Real-time interleaved spectroscopic photoacoustic and ultrasound (PAUS) scanning with simultaneous fluence compensation and motion correction.

For over two decades photoacoustic imaging has been tested clinically, but successful human trials have been limited. To enable quantitative clinical spectroscopy, the fundamental issues of wavelength-dependent fluence variations and inter-wavelength motion must be overcome. Here we propose a real-time, spectroscopic photoacoustic/ultrasound (PAUS) imaging approach using a compact, 1-kHz rate wavelength-tunable laser. Instead of illuminating tissue over a large area, the fiber-optic delivery system surrounding an US array sequentially scans a narrow laser beam, with partial PA image reconstruction for each laser pulse. The final image is then formed by coherently summing partial images. This scheme enables (i) automatic compensation for wavelength-dependent fluence variations in spectroscopic PA imaging and (ii) motion correction of spectroscopic PA frames using US speckle tracking in real-time systems. The 50-Hz video rate PAUS system is demonstrated in vivo using a murine model of labelled drug delivery.

Author Correction: Miniature gold nanorods for photoacoustic molecular imaging in the second near-infrared optical window.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Miniature gold nanorods for photoacoustic molecular imaging in the second near-infrared optical window.

In photoacoustic imaging, the second near-infrared (NIR-II) window is where tissue generates the least background signal. However, the large size of the few available contrast agents in this spectral range impedes their pharmacokinetics and decreases their thermal stability, leading to unreliable photoacoustic imaging. Here, we report the synthesis of miniaturized gold nanorods absorbing in the NIR-II that are 5-11 times smaller than regular-sized gold nanorods with a similar aspect ratio. Under nanosecond pulsed laser illumination, small nanorods are about 3 times more thermally stable and generate 3.5 times stronger photoacoustic signal than their absorption-matched larger counterparts. These unexpected findings are confirmed using theoretical and numerical analysis, showing that photoacoustic signal is not only proportional to the optical absorption of the nanoparticle solution but also to the surface-to-volume ratio of the nanoparticles. In living tumour-bearing mice, these small targeted nanorods display a 30% improvement in efficiency of agent delivery to tumours and generate 4.5 times greater photoacoustic contrast.

Molecular fingerprinting of nanoparticles in complex media with non-contact photoacoustics: beyond the light scattering limit.

Optical instruments can probe physical systems even to the level of individual molecules. In particular, every molecule, solution, and structure such as a living cell has a unique absorption spectrum representing a molecular fingerprint. This spectrum can help identify a particular molecule from others or quantify its concentration; however, scattering limits molecular fingerprinting within a complex compound and must be overcome. Here, we present a new, non-contact photoacoustic (PA)-based method that can almost completely remove the influence of background light scattering on absorption measurements in heterogeneous highly scattering solutions and, furthermore, separate the intrinsic absorption of nanoscale objects from their scattering. In particular, we measure pure absorption spectra for solutions of gold nanorods (GNRs) as an example of a plasmonic agent and show that these spectra differ from the extinction measured with conventional UV-VIS spectrophotometry. Finally, we show how the original GNR absorption changes when nanoparticles are internalized by cells.

Synthesis of hybrid magneto-plasmonic nanoparticles with potential use in photoacoustic detection of circulating tumor cells.

This article describes a novel synthetic route to obtain hybrid nanostructures that combine the plasmonic properties of gold nanorods with the magnetic properties of iron oxide nanoparticles in a robust silica nanostructure. The silica matrix enhances the physico-chemical stability of the nanostructure and preserves its magneto-plasmonic properties by avoiding the interface between gold and iron oxide. In addition, the magneto-plasmonic features of the nanohybrids can be tuned due to the independent synthesis of each component. The magnetic and plasmonic properties of these nanostructures can potentially enhance the photoacoustic detection of circulating tumor cells. Graphical abstract Schematic presentation of a hybrid magneto-plasmonic nanoparticle with an Au@Fe3O4@SiO2 core-satellite-shell arrangement. The magnetic and plasmonic responses of this kind of nanostructure enable magnetic trapping and photoacoustic detection of circulating tumor cells.

Optical coherence elastography in ophthalmology.

Optical coherence elastography (OCE) can provide clinically valuable information based on local measurements of tissue stiffness. Improved light sources and scanning methods in optical coherence tomography (OCT) have led to rapid growth in systems for high-resolution, quantitative elastography using imaged displacements and strains within soft tissue to infer local mechanical properties. We describe in some detail the physical processes underlying tissue mechanical response based on static and dynamic displacement methods. Namely, the assumptions commonly used to interpret displacement and strain measurements in terms of tissue elasticity for static OCE and propagating wave modes in dynamic OCE are discussed with the ultimate focus on OCT system design for ophthalmic applications. Practical OCT motion-tracking methods used to map tissue elasticity are also presented to fully describe technical developments in OCE, particularly noting those focused on the anterior segment of the eye. Clinical issues and future directions are discussed in the hope that OCE techniques will rapidly move forward to translational studies and clinical applications.

