Structural and functional photoacoustic molecular tomography aided by emerging contrast agents.

Photoacoustic (PA) imaging is a hybrid imaging technology which combines the best of optical (contrast) and ultrasound (resolution) imaging. PA imaging uses intrinsic contrast agents in the body like blood (hemoglobin), melanin, etc. But the contrast from the intrinsic contrast agents might not be sufficient for different applications. So, external contrast agents are needed for improving the contrast of PA images. Organic dyes, inorganic dyes, and nanomaterials can be used as photoacoustic contrast agents. The major issue with using external contrast agent is that they often need FDA approval to be used for in-vivo studies. Availability of FDA approved contrast agents for PA imaging is very limited. In this work, we present the feasibility of using food and food-based dyes as photoacoustic contrast agents. We use commonly used foods like coffee, tea, chocolate and food colorants as contrast agents. We use alpinion’s E-CUBE dual mode ultrasound and photoacoustic imaging system with mobile Nd:YAG laser pumped by OPO laser to demonstrate the efficiency of the contrast agents. The contrast agents were compared with methylene blue. Out of the many different agents we tested, coffee, chocolate and few other dyes proved as efficient photoacoustic contrast agents. We performed photoacoustic spectroscopy to identify at which wavelength the dye performed best. We also tested the contrast agents for imaging sentinel lymph nodes in rats and the results are very similar to methylene blue. This will enable the transition of photoacoustic imaging to the clinics more easily.

As a molecular imaging modality, photoacoustic (PA) imaging has been in the spotlight because it can provide an optical contrast image of physiological information and a relatively deep imaging depth. However, its sensitivity is limited despite the use of exogenous contrast agents due to the background PA signals generated from nontargeted absorbers, such as blood and boundaries between different biological tissues. In addition, clutter artifacts generated in both in-plane and out-of-plane imaging region degrade the sensitivity of PA imaging. We propose a method to eliminate the nontargeted PA signals. For this study, we used a dual-modal ultrasound (US)-PA contrast agent that is capable of generating both the backscattered US and PA signals in response to the transmitted US and irradiated light, respectively. The US images of the contrast agents are used to construct a masking image that contains the location information about the target site and is applied to the PA image acquired after contrast agent injection. In vitro and in vivo experimental results demonstrated that the masking image constructed using the US images makes it possible to completely remove nontargeted PA signals. The proposed method can be used to enhance the clear visualization of the target area in PA images.

Photoacoustic (PA) imaging holds great promise for preclinical research and clinical practice. However, most studies rely on the laser wavelength in the first near-infrared (NIR) window (NIR-I, 650-950 nm), while few studies have been exploited in the second NIR window (NIR-II, 1000-1700 nm), mainly due to the lack of NIR-II absorbing contrast agents. We herein report the synthesis of a broadband absorbing PA contrast agent based on semiconducting polymer nanoparticles (SPN-II) and apply it for PA imaging in NIR-II window. SPN-II can absorb in both NIR-I and NIR-II regions, providing the feasibility to directly compare PA imaging at 750 nm with that at 1064 nm. Because of the weaker background PA signals from biological tissues in NIR-II window, the signal-to-noise ratio (SNR) of SPN-II resulted PA images at 1064 nm can be 1.4-times higher than that at 750 nm when comparing at the imaging depth of 3 cm. The proof-of-concept application of NIR-II PA imaging is demonstrated in in vivo imaging of brain vasculature in living rats, which showed 1.5-times higher SNR as compared with NIR-I PA imaging. Our study not only introduces the first broadband absorbing organic contrast agent that is applicable for PA imaging in both NIR-I and NIR-II windows but also reveals the advantages of NIR-II over NIR-I in PA imaging.

