7×10 −19 mol ) and 86,000 oxygenated hemoglobin molecules (140 zeptomol) using narrowband continuous-wave photoacoustics. The ultimate sensitivity of photoacoustics is fundamentally limited by thermal noise, which can present in the acoustic detection system as well as in the medium itself. Under the optimized conditions described herein and using commercially available detectors, photoacoustic microscopy can detect as few as 100s of oxygenated hemoglobin molecules. Realizable improvements to the detector may enable single molecule detection of select molecules.
A cross-correlation-based method is proposed to quantitatively measure transverse flow velocity using optical resolution photoacoustic (PA) microscopy enhanced with a digital micromirror device (DMD). The DMD is used to alternately deliver two spatially separated laser beams to the target. Through cross-correlation between the slow-time PA profiles measured from the two beams, the speed and direction of transverse flow are simultaneously derived from the magnitude and sign of the time shift, respectively. Transverse flows in the range of 0.50 to 6.84 mm/s are accurately measured using an aqueous suspension of 10-μm-diameter microspheres, and the root-mean-squared measurement accuracy is quantified to be 0.22 mm/s . The flow measurements are independent of the particle size for flows in the velocity range of 0.55 to 6.49 mm/s , which was demonstrated experimentally using three different sizes of microspheres (diameters: 3, 6, and 10 μm). The measured flow velocity follows an expected parabolic distribution along the depth direction perpendicular to the flow. Both maximum and minimum measurable velocities are investigated for varied distances between the two beams and varied total time for one measurement. This technique shows an accuracy of 0.35 mm/s at 0.3-mm depth in scattering chicken breast, making it promising for measuring flow in biological tissue.
Förster resonance energy transfer (FRET) is a distance-dependent process that transfers excited state energy from a donor molecule to an acceptor molecule without the emission of a photon. The FRET rate is determined by the proximity between the donor and the acceptor molecules; it becomes significant only when the proximity is within several nanometers. Therefore, FRET has been applied to visualize interactions and conformational changes of biomolecules, such as proteins, lipids, and nucleic acids that cannot be resolved by optical microscopy. Here, we report photoacoustic tomography of FRET efficiency at a 1-cm depth in chicken breast tissue, whereas conventional high-resolution fluorescence imaging is limited to <0.1 cm . Photoacoustic tomography is expected to facilitate the examination of FRET phenomena in living organisms.
For both ultrasound and photoacoustic microscopic imaging, a fast scanning ability is required, whereas the liquid
environment for acoustic propagation limits the usage of traditional MEMS scanning mirrors. In this paper, a new waterimmersible
scanning mirror microsystem has been designed, fabricated and tested. To achieve reliable underwater
scanning, flexible polymer torsion hinges fabricated by laser micromachining were used to support the reflective silicon
mirror plate. Two efficient electromagnetic microactuators consisting of compact RF choke inductors and high-strength
neodymium magnet disc were constructed to drive the silicon mirror plate around a fast axis and a slow axis,
respectively. The performance of the water-immersible scanning mirror microsystem in both air and water were tested
using the laser tracing method. For the fast axis, the resonance frequency reached 224 Hz in air and 164 Hz in water,
respectively. The scanning angles in air and water under ±10 V AC driving (at the resonance frequencies) were ±13.6°
and ±10°. The scanning angles in both air and water under ±16 V DC driving were ±12°. For the slow axis, the resonance
frequency reached 55 Hz in air and 38 Hz in water, respectively. The scanning angles in air and water under ±10 V AC
driving (at the resonance frequencies) were ±8.5° and ±6°. The scanning angles in both air and water under ±10 V DC
driving were ± 6.5°. The feasibility of using such a water-immersible scanning mirror microsystem for scanning
ultrasound microscopic (SAM) imaging has been demonstrated with a 25-MHz ultrasound pulse/echo system and a
target consisting of three optical fibers.
Achieving real-time photoacoustic (PA) tomography typically requires massive ultrasound transducer arrays and data
acquisition (DAQ) electronics to receive PA waves simultaneously. In this paper, we report the first demonstration of a
photoacoustic tomography (PAT) system using optical fiber-based parallel acoustic delay lines (PADLs). By employing
PADLs to introduce specific time delays, the PA signals (on the order of a few micro seconds) can be forced to arrive at
the ultrasonic transducers at different times. As a result, time-delayed PA signals in multiple channels can be ultimately
received and processed in a serial manner with a single-element transducer, followed by single‐channel DAQ electronics. Our results show that an optically absorbing target in an optically scattering medium can be photoacoustically imaged using the newly developed PADL-based PAT system. Potentially, this approach could be adopted to significantly reduce the complexity and cost of ultrasonic array receiver systems.
A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature.
With 3 seconds per frame imaging speed, a temperature resolution of 0.2 °C was achieved in a photo-thermal cell heating
experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring
custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell
thermometry into a routine lab tool and make it accessible to a much broader biological research community.