Polypyrrole-Coated Perfluorocarbon Nanoemulsions as a Sono-Photoacoustic Contrast Agent.

A new contrast agent for combined photoacoustic and ultrasound imaging is presented. It has a liquid perfluorocarbon (PFC) core of about 250 nm diameter coated by a 30 nm thin polypyrrole (PPy) doped polymer shell emulsion that represents a broadband absorber covering the visible and near-infrared ranges (peak optical extinction at 1050 nm). When exposed to a sufficiently high intensity optical or acoustic pulse, the droplets vaporize to form microbubbles providing a strong increase in imaging sensitivity and specificity. The threshold for contrast agent activation can further drastically be reduced by up to 2 orders of magnitude if simultaneously exposing them with optical and acoustic pulses. The selection of PFC core liquids with low boiling points (i.e., perfluorohexane (56 °C), perfluoropentane (29 °C), and perfluorobutane (-2 °C)) facilitates activation and reduces the activation threshold of PPy-coated emulsion contrast agents to levels well within clinical safety limits (as low as 0.2 MPa at 1 mJ/cm2). Finally, the potential use of these nanoemulsions as a contrast agent is demonstrated in a series of phantom imaging studies.

Dynamic contrast-enhanced photoacoustic imaging using photothermal stimuli-responsive composite nanomodulators.

Molecular photoacoustic imaging has shown great potential in medical applications; its sensitivity is normally in pico-to-micro-molar range, dependent on exogenous imaging agents. However, tissue can produce strong background signals, which mask the signals from the imaging agents, resulting in orders of magnitude sensitivity reduction. As such, an elaborate spectral scan is often required to spectrally un-mix the unwanted background signals. Here we show a new single-wavelength photoacoustic dynamic contrast-enhanced imaging technique by employing a stimuli-responsive contrast agent. Our technique can eliminate intrinsic background noises without significant hardware or computational resources. We show that this new contrast agent can generate up to 30 times stronger photoacoustic signals than the concentration-matched inorganic nanoparticle counterparts. By dynamically modulating signals from the contrast agents with an external near-infrared optical stimulus, we can further suppress the background signals leading to an additional increase of more than five-fold in imaging contrast in vivo.

Acoustic micro-tapping for non-contact 4D imaging of tissue elasticity.

Elastography plays a key role in characterizing soft media such as biological tissue. Although this technology has found widespread use in both clinical diagnostics and basic science research, nearly all methods require direct physical contact with the object of interest and can even be invasive. For a number of applications, such as diagnostic measurements on the anterior segment of the eye, physical contact is not desired and may even be prohibited. Here we present a fundamentally new approach to dynamic elastography using non-contact mechanical stimulation of soft media with precise spatial and temporal shaping. We call it acoustic micro-tapping (AµT) because it employs focused, air-coupled ultrasound to induce significant mechanical displacement at the boundary of a soft material using reflection-based radiation force. Combining it with high-speed, four-dimensional (three space dimensions plus time) phase-sensitive optical coherence tomography creates a non-contact tool for high-resolution and quantitative dynamic elastography of soft tissue at near real-time imaging rates. The overall approach is demonstrated in ex-vivo porcine cornea.

Moving-source elastic wave reconstruction for high-resolution optical coherence elastography.

Optical coherence tomography (OCT)-based elasticity imaging can map soft tissue elasticity based on speckle-tracking of elastic wave propagation using highly sensitive phase measurements of OCT signals. Using a fixed elastic wave source and moving detection, current imaging sequences have difficulty in reconstructing tissue elasticity within speckle-free regions, for example, within the crystalline lens of the eye. We present a moving acoustic radiation force imaging sequence to reconstruct elastic properties within a speckle-free region by tracking elastic wave propagation from multiple laterally moving sources across the field of view. We demonstrate the proposed strategy using heterogeneous and partial speckle-free tissue-mimicking phantoms. Harder inclusions within the speckle-free region can be detected, and the contrast-to-noise ratio slightly enhanced compared to current OCE imaging sequences. The results suggest that a moving source approach may be appropriate for OCE studies within the large speckle-free regions of the crystalline lens.

Air-coupled acoustic radiation force for non-contact generation of broadband mechanical waves in soft media.