Urinary bladder imaging is critical to diagnose urinary tract disorders, and bladder cancer. There is a great need for safe, non-invasive, and sensitive imaging technique which enables bladder imaging. Photoacoustic imaging is a rapidly growing imaging technique for various biological applications. It can be combined with clinical ultrasound imaging system for hand-held, dual modal ultrasound-photoacoustic real-time imaging. Structural (bladder wall) and functional (accretion of nanoparticles) bladder imaging is shown here with combined ultrasound and photoacoustic imaging in rats. Photoacoustic images of bladder wall is shown using black ink as the contrast agent. Chicken tissues were stacked on the abdomen of the animal to demonstrate the feasibility of photoacoustic imaging till a depth of 2 cm. Also, the feasibility of photoacoustic imaging for a common bladder disorder, vesicoureteral reflux is studied using urinary tract mimicking phantom. It is also shown that a clinical ultrasound system can be used for photoacoustic imaging of non-invasive clearance study of gold nanorods from circulation by monitoring the gradual accumulation of the gold nanorods in the bladder. The time taken for accumulation of nanorods in the bladder can be used as an indicator of the clearance rate of the nanoparticle circulation from the body.

The growing prevalence of brain diseases and the limitations of existing imaging techniques highlight the urgent demand for advanced, biocompatible, and high-resolution brain imaging approaches. Photoacoustic imaging (PAI) uniquely integrates scalable spatial resolution, deep penetration, and non-ionizing excitation, which enables multi-scale visualization from subcellular structures to the whole brain. This review systematically summarizes the fundamental principles of PAI, commonly used contrast agents, and key system components, and provides a comparative analysis of the major imaging modalities-optical-resolution and acoustic-resolution photoacoustic microscopy (OR-/AR-PAM), photoacoustic computed tomography (PACT), and photoacoustic endoscopy (PAE). PACT offers superior penetration depth and field of view, whereas OR-/AR-PAM provides higher spatial resolution at molecular scales. By aligning disease-specific imaging requirements with the functional strengths of each modality, PAI has enabled integrated structural and functional imaging in glioblastoma, neurodegenerative disorders, traumatic brain injury, and cerebrovascular diseases, highlighting its integrated advantages for multiscale brain imaging. Finally, the review discusses key challenges for clinical translation of PAI, including motion artifacts, blood-brain barrier constraints, contrast agent approval, limitations of single-modality approaches, and technical limitations, and proposes corresponding strategies to address these obstacles. This review provides forward-looking guidance for the clinical translation of multiscale brain imaging technologies.

Photoacoustic (PA) imaging provides excellent optical contrast with decent penetration and high spatial resolution, making it attractive for a variety of neural applications. We evaluated optical contrast agents with high absorption in the near infrared (NIR) as potential enhancers for PA neuroimaging: optical dyes, gold nanorods (GNRs) and PEBBLEs loaded with indocyanine green. Two PA systems were developed to test these agents in excised neural tissue and in vivo mouse brain. Lobster nerves were stained with the agents for 30 minutes and placed in a hybrid nerve chamber capable of electrical stimulation and recording, optical spectroscopy and PA imaging. Contrast agents boosted the PA signal by at least 30 dB using NIR illumination from a tunable pulsed laser. Photobleaching may be a limiting factor for optical dyes-the PA signal decreased steadily with laser illumination. The second setup enabled in vivo transcranial imaging of the mouse brain. A custom clinical ultrasound scanner and a 10-MHz linear array provided near real-time images during and after an injection of 2 nM gold nanorods into the tail vein. The peak PA signal from the brain vasculature was enhanced by up to 2 dB at 710 nm. Temporal dynamics of the PA signal were also consistent with mixing of the GNRs in the blood. These studies provide a baseline for enhanced PA imaging in neural tissue. The smart contrast agents employed in this study can be further engineered for molecular targeting and controlled drug delivery with potential treatment for a myriad of neural disorders.