Recently, a number of optical imaging modalities have achieved single molecule sensitivity, including photothermal
imaging, stimulated emission microscopy, ground state depletion microscopy, and transmission microscopy. These
optical techniques are based on optical absorption contrast, extending single-molecule detection to non-fluorescent
chromophores. Photoacoustics is a hybrid technique that utilizes optical excitation and ultrasonic detection, allowing it to
scale both the optical and acoustic regimes with 100% sensitivity to optical absorption. However, the sensitivity of
photoacoustics is limited by thermal noise, inherent in the medium itself in the form of acoustic black body radiation. In
this paper, we investigate the molecular sensitivity of photoacoustics in the context of the thermal noise limit. We show
that single molecule sensitivity is achievable theoretically at room temperature for molecules with sufficiently fast
relaxation times. Hurdles to achieve single molecule sensitivity in practice include development of detection schemes
that work at short working distance, <100 microns, high frequency, <100 MHz, and low loss, <10 dB.
Proc. SPIE. 8581, Photons Plus Ultrasound: Imaging and Sensing 2013
KEYWORDS: Biomedical optics, Luminescence, Energy transfer, Resonance energy transfer, Photoacoustic microscopy, Photoacoustic spectroscopy, In vivo imaging, Fluorescence resonance energy transfer, Tissue optics, Rhodamine
Förster resonance energy transfer (FRET) provides fluorescence signals sensitive to intra- and inter-molecular
distances in the 1-10 nm range. Widely applied in the optical imaging environment, FRET enables visualization of
physicochemical processes in molecular interactions and conformation changes. We reported photoacoustic imaging
of FRET, based on non-radiative decay that produces heat and subsequent acoustic waves. The experimental results
show that photoacoustic imaging offers better penetration into scattering biological tissue. Through its ability to
three-dimensionally image tissue with scalable resolution, photoacoustic microscopy provides a beneficial
biomedical tool to broaden the in vivo application of the FRET technique.
The Grüneisen parameter of tissue is a constitutive parameter in photoacoustic tomography. Here, we applied
photoacoustic spectrometry (PAS) to directly measure the Grüneisen parameter. In our PAS system, laser pulses at
wavelengths between 460 and 1600 nm were delivered to tissue samples, and photoacoustic signals were detected by a 20 MHz flat water-immersion ultrasonic transducer. By fitting photoacoustic spectra to light absorption spectra, we
found that the Grüneisen parameter was 0.73 for porcine subcutaneous fat tissue, and 0.15 for oxygenated bovine red
blood cells at room temperature (24°C).
Blood pulse wave velocity (PWV) is an important indicator for vascular stiffness. In this letter, we present
electrocardiogram-synchronized photoacoustic microscopy for in vivo noninvasive quantification of the PWV in the
peripheral vessels of mice. Interestingly, strong correlation between blood flow speed and ECG were clearly
observed in arteries but not in veins. PWV is measured by the pulse travel time and the distance between two spot of
a chose vessel, where simultaneously recorded electrocardiograms served as references. Statistical analysis shows a
linear correlation between the PWV and the vessel diameter, which agrees with known physiology.
Keywords: photoacoustic microscopy, photoacoustic spectroscopy, bilirubin, scattering medium.
We demonstrated the potential of carbon nanoparticles (CNPs) as exogenous contrast agents for both thermoacoustic
(TA) tomography (TAT) and photoacoustic (PA) tomography (PAT). In comparison to deionized water, the CNPs
provided a four times stronger signal in TAT at 3 GHz. In comparison to blood, The CNPs provided a much stronger
signal in PAT over a broad wavelength range of 450-850 nm. Specifically, the maximum signal enhancement in PAT
was 9.4 times stronger in the near-infrared window of 635-670 nm. In vivo blood-vessel PA imaging was performed
non-invasively on a mouse femoral area. The images, captured after the tail vein injection of CNPs, show a gradual
enhancement of the optical absorption in the vessels by up to 230%. The results indicate that CNPs can be potentially
used as contrast agents for TAT and PAT to monitor the intravascular or extravascular pathways in clinical applications.
It is a challenge to non-invasively visualize in vivo the neovascularization in a three-dimensional (3D) scaffold with high
spatial resolution and deep penetration depth. Here we used photoacoustic microscopy (PAM) to chronically monitor
neovascularization in an inverse opal scaffold implanted in a mouse model for up to six weeks. The neovasculature was
observed to develop gradually in the same mouse. These blood vessels not only grew on top of the implanted scaffold
but also penetrated into the scaffold. The PAM system offered a lateral resolution of ~45 μm and a penetration depth of ~3 mm into the scaffold/tissue construct. By using the 3D PAM data, we further quantified the vessel area as a function
Photoacoustic computed tomography (PACT) is an emerging imaging technique which is based on the acoustic detection
of optical absorption from tissue chromophores, such as oxy-hemoglobin and deoxy-hemoglobin. An important
application of PACT is functional brain imaging of small animals. The conversion of light to acoustic waves allows
PACT to provide high resolution images of cortical vasculatures through the intact scalp. Here, PACT was utilized to
study the activated areas of the mouse brain during forepaw and hindpaw stimulations. Temporal PACT images were
acquired enabling computation of hemodynamic changes during stimulation. The stimulations were performed by trains
of pulses at different stimulation currents (between 0.1 to 2 mA) and pulse repetition rates (between 0.05 Hz to 0.01Hz).
The response at somatosensory cortex-forelimb, and somatosensory cortex-hindlimb, were investigated. The Paxinos
mouse brain atlas was used to confirm the activated regions. The study shows that PACT is a promising new technology
that can be used to study brain functionality with high spatial resolution.
