In-vivo assessment of a rat rectal tumor using optical-resolution photoacoustic endoscopy.

Optical-resolution photoacoustic endoscopy (OR-PAE) has been proven to realize imaging on the vascular network in the gastrointestinal (GI) tract with high sensitivity and spatial resolution, providing morphological information. Various photoacoustic endoscopic catheters were developed to improve the resolution and adaptivity of in-vivo imaging. However, this technology has not yet been validated on in-vivo GI tumors, which generally feature angiogenesis. The tumor causes thickened mucosa and neoplasia, requiring large depth-of-field (DOF) in imaging, which contradicts to high-resolution imaging. In this work, a novel catheter was developed with a high resolution of ∼27 µm, providing a matched DOF of ∼400 µm to cover the vessels up to the submucosa layer. Optical-resolution photoacoustic endoscopic imaging was first performed on in-vivo rat rectal tumors. In addition, to further characterize the vessel morphology, tumor-suspected regions and normal regions were selected for quantification and analysis of vessel dimension distribution and tortuosity. All the results suggest that the OR-PAE has great application potential in tumor diagnosis, evaluation, and monitoring of therapeutic efficacy.

Saliency enhancement method for photoacoustic molecular imaging based on Grüneisen relaxation nonlinear effect.

Photoacoustic molecular imaging technology has a wide range of applications in biomedical research. In practical scenarios, both the probes and blood generate signals, resulting in the saliency of the probes in the blood environment being diminished, impacting imaging quality. Although several methods have been proposed for saliency enhancement, they inevitably suffer from moderate generality and detection speed. The Grüneisen relaxation (GR) nonlinear effect offers an alternative for enhancing saliency and can improve generality and speed. In this article, the excitation and detection efficiencies are optimized to enhance the GR signal amplitude. Experimental studies show that the saliency of the probe is enhanced. Moreover, the issue of signal aliasing is studied to ensure the accuracy of enhancement results in the tissues. In a word, the feasibility of the GR-based imaging method in saliency enhancement is successfully demonstrated in the study, showing the superiorities of good generality and detection speed.

Non-invasive evaluation of endometrial microvessels via in vivo intrauterine photoacoustic endoscopy.

The endometrium microvessel system, responsible for supplying oxygen and nutrients to the embryo, holds significant importance in evaluating endometrial receptivity (ER). Visualizing this system directly can significantly enhance ER evaluation. Currently, clinical methods like Narrow-band hysteroscopy and Color Doppler ultrasound are commonly used for uterine blood vessel examination, but they have limitations in depth or resolution. Endoscopic Photoacoustic Imaging (PAE) has proven effective in visualizing microvessels in the digestive tract, while its adaptation to uterine imaging faces challenges due to the uterus's unique physiological characteristics. This paper for the first time that uses high-resolution PAE in vivo to capture a comprehensive network of endometrial microvessels non-invasively. Followed by continuous observation and quantitative analysis in the endometrial injury model, we further corroborated that PAE detection of endometrial microvessels stands as a valuable indicator for evaluating ER. The PAE system showcases its promising potential for integration into reproductive health assessments.

Broadband transparent ultrasound transducer with polymethyl methacrylate as matching layer for in vivo photoacoustic microscopy.

Photoacoustic imaging (PAI) uniquely combines optics and ultrasound, presenting a promising role in biomedical imaging as a non-invasive and label-free imaging technology. As the traditional opaque ultrasound (US) transducers could hinder the transportation of the excitation light and limit the performance of PAI system, piezoelectric transparent ultrasonic transducers (TUTs) with indium tin oxide (ITO) electrodes have been developed to allow light transmission through the transducer and illuminate the sample directly. Nevertheless, without having transparent matching materials with appropriate properties, the bandwidth of those TUTs was generally narrow. In this work, we propose to employ polymethyl methacrylate (PMMA) as the matching layer material to improve the bandwidth of lithium niobate (LN)-based TUTs. The effects of PMMA matching layer on the performance of TUTs have been systematically studied. With the optimized PMMA matching layer, the very wide bandwidth of > 50 % could be achieved for the TUTs even with different transducer frequencies, leading to the great enhancement of axial resolution when compared to the similar reported work. In addition, the imaging performance of the developed TUT prototype has been evaluated in a PAI system and demonstrated by both phantom and in vivo small animal imaging.

A Miniature Multi-Functional Photoacoustic Probe.