A three-dimensional laser optoacoustic imaging system was developed, which combines the advantages of optical spectroscopy and high resolution ultrasonic detection, to produce high contrast maps of optical absorbance in tissues. This system was tested in a nude mouse model of breast cancer and produced tissue images of tumors and vasculature. The imaging can utilize either optical properties of hemoglobin and oxyhemoglobin, which are the main endogenous tissue chromophores in the red and near-infrared spectral ranges, or exogenous contrast agent based on gold nanorods. Visualization of tissue molecules targeted by the contrast agent provides molecular information. Visulization of blood at multiple colors of light provides functional information on blood concentration and oxygen saturation. Optoacoustic imaging, using two or more laser illumination wavelengths, permits an assessment of the angiogenesis-related microvasculature, and thereby, an evaluation of the tumor stage and its metastatic potential. The optoacoustic imaging system was also used to generate molecular images of the malignancy-related receptors induced by the xenografts of BT474 mammary adenocarcinoma cells in nude mice. The development of the latter images was facilitated by the use of an optoacoustic contrast agent that utilizes gold nanorods conjugated to monoclonal antibody raised against HER2/neu antigens. These nanorods possess a very strong optical absorption peak that can be tuned in the near-infrared by changing their aspect ratio. The effective conversion of the optical energy into heat by the gold nanorods, followed by the thermal expansion of the surrounding water, makes these nanoparticles an effective optoacoustic contrast agent. Optical scattering methods and x-ray tomography may also benefit from the application of this contrast agent. Administration of the gold nanorod bioconjugates to mice resulted in an enhanced contrast of breast tumors relative the background of normal tissues in the nude mouse model. The combination of this novel contrast agent and optoacoustic imaging has the potential to become a useful imaging modality, for preclinical research in murine models of cancer and other human diseases.

Optoacoustic imaging (OAI) is a unique in vivo imaging technique combining deep tissue penetration with high resolution and molecular sensitivity. OAI relying on strong intrinsic contrast, such as blood hemoglobin, already shows its value in medical diagnostics. However, OAI sensitivity to current extrinsic contrast agents is insufficient and limits its role in detecting disease-related biomarkers. The recently introduced concept of photoswitching and temporal unmixing techniques for OAI allows detecting extrinsic contrast with high sensitivity, allowing the visualization of small populations of cells labeled with photoswitching proteins deep within the tissue. However, transgene modification might not be permitted in some cases, such as for diagnostic use. Therefore, it is desirable to leverage the concept of photoswitching OAI towards injectable formulations. Since photoswitchable synthetic dyes are mainly excited by blue wavelengths unsuited for imaging in tissue, we propose exploiting the addition of XTENs to photoswitching proteins towards yielding injectable agents. The addition of XTEN to a protein enhances its plasma half-life and bioavailability, thus allowing its use, for example, in targeted labeling approaches. In this pilot study, we show that intravenously injected near-infrared absorbing photoswitchable proteins, ReBphP-PCM, coupled to XTEN, allow highly sensitive optoacoustic visualization of a tumor xenograft in vivo. The sensitivity to XTENs-ReBphP-PCM determined by ex vivo analysis of labeled cells is one to two orders of magnitude beyond conventional synthetic dyes used currently in OAI. The enhanced sensitivity afforded by photoswitching OAI, in combination with the increased bioavailability and biocompatibility of XTENs-ReBphP-PCM, makes this fusion protein a promising tool for facilitating sensitive detection of biomarkers in OAI with a potential for future use in diagnostics. STATEMENT OF SIGNIFICANCE: Optoacoustic imaging (OAI) is a unique in vivo imaging technique that combines deep tissue penetration with high resolution. OAI, which relies on intrinsic contrast, such as blood hemoglobin, could already be valuable in medical diagnostics. However, the use of extrinsic contrast agents to augment disease-related biomarkers in research and diagnostics suffers from very limited sensitivity of the generated contrast agent. We present an intravenously injected photoswitchable protein, ReBphP-PCM, coupled to XTEN, allowing highly sensitive OAI. The sensitivity is one to two orders of magnitude greater than that of conventional synthetic dyes used currently in OA imaging. The high sensitivity afforded by photoswitching together with the enhanced bioavailability and biocompatibility of the XTENs-ReBphP-PCM make this a standard agent for high-quality detection of OAI with potential for clinical use.