Like ultrasound endoscopy, photoacoustic endoscopy (PAE) could become a valuable addition to clinical practice due
to its deep imaging capability. Results from our recent in vivo transesophageal endoscopic imaging study on rabbits
demonstrate the technique’s capability to image major organs in the mediastinal region, such as the lung, trachea, and
cardiovascular systems. Here, we present various features from photoacoustic images from the mediastinal region of
several rabbits and discuss possible clinical contributions of this technique and directions of future technology
Due to the wide use of animals for human disease studies, small animal whole-body imaging plays an increasingly
important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical
and glucose metabolic information, leading to higher costs of building dual-modality systems. Even with image coregistration,
the spatial resolution of the metabolic imaging modality is not improved. We present a ring-shaped confocal
photoacoustic computed tomography (RC-PACT) system that can provide both assessments in a single modality.
Utilizing the novel design of confocal full-ring light delivery and ultrasound transducer array detection, RC-PACT
provides full-view cross-sectional imaging with high spatial resolution. Scanning along the orthogonal direction provides
three-dimensional imaging. While the mouse anatomy was imaged with endogenous hemoglobin contrast, the glucose
metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose. Through mouse tumor models, we
demonstrate that RC-PACT may be a paradigm shifting imaging method for preclinical research.
Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we
implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM
system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking
samples, both without and with blood mixed. Our results show that PAM has the potential to
quantitatively image bilirubin in vivo for clinical applications.
Time-reversed ultrasonically encoded (TRUE) optical focusing focuses light beyond one transport mean free
path by phase-conjugating the ultrasonically tagged light. However, in previous works, only a small portion of the tagged
light was phase-conjugated by using a photorefractive Bi12SiO20 crystal, due to its small active area (1x1 cm2). In this work, we report high-efficiency TRUE focusing using a large-area photorefractive polymer (5x5 cm2), which
demonstrated ~40 times increase in focused energy. Further, we imaged absorbers embedded in a turbid sample of
thickness of ~12 transport mean free paths.
We report the development of photoacoustic microscopy capable of video-rate high-resolution in-vivo
imaging in deep tissue. A lightweight photoacoustic probe is made of a single-element
broadband ultrasound transducer, a compact photoacoustic beam combiner, and a bright-field light
delivery system. Focused broadband ultrasound detection provides a 44-μm lateral resolution and a
28-μm axial resolution. A multimode optical fiber is used to deliver laser pulses. The bright-field
light delivery system can effectively improve the illumination efficiency. The photoacoustic probe
weighs less than 40 grams and is mounted on a voice-coil scanner to acquire 40 cross-sectional
images per second over several-mm range. The fast speed can effectively improve imaging
throughput, reduce motion artifacts, and enable the visualization of highly dynamic biomedical
processes. High-resolution micro-vascular imaging is successfully demonstrated.
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
There remains an urgent need to develop effective photoacoustic computed tomography (PACT) image recon-
struction methods for use with acoustically inhomogeneous media. Transcranial PACT brain imaging is an im-
portant example of an emerging imaging application that would benefit greatly from this. Existing approaches
to PACT image reconstruction in acoustically heterogeneous media are limited to weakly varying media, are
computationally burdensome, and/or make impractical assumptions regarding the measurement geometry. In
this work, we develop and investigate a full-wave approach to iterative image reconstruction in PACT for media
possessing inhomogeneous speed-of-sound and mass density distributions. A key contribution of the work is the
formulation of a procedure to implement a matched discrete forward and backprojection operator pair, which
facilitates the application of a wide range of modern iterative image reconstruction algorithms. This presents
the opportunity to employ application-specific regularization methods to mitigate image artifacts due to mea-
surement data incompleteness and noise. Our results establish that the proposed image reconstruction method
can effectively compensate for acoustic aberration and reduces artifacts in the reconstructed image.
We have developed a new photoacoustic endoscopic probe specifically designed for human urogenital imaging. The
endoscopic probe uses a parabolic mirror-based mechanical scanning mechanism, providing an angular field of view of 270°. And it has a rigid, dome shaped end section for smooth cavity introduction. Here we introduce the new
endoscope’s design and imaging principle, and present experimental results validating its in vivo imaging ability.
The axial resolution of photoacoustic microscopy (PAM) is much lower than its lateral resolution, which resolves down
to the submicron level. Here we achieved so far the highest axial resolution of 7.6 μm by using a commercial 125 MHz
ultrasonic transducer for signal detection, followed by the Wiener deconvolution for signal processing. The axial
resolution was validated by imaging two layers of red ink in a wedge shape. Melanoma cells were imaged ex vivo with
high axial resolution. Compared with a PAM system with a 50 MHz ultrasonic transducer, our high-axial-resolution
PAM system resolved the blood vessels in mouse ears in vivo much more clearly in the depth direction.