Photoacoustic technology is a promising tool to provide morphological and functional information in biomedical research. To enhance the imaging efficiency, the reported photoacoustic probes have been designed coaxially involving complicated optical/acoustic prisms to bypass the opaque piezoelectric layer of ultrasound transducers, but this has led to bulky probes and has hindered the applications in limited space. Though the emergence of transparent piezoelectric materials helps to save effort on the coaxial design, the reported transparent ultrasound transducers were still bulky. In this work, a miniature photoacoustic probe with an outer diameter of 4 mm was developed, in which an acoustic stack was made with a combination of transparent piezoelectric material and a gradient-index lens as a backing layer. The transparent ultrasound transducer exhibited a high center frequency of ~47 MHz and a -6 dB bandwidth of 29.4%, which could be easily assembled with a pigtailed ferrule of a single-mode fiber. The multi-functional capability of the probe was successfully validated through experiments of fluid flow sensing and photoacoustic imaging.

Circular array transducer based-photoacoustic/ultrasonic endoscopic imaging with tunable ring-beam excitation.

Photoacoustic/ultrasound endoscopic imaging is regarded as an effective method to achieve accurate detection of intestinal disease by offering both the functional and structural information, simultaneously. Compared to the conventional endoscopy with single transducer and laser spot for signal detection and optical excitation, photoacoustic/ultrasound endoscopic probe using circular array transducer and ring-shaped laser beam avoids the instability brought by the mechanical scanning point-to-point, offering the dual-modality imaging with high accuracy and efficiency. Meanwhile, considering the complex morphological environments of intestinal tracts in clinics, developing the probe having sufficient wide imaging distance range is especially important. In this work, we develop a compact circular photoacoustic/ultrasonic endoscopic probe, using the group of fiber, lens and home-made axicon, to generate relatively concentrated ring-shaped laser beam for 360° excitation with high efficiency. Furthermore, the laser ring size can be tuned conveniently by changing the fiber-lens distance to ensure the potential applicability of the probe in various and complex morphological environments of intestines. Phantom experimental results demonstrate imaging distance range wide enough to cover from 12 mm to 30 mm. In addition, the accessibility of the photoacoustic signals of molecular probes in ex vivo experiments at the tissue depth of 7 mm using excitation energy of 5 mJ has also been demonstrated, showing a high optical excitation efficiency of the probe.

Miniature intravascular photoacoustic endoscopy with coaxial excitation and detection.

Recent research pointed out that the degree of inflammation in the adventitia could correlate with the severity of atherosclerotic plaques. Intravascular photoacoustic endoscopy can provide the information of arterial morphology and plaque composition, and even detecting the inflammation. However, most reported work used a noncoaxial configuration for the photoacoustic catheter design, which formed a limited light-sound overlap area for imaging so as to miss the adventitia information. Here we developed a novel 0.9 mm-diameter intravascular photoacoustic catheter with coaxial excitation and detection to resolve the aforementioned issue. A miniature hollow ultrasound transducer with a 0.18 mm-diameter orifice in the center was successfully fabricated. To show the significance and merits of our design, phantom and ex vivo imaging experiments were conducted on both coaxial and noncoaxial catheters for comparison. The results demonstrated that the coaxial catheter exhibited much better photoacoustic/ultrasound imaging performance from the intima to the adventitia.

Multi-spectral intravascular photoacoustic/ultrasound/optical coherence tomography tri-modality system with a fully-integrated 0.9-mm full field-of-view catheter for plaque vulnerability imaging.

Myocardial infarctions are most often caused by the so-called vulnerable plaques, usually featured as non-obstructive lesions with a lipid-rich necrotic core, thin-cap fibroatheroma, and large plaque size. The identification and quantification of these characteristics are the keys to evaluate plaque vulnerability. However, single modality intravascular methods, such as intravascular ultrasound, optical coherence tomography and photoacoustic, can hardly achieve all the comprehensive information to satisfy clinical needs. In this paper, for the first time, we developed a novel multi-spectral intravascular tri-modality (MS-IVTM) imaging system, which can perform 360° continuous rotation and pull-backing with a 0.9-mm miniature catheter and achieve simultaneous acquisition of both morphological characteristics and pathological compositions. Intravascular tri-modality imaging demonstrates the ability of our MS-IVTM system to provide macroscopic and microscopic structural information of the vessel wall, with identity and quantification of lipids with multi-wavelength excitation. This study offers clinicians and researchers a novel imaging tool to facilitate the accurate diagnosis of vulnerable atherosclerotic plaques. It also has the potential of clinical translations to help better identify and evaluate high-risk plaques during coronary interventions.

Full three-dimensional segmentation and quantification of tumor vessels for photoacoustic images.