ObjectiveTo evaluate in vivo a fully integrated photoacoustic tomography imaging system based on Fabry–Pérot ultrasound sensing method applied on porcine abdominal organs. This approach could be used by surgeons during intraoperative clinical procedures.MethodsThe photoacoustic imaging system was fully integrated into a single structure, and the detection technology was based on a Fabry–Pérot interferometer. The detection probe connected to the imaging system was applied directly to the organs of a male “large white” Sus scrofa pig weighing 80 kg, either manually or using a stand, with or without a gel interface. All experiments were performed in compliance with EU Directive 2010/63/EU on animal experimentation (APAFiS #31507).ResultsAll intraperitoneal and retroperitoneal organs were evaluated using photoacoustic imaging. The evaluation of both hollow and solid organs was successfully conducted with consistent three-dimensional image quality. We demonstrate the system’s ability to image blood vessels with diameters ranging from several millimeters down to less than 100 µm. Macroscopic evaluation of the organs using photoacoustic tomography imaging did not reveal any damage or burns caused by the excitation laser.ConclusionTo our knowledge, this is the first reported imaging session of abdominal organs in an in vivo porcine model, performed using a photoacoustic tomography system with Fabry–Pérot interferometer detection. We present a high-resolution photoacoustic tomography system that is closer to routine clinical translation, thanks to a fully integrated system.Relevance statementPhotoacoustic evaluation of organs using a fully integrated system could become a valuable tool for surgical teams for intraprocedural assessment of vascularization.Key PointsPhotoacoustic imaging visualizes blood vessels without contrast agents or ionizing radiation.Photoacoustic imaging systems detect blood vessels ranging from millimeters to 100 µm.Fully integrated photoacoustic imaging systems are autonomously operable by surgical teams.Graphical

A targeted gold nanoparticle has been developed as a contrast agent for photoacoustic medical imaging. We have studied cancer cell targeting by antibody conjugated gold nanorods for high contrast photoacoustic imaging. By changing the aspect ratio of the elongated “rod” shape of the gold nanoparticle, its plasmon peak absorption wavelength can be tuned to the near IR (700–900nm) for an increased penetration depth into biological tissue. Effective cell targeting and sensitive photoacoustic detection of a single layer of cells are demonstrated. Combining ultrasound with contrast agent based photoacoustic imaging is proposed as a visual tool to compound molecular and structural information for early stage prostate cancer detection.

Photoacoustic (PA) imaging offers depth-resolved images of optical absorbers with the spatial resolution of ultrasound imaging. To enhance tumour contrast, tumour-specific probes are used as contrast agents. We synthesised a colourless PA probe that is activated in the presence of γ-glutamyltranspeptidase, a cancer-associated enzyme, to show its original colour and fluorescence. We have acquired high specificity fluorescence images of small tumours, using a fluorescent probe based on similar enzymatic reactions. Here, we developed a PA imaging technique to detect the PA probe. In PA imaging, depending on the concentration and excitation wavelength of the probe, the intensities of the probe signals may be lower than those of the background signals produced by intrinsic optical absorbers such as haemoglobin. For probe imaging in the presence of strong background signals, multispectral photoacoustic (MS-PA) imaging was evaluated. In MS-PA imaging, the spectral fitting method, which distinguishes the probe signals from background signals using reference spectra, has been widely used. To compensate for the decrease of fluence due to optical attenuation in biological tissue, we used a simplified compensation method that calculates fluence inside biological tissues by the Monte-Carlo model using published data on optical properties of biological tissues. The validity of the method was confirmed using tissue-mimicking phantoms. Finally, MS-PA imaging of a mouse subcutaneous tumour injected with the activatable probe was demonstrated. In conclusion, our MS-PA imaging technique afforded successful detection of the activated probe in the tumour, and time-increase of PA signals were successfully observed.