In current photoacoustic tomography (PAT) systems, ultrasound transducer arrays and multi-channel data acquisition (DAQ) electronics are used to receive the PA signals. To achieve real-time PA imaging, massive 1D or even 2D transducer arrays and large number of DAQ channels are necessary. As a result, the ultrasound receiver becomes very complex, bulky and also costly. In this paper, we report the development of novel micromachined silicon acoustic delay line systems, which are expected to provide a new approach to address the above issue. First, fundamental building block structures of the acoustic delay line systems were designed and fabricated. Their acoustic properties were characterized with ultrasound and photoacoustic measurements. Second, two different acoustic delay line systems (parallel and serial) were designed and fabricated using advanced micromachining processes to ensure compact size, high accuracy, and good repeatability. The transmission of multiple acoustic signals in the acoustic delay line systems were studied with ultrasound experiment. Experimental results show that the silicon acoustic delay line systems can guide multiple channels of acoustic signals with low loss and distortion. With the addition of a set of suitable time delays, the time-delay acoustic signals arrived at a single-element transducer at different times and were unambiguously received and processed by the following DAQ electronics. Therefore, the micromachined silicon acoustic delay line systems could be used to combine multiple signal channels into a single one (without the involvement of electronic multiplexing), thereby reducing the complexity and cost of the ultrasound receiver for real-time PAT application.
We performed a photoacoustic endoscopic imaging study of melanoma tumor growth in a nude rat in vivo. After
inducing the tumor at the colorectal wall of the animal, we monitored the tumor development in situ by using a
photoacoustic endoscopic system. This paper introduces our experimental method for tumor inoculation and presents
imaging results showing the morphological changes of the blood vasculature near the tumor region according to the
tumor progress. Our study could provide insights for future studies on tumor development in small animals.
Compared with single-focus optical-resolution photoacoustic microscopy (OR-PAM), multifocal OR-PAM utilizes both
multifocal optical illumination and an ultrasonic array transducer, significantly increasing the imaging speed. Here we
present a reflection-mode multifocal OR-PAM system based on a microlens array that provides multiple foci and an
ultrasonic array transducer that receives the excited photoacoustic waves from all foci simultaneously. By using a
customized microprism to reflect the incident laser beam to the microlens array, we align the multiple optical foci
confocally with the focal zone of the ultrasonic array transducer. Experiments show our reflection-mode multifocal ORPAM
system is capable of imaging microvessels in vivo, and it can image a 9 mm x 5 mm x 2.5 mm volume at 16 μm
lateral resolution in ~4 min, limited by the signal multiplexing ratio and laser pulse repetition rate.
For years, ultrasound-modulated optical tomography (UOT) has been proposed to image optical contrasts deep inside
turbid media (such as biological tissue) at an ultrasonic spatial resolution. The reported imaging depth so far, however,
has been limited, preventing this technique from finding broader applications. In this work, we present our latest
experimental explorations that push UOT to clinically useful imaging depths, achieved through optimizing from different
aspects. One improvement is the use of a large aperture fiber bundle, which more effectively collects the diffused light,
including both ultrasound-modulated and unmodulated portions, from the turbid sample and then sends it to the
photorefractive material. Another endeavor is employment of a large aperture photorefractive polymer film for
demodulating the ultrasound-induced phase modulation. Compared with most UOT detection schemes, the polymer film
based setup provides a much higher etendue as well as photorefractive two-beam-coupling gain. Experimentally, we have
demonstrated enhanced sensitivity and have imaged through tissue-mimicking samples up to 9.4 cm thick at the
ultrasonically-determined spatial resolutions.
To control the overall action of the body, brain consumes a large amount of energy in proportion to its volume. In
humans and many other species, the brain gets most of its energy from oxygen-dependent metabolism of glucose. An
abnormal metabolic rate of glucose and/or oxygen usually reflects a diseased status of brain, such as cancer or
Alzheimer’s disease. We have demonstrated the feasibility of imaging mouse brain metabolism using photoacoustic
computed tomography (PACT), a fast, noninvasive and functional imaging modality with optical contrast and acoustic
resolution. Brain responses to forepaw stimulations were imaged transdermally and transcranially. 2-NBDG, which
diffuses well across the blood-brain-barrier, provided exogenous contrast for photoacoustic imaging of glucose response.
Concurrently, hemoglobin provided endogenous contrast for photoacoustic imaging of hemodynamic response. Glucose
and hemodynamic responses were quantitatively unmixed by using two-wavelength measurements. We found that
glucose uptake and blood perfusion around the somatosensory region of the contralateral hemisphere were both
increased by stimulations, indicating elevated neuron activity. The glucose response amplitude was about half that of the
hemodynamic response. While the glucose response area was more homogenous and confined within the somatosensory
region, the hemodynamic response area showed a clear vascular pattern and spread about twice as wide as that of the
glucose response. The PACT of mouse brain metabolism was validated by high-resolution open-scalp OR-PAM and
fluorescence imaging. Our results demonstrate that 2-NBDG-enhanced PACT is a promising tool for noninvasive studies
of brain metabolism.
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.
Compared with single-focus optical-resolution photoacoustic microscopy (OR-PAM), multifocal OR-PAM utilizes both multifocal optical illumination and an ultrasonic array transducer, significantly increasing the imaging speed. A reflection-mode multifocal OR-PAM system based on a microlens array that provides multiple foci as well as an ultrasonic array transducer that receives the excited photoacoustic waves from all foci simultaneously is presented. Using a customized microprism to reflect the incident laser beam to the microlens array, the multiple optical foci are aligned confocally with the focal zone of the ultrasonic array transducer. Experiments show the reflection-mode multifocal OR-PAM is capable of imaging microvessels in vivo, and it can image a 6×5×2.5 mm 3 volume at 16 μm lateral resolution in ∼2.5 min , which was limited by the signal multiplexing ratio and laser pulse repetition rate.
JBO sustained its healthy growth in 2012. The number of manuscript submissions increased by 5% to 788. JBO’s success is attributed to the contributions of the authors, reviewers, guest editors, and editorial board members.
