Photoacoustic tomography (PAT) can create multiscale multicontrast images of living biological structures ranging from organelles to organs. This emerging technology overcomes the high degree of scattering of optical photons in biological tissue by making use of the photoacoustic effect. Light absorption by molecules creates a thermally induced pressure jump that launches ultrasonic waves, which are received by acoustic detectors to form images. Different implementations of PAT allow the spatial resolution to be scaled with the desired imaging depth in tissue while a high depth-to-resolution ratio is maintained. As a rule of thumb, the achievable spatial resolution is on the order of 1/200 of the desired imaging depth, which can reach up to 7 centimeters. PAT provides anatomical, functional, metabolic, molecular, and genetic contrasts of vasculature, hemodynamics, oxygen metabolism, biomarkers, and gene expression. We review the state of the art of PAT for both biological and clinical studies and discuss future prospects.

Photoacoustic Tomography: In Vivo Imaging from Organelles to Organs

Photoacoustic (PA) imaging, also called optoacoustic imaging, is a new biomedical imaging modality based on the use of laser-generated ultrasound that has emerged over the last decade. It is a hybrid modality, combining the high-contrast and spectroscopic-based specificity of optical imaging with the high spatial resolution of ultrasound imaging. In essence, a PA image can be regarded as an ultrasound image in which the contrast depends not on the mechanical and elastic properties of the tissue, but its optical properties, specifically optical absorption. As a consequence, it offers greater specificity than conventional ultrasound imaging with the ability to detect haemoglobin, lipids, water and other light-absorbing chomophores, but with greater penetration depth than purely optical imaging modalities that rely on ballistic photons. As well as visualizing anatomical structures such as the microvasculature, it can also provide functional information in the form of blood oxygenation, blood flow and temperature. All of this can be achieved over a wide range of length scales from micrometres to centimetres with scalable spatial resolution. These attributes lend PA imaging to a wide variety of applications in clinical medicine, preclinical research and basic biology for studying cancer, cardiovascular disease, abnormalities of the microcirculation and other conditions. With the emergence of a variety of truly compelling in vivo images obtained by a number of groups around the world in the last 2–3 years, the technique has come of age and the promise of PA imaging is now beginning to be realized. Recent highlights include the demonstration of whole-body small-animal imaging, the first demonstrations of molecular imaging, the introduction of new microscopy modes and the first steps towards clinical breast imaging being taken as well as a myriad of in vivo preclinical imaging studies. In this article, the underlying physical principles of the technique, its practical implementation, and a range of clinical and preclinical applications are reviewed.

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Biomedical photoacoustic imaging

The nonradiative conversion of light energy into heat (photothermal therapy, PTT) or sound energy (photoacoustic imaging, PAI) has been intensively investigated for the treatment and diagnosis of cancer, respectively. By taking advantage of nanocarriers, both imaging and therapeutic functions together with enhanced tumour accumulation have been thoroughly studied to improve the pre-clinical efficiency of PAI and PTT. In this review, we first summarize the development of inorganic and organic nano photothermal transduction agents (PTAs) and strategies for improving the PTT outcomes, including applying appropriate laser dosage, guiding the treatment via imaging techniques, developing PTAs with absorption in the second NIR window, increasing photothermal conversion efficiency (PCE), and also increasing the accumulation of PTAs in tumours. Second, we introduce the advantages of combining PTT with other therapies in cancer treatment. Third, the emerging applications of PAI in cancer-related research are exemplified. Finally, the perspectives and challenges of PTT and PAI for combating cancer, especially regarding their clinical translation, are discussed. We believe that PTT and PAI having noteworthy features would become promising next-generation non-invasive cancer theranostic techniques and improve our ability to combat cancers.

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Photothermal therapy and photoacoustic imaging via nanotheranostics in fighting cancer

Optical microscopy has been a fundamental tool of biological discovery for more than three centuries, but its in vivo tissue imaging ability has been restricted by light scattering to superficial investigations, even when confocal or multiphoton methods are used. Recent advances in optical and optoacoustic (photoacoustic) imaging now allow imaging at depths and resolutions unprecedented for optical methods. These abilities are increasingly important to understand the dynamic interactions of cellular processes at different systems levels, a major challenge of postgenome biology. This Review discusses promising photonic methods that have the ability to visualize cellular and subcellular components in tissues across different penetration scales. The methods are classified into microscopic, mesoscopic and macroscopic approaches, according to the tissue depth at which they operate. Key characteristics associated with different imaging implementations are described and the potential of these technologies in biological applications is discussed.

Going deeper than microscopy: the optical imaging frontier in biology

Photoacoustic tomography (PAT) is probably the fastest-growing area of biomedical imaging technology, owing to its capacity for high-resolution sensing of rich optical contrast in vivo at depths beyond the optical transport mean free path (~1 mm in human skin). Existing high-resolution optical imaging technologies, such as confocal microscopy and two-photon microscopy, have had a fundamental impact on biomedicine but cannot reach the penetration depths of PAT. By utilizing low ultrasonic scattering, PAT indirectly improves tissue transparency up to 1000-fold and consequently enables deeply penetrating functional and molecular imaging at high spatial resolution. Furthermore, PAT promises in vivo imaging at multiple length-scales; it can image subcellular organelles to organs with the same contrast origin — an important application in multiscale systems biology research.