Photoacoustic imaging is a hybrid imaging technique based on the photoacoustic effect. As a non-invasive and non-ionizing modality, photoacoustic imaging takes the both merits of the conventional acoustic imaging and optical imaging. Firstly, the contrast of photoacoustic imaging primarily depends on the optical absorption. The unique optical spectra of atoms and molecules makes optical methods to be a widely used modality to probe the molecular and chemical information of biological tissue. Therefore, photoacoustic imaging has its inherent advantage in high-contrast functional and physiological imaging of biological tissue, as well as the optical imaging method. Secondly, photoacoustic imaging has the high spatial resolution in deep tissue in comparison with the pure optical imaging method. Since the strongly optical scattering in biological tissue, pure optical imaging method is difficult to obtain the high-resolution image in the tissue deeper than ～1 mm. Whereas, acoustic wave suffers much less from scattering than optical wave, the acoustic scattering coefficient is about 2-3 orders of magnitude less than the optical scattering coefficient. Photoacoustic imaging can achieve a fine resolution in deep tissue, which equivalent to 1/200 of the imaging depth. Thirdly, non-ionizing radiation used for photoacoustic imaging is much safer than X-ray. Moreover, the low-temperature rises make photoacoustic imaging be safely used in live tissue. A laser-induced temperature rise of 1 mK yields an initial pressure of ～800 Pa in soft tissue. Such a sound pressure level has reached the sensitivities of typical ultrasonic transducers. Fourthly, photoacoustic imaging has the ability of extracting multiple contrasts, including biochemical parameter, biomechanical parameter, blood velocity distribution, tissue temperature, and microstructure information. Photoacoustic imaging can capture more specific and reliable information about the tissue structure, function, metabolism, molecule, and gene. As a result, photoacoustic imaging has become one of the fastest growing biomedical imaging techniques in the past decade.#br#In this review, we will explain photoacoustic effect and the principle of photoacoustic imaging. Then, we introduce the two classical photoacoustic imaging schemes, including photoacoustic tomography and photoacoustic microscopy. Their main specifications, such as resolution, are also preflents. We review the ability of photoacoustic imaging in extracting multiple contrasts and discuss their biomedicine applications. In addition, we also introduce the remarkable breakthroughs in super-resolution photoacoustic imaging. Finally, we look the further development and the limitations of photoacoustic imaging.

Bioconjugated gold nanoparticles as a molecular based contrast agent: implications for imaging of deep tumors using optoacoustic tomography

Photoacoustic tomography (PAT) offers structural and functional imaging of living biological tissue with highly sensitive optical absorption contrast and excellent spatial resolution comparable to medical ultrasound (US) imaging. We report the development of a fully integrated PAT and US dual-modality imaging system, which performs signal scanning, image reconstruction and display for both photoacoustic (PA) and US imaging all in a truly real-time manner. The backprojection (BP) algorithm for PA image reconstruction is optimized to reduce the computational cost and facilitate parallel computation on a state of the art graphics processing unit (GPU) card. For the first time, PAT and US imaging of the same object can be conducted simultaneously and continuously, at a real time frame rate, presently limited by the laser repetition rate of 10 Hz. Noninvasive PAT and US imaging of human peripheral joints in vivo were achieved, demonstrating the satisfactory image quality realized with this system. Another experiment, simultaneous PAT and US imaging of contrast agent flowing through an artificial vessel was conducted to verify the performance of this system for imaging fast biological events. The GPU based image reconstruction software code for this dual-modality system is open source and available for download from http://sourceforge.net/projects/pat realtime .

Photoacoustic imaging (PAI), combining the advantages of optical excitation and of acoustic detection, has been widely used for both structural and functional imaging with scalable resolution and penetration in biological tissues. However, due to the strong light scattering in tissue, the maximum penetration depth of PAI, especially microscopy, is limited. Meanwhile, strong light scattering also introduces strong spatial resolution degradation along the depth direction in optical resolution PAI. As an effective method to reduce optical scattering, the optical clearing technique has been widely used in optical imaging modalities. In this chapter, the status of applying optical clearing in PAI is summarized. Examples of both optical resolution and acoustic resolution PAI are illustrated in detail. Future directions in the use of optical clearing in PAI are also discussed in this chapter.