A non-contact method for efficient, non-invasive excitation of mechanical waves in soft media is proposed, in which we focus an ultrasound (US) signal through air onto the surface of a medium under study. The US wave reflected from the air/medium interface provides radiation force to the medium surface that launches a transient mechanical wave in the transverse (lateral) direction. The type of mechanical wave is determined by boundary conditions. To prove this concept, a home-made 1 MHz piezo-ceramic transducer with a matching layer to air sends a chirped US signal centered at 1 MHz to a 1.6 mm thick gelatin phantom mimicking soft biological tissue. A phase-sensitive (PhS)-optical coherence tomography system is used to track/image the mechanical wave. The reconstructed transient displacement of the mechanical wave in space and time demonstrates highly efficient generation, thus offering great promise for non-contact, non-invasive characterization of soft media, in general, and for elasticity measurements in delicate soft tissues and organs in bio-medicine, in particular.

Functional Photoacoustic Imaging of Gastric Acid Secretion Using pH-Responsive Polyaniline Nanoprobes.

A stomach functional imaging technique based on photoacoustics achieves noninvasive gastric acid secretory assessment utilizing pH-responsive polyaniline nanoprobes. A testing protocol mimicking clinical practice is established using a mouse model. After imaging, the nanoprobes are excreted outside the body without inducing systematic toxicity. Further optimization and translation of this technology can help alleviate patients' suffering and side effects.

Stably Doped Conducting Polymer Nanoshells by Surface Initiated Polymerization.

Despite broad applications ranging from electronics to biomedical sensing and imaging, a long-standing problem of conducting polymers is the poor resistance to dedoping, which directly affects their signature electrical and optical properties. This problem is particularly significant for biomedical uses because of fast leaching of dopant ions in physiological environments. Here, we describe a new approach to engineer multimodal core-shell nanoparticles with a stably doped conductive polymer shell in biological environments. It was achieved by making a densely packed polymer brush rather than changing its molecular structure. Polyaniline (PANI) was used as a model compound due to its concentrated near-infrared (NIR) absorption. It was grafted onto a magnetic nanoparticle via a polydopamine intermediate layer. Remarkably, at pH 7 its conductivity is ca. 2000× higher than conventional PANI nanoshells. Similarly, its NIR absorption is enhanced by 2 orders of magnitude, ideal for photothermal imaging and therapy. Another surprising finding is its nonfouling property, even outperforming polyethylene glycol. This platform technology is also expected to open exciting opportunities in engineering stable conductive materials for electronics, imaging, and sensing.

Optimization of the laser irradiation pattern in a high frame rate integrated photoacoustic / ultrasound (PAUS) imaging system.

To integrate real-time photoacoustics (PA) into ultrasound (US) scanners and accelerate clinical translation of combined PAUS imaging, we previously developed a system using a portable, low-cost, low pulse energy, high-repetition rate laser (~1kHz) with a 1D galvo-mirror for rapid laser beam scanning over the imaging area. However, the frame rate and pulse energy are limited because of regulations on the radiance (1 W/cm2). Therefore, a laser scan scheme needs to be optimized to provide high frame rate within this safety limit. In addition, the laser light should be evenly distributed to minimize any artifacts caused by the scanning approach. In this paper, we calculated the laser light distribution using 3D Monte Carlo simulation and further developed the system to scan the laser beam in elevation as well as laterally using a 2-dimensional galvo-mirror scanner to achieve higher frame rates within the radiance safety limit. Insertion of a needle into chicken breast tissue was used to demonstrate our optimized scan scheme.

Development and optimization of near-IR contrast agents for immune cell tracking.

Gold nanorods (NRs) are attractive for in vivo imaging due to their high optical cross-sections and tunable absorbance. However, the feasibility of using NRs for cell tracking has not been fully explored. Here, we synthesized dye doped silica-coated NRs as multimodal contrast agents for imaging of macrophages - immune cells which play an important role in cancer and cardiovascular diseases. We showed the importance of silica coating in imaging of NR-labeled cells. Photoacoustic (PA) imaging of NRs labeled macrophages showed high sensitivity. Therefore, these results provide foundation for applications of silica-coated NRs and PA imaging in tracking of immune cells.

Photoacoustic Imaging for Cancer Detection and Staging.

Cancer is one of the leading causes of death in the world. Diagnosing a cancer at its early stages of development can decrease the mortality rate significantly and reduce healthcare costs. Over the past two decades, photoacoustic imaging has seen steady growth and has demonstrated notable capabilities to detect cancerous cells and stage cancer. Furthermore, photoacoustic imaging combined with ultrasound imaging and augmented with molecular targeted contrast agents is capable of imaging cancer at the cellular and molecular level, thus opening diverse opportunities to improve diagnosis of tumors, detect circulating tumor cells and identify metastatic lymph nodes. In this paper we introduce the principles of photoacoustic imaging, and review recent developments in photoacoustic imagingas an emerging imaging modality for cancer diagnosis and staging.