Quantitative analysis of tumor vessels is of great significance for tumor staging and diagnosis. Photoacoustic imaging (PAI) has been proven to be an effective way to visualize comprehensive tumor vascular networks in three-dimensional (3D) volume, while previous studies only quantified the vessels projected in one plane. In this study, tumor vessels were segmented and quantified in a full 3D framework. It had been verified in the phantom experiments that the 3D quantification results have better accuracy than 2D. Furthermore, in vivo vessel images were quantified by 2D and 3D quantification methods respectively. And the difference between these two results is significant. In this study, complete vessel segmentation and quantification method within a 3D framework was implemented, which showed obvious advantage in the analysis accuracy of 3D photoacoustic images, and potentially improve tumor study and diagnosis.

Quantitative analysis on in vivo tumor-microvascular images from optical-resolution photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) has been shown to be an excellent imaging modality for monitoring and study of tumor microvasculature. However, previous studies focused mainly on the normal tissues and did not quantify the tumor microvasculature. In this study, we present an in vivo OR-PAM imaging of the melanomas and hepatoma implanted in the mouse ear. We quantify the vessel growth by extracting the skeletons of both dense and thin branches of the tumor microvasculature obtained by Hessian matrix enhancement followed by improved two-step multistencils fast marching method. Compared with the previous methods of using OR-PAM for normal tissues, our method was more effective in extracting the binary vascular network in the tumor images and in obtaining the complete and continuous microvascular skeleton maps. Our demonstration of using OR-PAM in improving microvasculature of tumors and quantification of tumor growth would push deep this technology for the early diagnosis and treatment of cancers.

Motion Correction in Optical Resolution Photoacoustic Microscopy.

In this paper, we are proposing a novel motion correction algorithm for high-resolution OR-PAM imaging. Our algorithm combines a modified demons-based tracking approach with a newly developed multi-scale vascular feature matching method to track motion between adjacent B-scan images without needing any reference object. We first applied this algorithm to correct motion artifacts within one three-dimensional (3D) data segment of rat iris obtained with OR-PAM imaging. We then extended the application of this algorithm to correct motions to obtain vasculature imaging in the whole mouse back. In here, we stitched five adjacent 3D data segments (large field-of-view) obtained while changing the focus of OR-PAM differently for each subarea. The results showed that the motion artifacts of both large blood vessels and microvessels could be accurately corrected in both cases. Compared to the manually stitching method and the traditional SIFT algorithm, the algorithm proposed in this paper has better performance in stitching adjacent data segments. The high accuracy of the motion correction algorithm makes it valuable in OR-PAM for high-resolution imaging of large animals and for quantitative functional imaging.

Dual-foci detection in photoacoustic computed tomography with coplanar light illumination and acoustic detection: a phantom study.

A dual-foci transducer with coplanar light illumination and acoustic detection was applied for the first time. It overcame the small directivity angle, low-sensitivity, and large datasets in conventional circular scanning or array-based photoacoustic computed tomography (PACT). The custom-designed transducer is focused on both the scanning plane with virtual-point detection and the elevation direction for large field of view (FOV) cross-sectional imaging. Moreover, a coplanar light illumination and acoustic detection configuration can provide ring-shaped light irradiation with highly efficient acoustic detection, which in principle has a better adaptability when imaging samples of irregular surfaces. Phantom experiments showed that our PACT system can achieve high resolution (∼0.5 mm), enhanced signal-to-noise ratio (16-dB improvement), and a more complete structure in a greater FOV with an equal number of sampling points compared with the results from a flat aperture transducer. This study provides the proof of concept for the fabrication of a sparse array with the dual-foci property and large aperture size for high-quality, low-cost, and high-speed photoacoustic imaging.

Three-dimensional Hessian matrix-based quantitative vascular imaging of rat iris with optical-resolution photoacoustic microscopy in vivo.

For the diagnosis and evaluation of ophthalmic diseases, imaging and quantitative characterization of vasculature in the iris are very important. The recently developed photoacoustic imaging, which is ultrasensitive in imaging endogenous hemoglobin molecules, provides a highly efficient label-free method for imaging blood vasculature in the iris. However, the development of advanced vascular quantification algorithms is still needed to enable accurate characterization of the underlying vasculature. We have developed a vascular information quantification algorithm by adopting a three-dimensional (3-D) Hessian matrix and applied for processing iris vasculature images obtained with a custom-built optical-resolution photoacoustic imaging system (OR-PAM). For the first time, we demonstrate in vivo 3-D vascular structures of a rat iris with a the label-free imaging method and also accurately extract quantitative vascular information, such as vessel diameter, vascular density, and vascular tortuosity. Our results indicate that the developed algorithm is capable of quantifying the vasculature in the 3-D photoacoustic images of the iris in-vivo, thus enhancing the diagnostic capability of the OR-PAM system for vascular-related ophthalmic diseases in vivo.