A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With three-seconds-per-frame imaging speed, a temperature resolution of 0.2°C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community.
Photoacoustic microscopy (PAM) has achieved submicron lateral resolution in showing subcellular structures; however, relatively few endogenous subcellular contrasts have so far been imaged. Given that the hemeprotein, mostly cytochromes in general cells, is optically absorbing around the Soret peak (∼420 nm ), we implemented label-free PAM of cytochromes in cytoplasm for the first time. By measuring the photoacoustic spectra of the oxidized and reduced states of fibroblast lysate and fitting the difference spectrum with three types of cytochromes, we found that the three cytochromes account for more than half the optical absorption in the cell lysate at 420 nm wavelength. Fixed fibroblasts on slides were imaged by PAM at 422 and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard staining histology. PAM was also applied to label-free histology of mouse ear sections by showing cytoplasms and nuclei of various cells. PAM of cytochromes in cytoplasm is expected to be a high-throughput, label-free technique for studying live cell functions, which cannot be accomplished by conventional histology.
Determining both bilirubin's concentration and its spatial distribution are important in disease diagnosis. Here, for the first time, we applied quantitative multiwavelength photoacoustic microscopy (PAM) to detect bilirubin concentration and distribution simultaneously. By measuring tissue-mimicking phantoms with different bilirubin concentrations, we showed that the root-mean-square error of prediction has reached 0.52 and 0.83 mg/dL for pure bilirubin and for blood-mixed bilirubin detection (with 100% oxygen saturation), respectively. We further demonstrated the capability of the PAM system to image bilirubin distribution both with and without blood. Finally, by underlaying bilirubin phantoms with mouse skins, we showed that bilirubin can be imaged with consistent accuracy down to >400 μm in depth. Our results show that PAM has potential for noninvasive bilirubin monitoring in vivo, as well as for further clinical applications.
The imaging depth of ballistic optical imaging technologies is limited by light scattering. To study the effects of scattering on optical-resolution photoacoustic microscopy (OR-PAM), the signals were divided into target and background signals. A method to simulate the point spread function (PSF) of the PAM system considering both optical illumination and acoustic detection was proposed, then the PSF was used to calculate the contribution of each class of signal at different depths of the focal plane (zf). How image contrast is degraded when there is a uniformly absorbing background as well as when there are small targets densely packed in the acoustic resolution cell were studied. By using the hyperboloid-focusing-based Monte Carlo method, optical focusing into a scattering medium was simulated. It was found that the lateral resolution provided by optical focusing is degraded by only 14% when zf=1.1 transport mean free path (l′t), compared with the case of no scattering. When zf=1.7 l′t, the fluence at 50 μm radial distance away from the focal point is 93% of that at the focal point, which shows optical focusing is very weak at this depth. The method to simulate the PSF of PAM can be used in the future to optimize parameters so as to improve the system performance.
Achieving real-time photoacoustic (PA) tomography typically requires multi-element ultrasound transducer arrays and their associated multiple data acquisition (DAQ) electronics to receive PA waves simultaneously. We report the first demonstration of a photoacoustic tomography (PAT) system using optical fiber-based parallel acoustic delay lines (PADLs). By employing PADLs to introduce specific time delays, the PA signals (on the order of a few micro seconds) can be forced to arrive at the ultrasonic transducers at different times. As a result, time-delayed PA signals in multiple channels can be ultimately received and processed in a serial manner with a single-element transducer, followed by single-channel DAQ electronics. Our results show that an optically absorbing target in an optically scattering medium can be photoacoustically imaged using the newly developed PADL-based PAT system. Potentially, this approach could be adopted to significantly reduce the complexity and cost of ultrasonic array receiver systems.
Photoacoustic microscopy has achieved submicron lateral resolution, but its axial resolution is much lower. Here an axial resolution of 7.6 μm, the highest axial resolution validated by experimental data, has been achieved by using a commercial 125 MHz ultrasonic transducer for signal detection followed by the Wiener deconvolution for signal processing. Limited by the working distance, the high-frequency ultrasonic transducer can penetrate 1.2 mm into biological tissue from the ultrasound detection side. At this depth, the signal-to-noise ratio decreases by 11 dB, and the axial resolution degrades by 36%. The new system was demonstrated in imaging melanoma cells ex vivo and mouse ears in vivo.
Photoacoustic tomography (PAT) of the human brain is challenging due to the fact that the skull strongly absorbs and scatters light, and attenuates and distorts ultrasound as well. For the first time, we demonstrated the feasibility of PAT through a whole adult human skull. A photon recycler (PR) was built to increase light transmittance through the skull. Both a graphite target and a canine brain were imaged through the skull. Use of the PR was found to improve the photoacoustic signal-to-noise ratio by a factor of 2.4. In addition, subtraction of photoacoustic signals that arise from light absorption within the skull significantly improved the contrast of the target. Our results indicate that PAT can potentially be applied to in vivo human brain imaging.