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Multiscale photoacoustic microscopy and computed tomography.

Despite advancements in imaging and surgical methodology surgeons continue to face challenges in differentiating suitable margins in tumor resection sites and assessing the extent of cancer proliferation. Presently, histology is the gold standard used for determining margins post-operatively. However, currently no intraoperative tools can analyze entire resection sites. This results in unnecessary repeated surgeries and is especially critical to patient outcome for certain cancers. To address this critical need, we have developed a next-generation microscopy system intended to allow accurate intraoperative virtual histopathology for margin assessment. The modality, named ultraviolet photoacoustic remote sensing microscopy (UV-PARS), takes advantage of the intrinsic optical absorption contrast of DNA at 266 nm and a non-contact PARS technique. This approach measures time-dependent refractive index modulations at their subsurface origin resulting from thermo-elastic excitation from a pulsed excitation source. This enables the possibility of real time non-contact label-free visualization of cell nuclei in vivo. Preliminary results are presented including studies with live cell cultures, excised tissue samples, phantoms, and characterization of system parameters. Lateral-resolution was found to be 0.7 µm with a signal-to-noise ratio of 50 dB achieved in phantoms and 30 dB for HeLa cells. Simultaneously collected confocal reflectance microscopy using 1310 nm light provided cell body morphology. HeLa and PC3 cell cultures were imaged with good agreement to conventional H&E stained images of the same samples.

Label-free non-contact imaging of cell nuclei using ultraviolet photoacoustic remote sensing microscopy (Conference Presentation)

Optical-resolution photoacoustic microscopy (OR-PAM) can produce micron-scale, high-resolution images of optically-absorbing chromophores. The pressure rise of photoacoustic signals is proportional to the Grueneisen parameter, which is temperature dependent. High laser repetition-rates may cause overlapping of adjacent laser pulses on targets in laser-scanning OR-PAM. When laser-pulse-repetition intervals are shorter than thermal relaxation times, the zone of laser-spot overlap between pulses can generate higher photoacoustic signal than cases where beam-spots do not overlap or in cases where laser pulse-intervals are longer than the thermal relaxation time. This is because subsequent laser pulses experience higher Grueneisen parameters than previous pulses due to temperature rises induced by the previous pulses. Here, we present our recent studies on photoacoustic signals strength varying with scanning speed and laser-repetition-rate on black tape, human hair and rat blood respectively.

Investigation of photoacoustic signal strength as a function of scan-speed and laser-repetition-rate

Large and small CMUT membranes were fabricated in an interlaced fashion to create a multi-frequency array using a modified standard silicon-nitride sacrificial release process on a scale smaller than the wavelength to ensure the minimization of grating lobes. A 7 mm by 7 mm multi-frequency array with 20 elements was fabricated and wire-bonded to custom printed circuit boards (PCBs) mounted onto a custom interfacing circuitry with voltage protected pre-amplifiers, connected to a Verasonics programmable ultrasound platform. This system enabled real-time imaging and programmable control over low- and/or high-frequency transmission and reception. To demonstrate multi-band ultrasound imaging, three wire targets with the separation of 0.7 cm were imaged in an oil immersion medium. The low-frequency elements showed better signal to noise ratio (SNR) for the targets far from the transducer while high-frequency elements proved better resolution for shallower depths.

Multi-frequency CMUT imaging arrays for multi-scale imaging and imaging-therapy applications

We developed a unique reflection-mode photoacoustic technique sensitive to optical scattering in turbid media. We
focused a small laser spot on to the surface of a turbid medium and captured the photoacoustic signal by a focused
ultrasound transducer. The amplitude of the photoacoustic signal for different surface illumination spot locations is an
effective estimate of the Green's function of light transport in turbid media. Our results for different concentrations of
Intralipid indicate that this method is capable of distinguishing small changes in the reduced scattering coefficient. In
this work, we present experimental measurements for an Intralipid phantom with reduced scattering coefficients of 3, 4,
and 5 cm -1 , and show that Monte Carlo simulations of light transport accurately reproduce experimental curves. This
means that we can estimate transport-regime optical properties of the media given a suitable fitting algorithm.

Monte Carlo modeling for photoacoustic-based transport-regime optical property estimation

A novel all-optical non-contact photoacoustic microscopy system is introduced. The confocal configuration is used to ensure detection of initial pressure shock wave-induced intensity reflections at the subsurface origin where pressures are largest. Phantom studies confirm signal dependence on optical absorption, index-contrast, and excitation fluence. Taking advantage of a focused1310 nm interrogation beam, the penetration depth of the system is improved to ~ 2mm for an optical resolution system. High signal-to-noise ratios (>60dB) with ~ 2.5 cm working distance from the objective lens to the sample is achieved. Real-time in-vivo imaging of microvasculature and melanoma tumors are demonstrated.

Roger J. Zemp

Papers

Label-free non-contact imaging of cell nuclei using ultraviolet photoacoustic remote sensing microscopy (Conference Presentation)

Investigation of photoacoustic signal strength as a function of scan-speed and laser-repetition-rate

Multi-frequency CMUT imaging arrays for multi-scale imaging and imaging-therapy applications

Monte Carlo modeling for photoacoustic-based transport-regime optical property estimation

Non-interferometric deep optical resolution photoacoustic remote sensing microscopy (Conference Presentation)