Conjugation of antibodies to gold nanorods through Fc portion: synthesis and molecular specific imaging.

Anisotropic gold nanorods provide a convenient combination of properties, such as tunability of plasmon resonances and strong extinction cross sections in the near-infrared to red spectral region. These properties have created significant interest in the development of antibody conjugation methods for synthesis of targeted nanorods for a number of biomedical applications, including molecular specific imaging and therapy. Previously published conjugation approaches have achieved molecular specificity. However, the current conjugation methods have several downsides including low stability and potential cytotoxicity of bioconjugates that are produced by electrostatic interactions, as well as lack of control over antibody orientation during covalent conjugation. Here we addressed these shortcomings by introducing directional antibody conjugation to the gold nanorod surface. The directional conjugation is achieved through the carbohydrate moiety, which is located on one of the heavy chains of the Fc portion of most antibodies. The carbohydrate is oxidized under mild conditions to a hydrazide reactive aldehyde group. Then, a heterofunctional linker with hydrazide and dithiol groups is used to attach antibodies to gold nanorods. The directional conjugation approach was characterized using electron microscopy, zeta potential, and extinction spectra. We also determined spectral changes associated with nanorod aggregation; these spectral changes can be used as a convenient quality control of nanorod bioconjugates. Molecular specificity of the synthesized antibody targeted nanorods was demonstrated using hyperspectral, optical and photoacoustic imaging of cancer cell culture models. Additionally, we observed characteristic changes in optical spectra of molecular specific nanorods after their interactions with cancer cells; the observed spectral signatures can be explored for sensitive cancer detection.

Thermal stability of biodegradable plasmonic nanoclusters in photoacoustic imaging.

The photothermal stability of plasmonic nanoparticles is critically important to perform reliable photoacoustic imaging and photothermal therapy. Recently, biodegradable nanoclusters composed of sub-5 nm primary gold particles and a biodegradable polymer have been reported as clinically-translatable contrast agents for photoacoustic imaging. After cellular internalization, the nanoclusters degrade into 5 nm primary particles for efficient excretion from the body. In this paper, three different sizes of biodegradable nanoclusters were synthesized and the optical properties and photothermal stability of the nanoclusters were investigated and compared to that of gold nanorods. The results of our study indicate that 40 nm and 80 nm biodegradable nanoclusters demonstrate higher photothermal stability compared to gold nanorods. Furthermore, 40 nm nanoclusters produce higher photoacoustic signal than gold nanorods at a given concentration of gold. Therefore, the biodegradable plasmonic nanoclusters can be effectively used for photoacoustic imaging and photothermal therapy.

Utility of biodegradable plasmonic nanoclusters in photoacoustic imaging.

Plasmonic metal nanoparticles are used in photoacoustic imaging as contrast agents because of their resonant optical absorption properties in the visible and near-IR regions. However, the nanoparticles could accumulate and result in long-term toxicity in vivo, because they are generally not biodegradable. Recently, biodegradable plasmonic gold nanoclusters, consisting of sub-5 nm primary gold nanoparticles and biodegradable polymer stabilizer, were introduced. In this Letter, we demonstrate the feasibility of biodegradable nanoclusters as a photoacoustic contrast agent. We performed photoacoustic and ultrasound imaging of a tissue-mimicking phantom with inclusions containing nanoclusters at various concentrations. The results indicate that the biodegradable gold nanoclusters can be used as effective contrast agents in photoacoustic imaging.

Target dependence of the sensitivity in periodic nanowire-based localized surface plasmon resonance biosensors.

We investigate the target dependence of the sensitivity in a localized surface plasmon resonance (LSPR) biosensor and compare it with that of a conventional thin-film-based plasmon resonance structure. An LSPR biosensor was modeled as subwavelength periodic nanowires on a metal/dielectric substrate and targets either as bulk refractive index changes or as a biomolecular interaction that forms a monolayer. The results found that significant target-dependent variation arises in sensitivity and sensitivity enhancement by LSPR. The variation is attributed to the nonlinearity in the plasmon dispersion relation as well as the effective permittivity due to strong LSPR signals. The target dependence suggests that an LSPR structure be designed based on estimated index changes induced by target interactions. Associated broadening of resonance width can be controlled by way of profile engineering, which is discussed in connection with experimental data.