We report the development of functional photoacoustic microscopy capable of video-rate high-resolution in vivo imaging in deep tissue. A lightweight photoacoustic probe is made of a single-element broadband ultrasound transducer, a compact photoacoustic beam combiner, and a bright-field light delivery system. Focused broadband ultrasound detection provides a 44-μm lateral resolution and a 28-μm axial resolution based on the envelope (a 15-μm axial resolution based on the raw RF signal). Due to the efficient bright-field light delivery, the system can image as deep as 4.8 mm in vivo using low excitation pulse energy (28 μJ per pulse, 0.35 mJ/cm 2 on the skin surface). The photoacoustic probe is mounted on a fast-scanning voice-coil scanner to acquire 40 two-dimensional (2-D) B-scan images per second over a 9-mm range. High-resolution anatomical imaging is demonstrated in the mouse ear and brain. Via fast dual-wavelength switching, oxygen dynamics of mouse cardio-vasculature is imaged in realtime as well.
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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 to sacrifice 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 microelectromechanical systems (MEMS) scanning mirror (MEMS-OR-PAM). In MEMS-OR-PAM, the optical and acoustic beams are confocally configured and simultaneously steered, which ensures the uniform detection sensitivity. A B-scan imaging speed as high as 400 Hz can be achieved over a 3 mm scanning range. Using the system, we imaged the flow dynamics of both red blood cells and carbon particles in a mouse ear in vivo. Presented results show that MEMS-OR-PAM could be a powerful tool for studying highly dynamic and time-sensitive biological phenomena.
Time-reversed ultrasonically encoded (TRUE) optical focusing achieves light focusing into scattering media beyond one transport mean free path, which is desirable in biomedical optics. However, the focused optical energy needs to be increased for broad applications. Here, we report the use of a photorefractive polymer (PRP) as the phase conjugate mirror in TRUE optical focusing. The PRP boosted the focused optical energy by ∼ 40 times in comparison to the previously used photorefractive Bi12SiO20 crystal. As a result, we successfully imaged absorbing objects embedded in the middle plane of a tissue-mimicking phantom having an optical thickness of 120 scattering mean free paths.
KEYWORDS: Luminescence, Fluorescence resonance energy transfer, Rhodamine, Photoacoustic microscopy, Photoacoustic spectroscopy, Resonance energy transfer, Absorption, Tissue optics, Energy efficiency, Energy transfer
Förster, or fluorescence, resonance energy transfer (FRET) provides fluorescence signals sensitive to intra- and inter-molecular distances in the 1 to 10 nm range. Widely applied in the fluorescence imaging environment, FRET enables visualization of physicochemical processes in molecular interactions and conformations. In this paper, we report photoacoustic imaging of FRET, based on nonradiative decay that produces heat and subsequent acoustic waves. Estimates of the energy transfer efficiency by photoacoustic microscopy were compared to those obtained by fluorescence confocal microscopy. The experimental results in tissue phantoms show that photoacoustic microscopy is capable of FRET imaging with an enhanced penetration depth. Through its ability to three-dimensionally image tissue with scalable resolution, photoacoustic microscopy could be a beneficial biomedical tool to broaden the in vivo application of FRET.
With the increasing use of small animals for human disease studies, small-animal whole-body molecular imaging plays an important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical and glucose molecular information, leading to higher costs of building dual-modality systems. Even with image co-registration, the spatial resolution of the molecular imaging modality is not improved. Utilizing a ring-shaped confocal photoacoustic computed tomography system, we demonstrate, for the first time, that both anatomy and glucose uptake can be imaged in a single modality. Anatomy was imaged with the endogenous hemoglobin contrast, and glucose metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose.
Blood pulse wave velocity (PWV) is an important physiological parameter that characterizes vascular stiffness. In this letter, we present electrocardiogram-synchronized, photoacoustic microscopy for noninvasive quantification of the PWV in the peripheral vessels of living mice. Interestingly, blood pulse wave-induced fluctuations in blood flow speed were clearly observed in arteries and arterioles, but not in veins or venules. Simultaneously recorded electrocardiograms served as references to measure the travel time of the pulse wave between two cross sections of a chosen vessel and vessel segmentation analysis enabled accurate quantification of the travel distance. PWVs were quantified in ten vessel segments from two mice. Statistical analysis shows a linear correlation between the PWV and the vessel diameter which agrees with known physiology.
We have developed spectrally encoded photoacoustic microscopy using a digital mirror device for multi-wavelength tomography, which enables fast spectral imaging of optical absorption. The optical illumination wavelengths are multiplexed at a laser pulse repetition rate of ∼ 2 kHz. Liquid samples, whole blood, and blood vessels in mouse ears were imaged. Compared with internal wavelength tuning of a narrow-band laser, external wavelength tuning based on a digital mirror device improves the data acquisition speed of spectral photoacoustic microscopy. Compared with external wavelength scanning of a wide-band laser with the same pulse energy, spectral encoding improves the signal-to-noise ratio.
Cardiac myofibers are organized into sheet architectures, which contribute to up to 40% of the heart wall thickening for ejection of blood for circulation. It is important to delineate the sheet architecture for a better understanding of cardiac mechanisms. However, current sheet imaging technologies are limited by fixation-induced dehydration/deformation and low spatial resolution. Here we implemented high-resolution label-free photoacoustic microscopy (PAM) of the myocardial sheet architecture. With high endogenous optical-absorption contrast originating mainly from cytochrome, myoglobin, and melanin, PAM can image the unfixed, unstained and unsliced heart without introducing deformation artifacts. A fresh blood-free mouse heart was imaged by PAM ex vivo. The three-dimensional branching sheets were clearly identified within 150 µm depth. Various morphological parameters were derived from the PAM image. The sheet thickness (80±10 μm) and the cleavage height (11±1 μm) were derived from an undehydrated heart for the first time. Therefore, PAM has the potential for the functional imaging of sheet architecture in ex vivo perfused and viable hearts.