In vivo photoacoustic/ultrasonic dual-modality endoscopy with a miniaturized full field-of-view catheter.

Endoscopy is an essential clinical tool for the diagnosis of gastrointestinal (GI) tract cancer. A photoacoustic system that elegantly combines optical and ultrasound endoscopy advantages by providing high-sensitivity functional information and large imaging depth is a potentially powerful tool for GI tract imaging. Recently, several photoacoustic endoscopic imaging systems have been proposed and developed. However, the relatively large size and rigid length of the catheter make it difficult to translate them into wide clinical applications; while the existing system of a relatively small catheter, capable of in vivo animal imaging, is unable to acquire full (360°) field-of-view cross-section images. In this study, we developed a photoacoustic/ultrasonic dual-modality endoscopic system and a corresponding miniaturized, encapsulated imaging catheter, which provides a full 360° field-of-view. The diameter of the catheter is 2.5 mm, which is compatible with the 2.8-mm instrumental channel of a conventional clinical optical endoscope. Using this system, we demonstrate in vivo 3-dimensional endoscopic photoacoustic/ultrasonic imaging of the colorectum of a healthy Sprague Dawley rat, by depicting vasculature and morphology of the GI tract. The significantly improved imaging field of view, reduced catheter size, high-quality imaging results suggest that the developed photoacoustic/ultrasonic dual-modality endoscopy has a great potential to be translated into a broad range of clinical applications in gastroenterology.

Linear array-based real-time photoacoustic imaging system with a compact coaxial excitation handheld probe for noninvasive sentinel lymph node mapping.

We developed a linear ultrasound array-based real-time photoacoustic imaging system with a compact coaxial excitation handheld photoacoustic imaging probe for guiding sentinel lymph node (SLN) needle biopsy. Compared with previous studies, our system and probe have the following advantages: (1) the imaging probe is quite compact and user-friendly; (2) laser illumination and ultrasonic detection are achieved coaxially, enabling high signal-to-noise ratio; and (3) GPU-based image reconstruction enables real-time imaging and displaying at a frame rate of 20 Hz. With the system and probe, clear visualization of the SLN at the depth of 2 cm (~human SLN depth) was demonstrated on a living rat. A fine needle was pushed towards the SLN based on the guidance of real-time photoacoustic imaging. The proposed photoacoustic imaging system and probe was shown to have great potential to be used in clinics for guiding SLN needle biopsy, which may reduce the high morbidity rate related to the current gold standard clinical SLN biopsy procedure.

Advances in Imaging Techniques and Genetically Encoded Probes for Photoacoustic Imaging.

Photoacoustic (PA) imaging is a rapidly emerging biomedical imaging modality that is capable of visualizing cellular and molecular functions with high detection sensitivity and spatial resolution in deep tissue. Great efforts and progress have been made on the development of various PA imaging technologies with improved resolution and sensitivity over the past two decades. Various PA probes with high contrast have also been extensively developed, with many important biomedical applications. In comparison with chemical dyes and nanoparticles, genetically encoded probes offer easier labeling of defined cells within tissues or proteins of interest within a cell, have higher stability in vivo, and eliminate the need for delivery of exogenous substances. Genetically encoded probes have thus attracted increasing attention from researchers in engineering and biomedicine. In this review, we aim to provide an overview of the existing PA imaging technologies and genetically encoded PA probes, and describe further improvements in PA imaging techniques and the near-infrared photochromic protein BphP1, the most sensitive genetically encoded probe thus far, as well as the potential biomedical applications of BphP1-based PA imaging in vivo.

High-speed intravascular spectroscopic photoacoustic imaging at 1000 A-lines per second with a 0.9-mm diameter catheter.