A challenge in photoacoustic tomography (PAT) brain imaging is to compensate for aberrations in the measured photoacoustic data due to their propagation through the skull. By use of information regarding the skull morphology and composition obtained from adjunct x-ray computed tomography image data, we developed a subject-specific imaging model that accounts for such aberrations. A time-reversal-based reconstruction algorithm was employed with this model for image reconstruction. The image reconstruction methodology was evaluated in experimental studies involving phantoms and monkey heads. The results establish that our reconstruction methodology can effectively compensate for skull-induced acoustic aberrations and improve image fidelity in transcranial PAT.
Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3 % . We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed.
Ultrasound-modulated optical tomography (UOT) has the potential to reveal optical contrast deep inside soft biological tissues at an ultrasonically determined spatial resolution. The optical imaging depth reported so far has, however, been limited, which prevents this technique from broader applications. Our latest experimental exploration has pushed UOT to an unprecedented imaging depth. We developed and optimized a UOT system employing a photorefractive crystal-based interferometer. A large aperture optical fiber bundle was used to enhance the efficiencies for diffuse light collection and photorefractive two-wave-mixing. Within the safety limits for both laser illumination and ultrasound modulation, the system has attained the ability to image through a tissue-mimicking phantom of 9.4 cm in thickness, which has never been reached previously by UOT.
We used functional photoacoustic microscopy to image diabetes-induced damage to the microvasculature. To produce an animal model for Type 1 diabetes, we used streptozotocin (STZ), which is particularly toxic to the insulin-producing beta cells of the pancreas in mammals. A set number of ND4 Swiss Webster mice received intraperitoneal injections of STZ for five consecutive days at 50 mg/kg. Most mice developed a significant rise in blood glucose level ( ∼ 400 mg/dL) within three weeks of the first injection. Changes in vasculature and hemodynamics were monitored for six weeks. The mouse ear was imaged with an optical-resolution photoacoustic microscope at a main blood vessel branch from the root of the ear. There are noticeable and measurable changes associated with the disease, including decreased vessel diameter and possible occlusion due to vessel damage and polyurea. We also observed an increase in the blood flow speed in the vein and a decrease in the artery, which could be due to compensation for the dehydration and vessel diameter changes. Functional and metabolic parameters such as hemoglobin oxygen saturation, oxygen extraction fraction, and oxygen consumption rate were also measured, but showed no significant change.
Using a recently developed reconstruction method for photoacoustic tomography (PAT) valid for a planar measurement geometry parallel to a layered medium, we investigate the effects of shear wave propagation in the solid layer upon the ability to estimate Fourier components of the object. We examine this ability as a function of the thickness of the layer supporting shear waves as well as of the incidence angle of the field in the planewave representation. Examples are used to demonstrate the importance of accounting for shear waves in transcranial PAT. Error measures are introduced to quantify the error found when omitting shear waves from the forward model in PAT.
We report an investigation of image reconstruction in photoacoustic tomography for objects that possess heterogeneous material and acoustic attenuation distributions. When the object contains materials, such as bone and soft-tissue, that are modeled using power law attenuation models with distinct exponents, we demonstrate that the effects of acoustic attenuation due to the most strongly attenuating material can be compensated for if the attenuation of the other less attenuating material(s) are neglected. Experiments with phantom objects are presented to validated our findings.
We present an integrated photoacoustic and ultrasonic three-dimensional (3-D) volumetric imaging system based on a two-dimensional (2-D) matrix array ultrasound probe. A wavelength-tunable dye laser pumped by a Q-switched Nd:YAG laser serves as the light source and a modified commercial ultrasound imaging system (iU22, Philips Healthcare) with a 2-D array transducer (X7-2, Philips Healthcare) detects both the pulse-echo ultrasound and photoacoustic signals. A multichannel data acquisition system acquires the RF channel data. The imaging system enables rendering of co-registered 3-D ultrasound and photoacoustic images without mechanical scanning. The resolution along the azimuth, elevation, and axial direction are measured to be 0.69, 0.90 and 0.84 mm for photoacoustic imaging. In vivo 3-D photoacoustic mapping of the sentinel lymph node was demonstrated in a rat model using methylene blue dye. These results highlight the clinical potential of 3-D PA imaging for identification of sentinel lymph nodes for cancer staging in humans.
We report a novel small-animal whole-body imaging system called ring-shaped confocal photoacoustic computed tomography (RC-PACT). RC-PACT is based on a confocal design of free-space ring-shaped light illumination and 512-element full-ring ultrasonic array signal detection. The free-space light illumination maximizes the light delivery efficiency, and the full-ring signal detection ensures a full two-dimensional view aperture for accurate image reconstruction. Using cylindrically focused array elements, RC-PACT can image a thin cross section with 0.10 to 0.25 mm in-plane resolutions and 1.6 s/frame acquisition time. By translating the mouse along the elevational direction, RC-PACT provides a series of cross-sectional images of the brain, liver, kidneys, and bladder.