Intravascular spectroscopic photoacoustic technology can image atherosclerotic plaque composition with high sensitivity and specificity, which is critical for identifying vulnerable plaques. Here, we designed and engineered a catheter of 0.9 mm in diameter for intravascular photoacoustic (IVPA) imaging, smaller than the critical size of 1 mm required for clinical translation. Further, a quasifocusing photoacoustic excitation scheme was developed for the catheter, producing well-detectable IVPA signals from stents and lipids with a laser energy as low as ~30 µJ/pulse. As a result, this design enabled the use of a low-energy, high-repetition rate, ns-pulsed optical parametric oscillator laser for high-speed spectroscopic IVPA imaging at both the 1.2-µm and 1.7-µm spectral bands for lipid detection. Specifically, for each wavelength, a 1-kHz IVPA A-line rate was achieved, ~100-fold faster than previously reported IVPA systems offering a similar wavelength tuning range. Using the system, spectroscopic IVPA imaging of peri-adventitial adipose tissue from a porcine aorta segment was demonstrated. The significantly improved imaging speed, together with the reduced catheter size and multiwavelength spectroscopic imaging ability, suggests that the developed high-speed IVPA technology is of great potential to be further translated for in vivo applications.

Longitudinal label-free optical-resolution photoacoustic microscopy of tumor angiogenesis in vivo.

BACKGROUND: Optical-resolution photoacoustic microscopy (OR-PAM) is a high-resolution imaging technology capable of label-free imaging of the morphology and functions of the microvasculature in vivo. Previous studies of angiogenesis by OR-PAM were carried out primarily with transgenic mice and the mouse ear model. While important findings have been generated using this approach, the application of OR-PAM to the more widely used subcutaneous dorsal tumor models remains challenging, largely due to the respiratory and cardiac motion artifacts, as well as the protruding tumor contours. METHODS AND MATERIALS: A noninvasive dorsal skin-fold (N-DSF) model, along with adaptive z-scanning and a corresponding experimental protocol, is developed. Mammary carcinoma cells (4T1) were administered subcutaneously to the backs of female BALB/c mice for tumor inoculation. The mice were anesthetized using a mixture of isofluorane and oxygen. RESULTS: In vivo OR-PAM of angiogenesis with subcutaneous dorsal tumor models in mice has been demonstrated. To test the performance of this method, we have monitored the growth of 4T1 mouse mammary carcinoma in BALB/c mice over a period of 9 days. The major features of tumor angiogenesis, including the change of vascular tortuosity, the dilation of vessel diameters, and the increase of blood supply, have been clearly captured with OR-PAM. CONCLUSIONS: In combination with N-DSF model, OR-PAM has demonstrated outstanding capacity to provide label-free monitoring of angiogenesis in tumor. Thus, OR-PAM is of great potential to find broad biomedical applications in the pathophysiological studies of tumor and the treatments for anti-angiogenesis.

In vivo photoacoustic molecular imaging of breast carcinoma with folate receptor-targeted indocyanine green nanoprobes.

As an optical-acoustic hybrid imaging technology, photoacoustic imaging uniquely combines the advantages of rich optical contrast with high ultrasonic resolution in depth, opening up many new possibilities not attainable with conventional pure optical imaging technologies. To perform photoacoustic molecular imaging, optically absorbing exogenous contrast agents are needed to enhance the signals from specifically targeted disease activity. In this work, we designed and developed folate receptor targeted, indocyanine green dye doped poly(d,l-lactide-co-glycolide) lipid nanoparticles (FA-ICG-PLGA-lipid NPs) for molecular photoacoustic imaging of tumor. The fabricated FA-ICG-PLGA-lipid NPs exhibited good aqueous stability, a high folate-receptor targeting efficiency, and remarkable optical absorption in near-infrared wavelengths, providing excellent photoacoustic signals in vitro. Furthermore, after intravenous administration of FA-ICG-PLGA-lipid NPs, mice bearing MCF-7 breast carcinomas showed significantly enhanced photoacoustic signals in vivo in the tumor regions, compared with those using non-targeted ICG-PLGA-lipid NPs. Given the existing wide clinical use of ICG and PLGA, the developed FA-ICG-PLGA-lipid NPs, in conjunction with photoacoustic imaging technology, offer a great potential to be translated into the clinic for non-ionizing molecular imaging of breast cancer in vivo.

Compressed sensing based virtual-detector photoacoustic microscopy in vivo.

Photoacoustic microscopy (PAM) is becoming a vital tool for various biomedical studies, including functional and molecular imaging of cancer. However, due to the use of a focused ultrasonic transducer for photoacoustic detection, the image quality of conventional PAM degrades rapidly away from the ultrasonic focal zone. To improve the image quality of PAM for out-of-focus regions, we have developed compressed sensing based virtual-detector photoacoustic microscopy (CS-PAM). Through phantom and in vivo experiments, it has been demonstrated that CS-PAM can effectively extend the depth of focus of PAM, and thus may greatly expand its potential biomedical applications.