In order to image noninvasively cell nuclei in vivo without staining, we have developed ultraviolet photoacoustic microscopy (UV-PAM), in which ultraviolet light excites nucleic acids in cell nuclei to produce photoacoustic waves. Equipped with a tunable laser system, the UV-PAM was applied to in vivo imaging of cell nuclei in small animals. We found that 250 nm was the optimal wavelength for in vivo photoacoustic imaging of cell nuclei. The optimal wavelength enables UV-PAM to image cell nuclei using as little as 2 nJ laser pulse energy. Besides the optimal wavelength, application of a wavelength between 245 and 275 nm can produce in vivo images of cell nuclei with specific, positive, and high optical contrast.
We developed a novel trimodality system for human breast imaging by integrating photoacoustic (PA) and thermoacoustic (TA) imaging techniques into a modified commercial ultrasound scanner. Because light was delivered with an optical assembly placed within the microwave antenna, no mechanical switching between the microwave and laser sources was needed. Laser and microwave excitation pulses were interleaved to enable PA and TA data acquisition in parallel at a rate of 10 frames per second. A tube (7 mm inner diameter) filled with oxygenated bovine blood or 30 mM methylene blue dye was successfully detected in PA images in chicken breast tissue at depths of 6.6 and 8.4 cm, respectively, for the first time. The SNRs at these depths reached ∼ 24 and ∼ 15 dB, respectively, by averaging 200 signal acquisitions. Similarly, a tube (13 mm inner diameter) filled with saline solution (0.9%) at a depth of 4.4 cm in porcine fat tissue was successfully detected in TA images. The PA axial, lateral, and elevational resolutions were 640 μm, 720 μm, and 3.5 mm, respectively, suitable for breast cancer imaging. A PA noise-equivalent sensitivity to methylene blue solution of 260 nM was achieved in chicken tissue at a depth of 3.4 cm.
Speckles have been considered ubiquitous in all scattering-based coherent imaging technologies. However, as an optical-absorption-based coherent imaging technology, photoacoustic (PA) tomography (PAT) suppresses speckles by building up prominent boundary signals. We theoretically study the dependence of PAT speckles on the boundary roughness, which is quantified by the root-mean-squared value and the correlation length of the boundary height. Both the speckle visibility and the correlation coefficient between the reconstructed and actual boundaries are quantified. If the root-mean-squared height fluctuation is much greater than, and the height correlation length is much smaller than the imaging resolution, the reconstructed boundaries become fully developed speckles. In other words, speckle formation requires large uncorrelated height fluctuations within the resolution cell. The first- and second-order statistics of PAT speckles are also studied experimentally. While the amplitude of the speckles follows a Gaussian distribution, the autocorrelation of the speckle patterns tracks that of the system point spread function.
We developed multi-contrast photoacoustic microscopy (PAM) for in vivo anatomical, functional, metabolic, and
molecular imaging. This technical innovation enables comprehensive understanding of the tumor microenvironment.
With multi-contrast PAM, we longitudinally determined tumor vascular anatomy, blood flow, oxygen saturation of
hemoglobin, and oxygen extraction fraction.
2011 was another record year for JBO. The number of manuscript submissions reached nearly 750. The impact factor leaped to 3.188, with the 5-year combined impact factor at 3.659. The publication frequency changed to monthly starting in January. The manuscript review cycles have been shortened considerably without sacrificing quality of review. JBO is deeply indebted to the authors, reviewers, and editorial board members for their contributions to such marvelous achievements.
We report an experimental investigation of time-reversed ultrasonically encoded optical focusing in biological tissue. This technology combines the concepts of optical phase conjugation and ultrasound modulation of diffused coherent light. The ultrasonically encoded (or tagged) diffused light from a tissue sample is collected in reflection mode and interferes with a reference light in a photorefractive crystal (used as a phase conjugation mirror) to form a hologram. Then a time-reversed copy of the tagged light is generated and traces back the original trajectories to the ultrasonic focus inside the tissue sample. With our current setup, we can achieve a maximum penetration depth of 5 mm in a chicken breast sample and image optical contrasts within a tissue sample with a spatial resolution approximately equaling 1/2 of the ultrasound focal diameter.
We have applied optical-resolution photoacoustic microscopy (OR-PAM) for longitudinal monitoring of cerebral
metabolism through the intact skull of mice before, during, and up to 72 hours after a 1-hour transient middle cerebral
artery occlusion (tMCAO). The high spatial resolution of OR-PAM enabled us to develop vessel segmentation
techniques for segment-wise analysis of cerebrovascular responses.
As a new class of sentinel lymph node (SLN) tracers for photoacoustic (PA) imaging, Au nanocages offer the advantages
of noninvasiveness, strong optical absorption in the near-infrared region (for deep penetration), and accumulation in
higher concentrations than the initial injected solution. By monitoring the amplitude changes of PA signals in an animal
model, we quantified the accumulations of nanocages in SLNs over time. Based on this method, we quantitatively
evaluated the kinetics of gold nanocages in SLN in terms of concentration, size, and surface modification. We could
detect the SLN at an Au nanocage injection concentration of 50 pM and a dose of 100 μL in vivo. This concentration is
about 40 times less than the previously reported value. We also investigated the influence of nanocages' size (50 nm and
30 nm in edge length), and the effects of surface modification (with positive, or neutral, or negative surface charges).
The results are helpful to develop this AuNC-based PA imaging system for noninvasive lymph node mapping, providing
valuable information about metastatic cancer staging.