Development and validation of nomograms using photoacoustic imaging and 2D ultrasound to predict breast nodule benignity and malignancy.

BACKGROUND: The application of photoacoustic imaging (PAI), utilizing laser-induced ultrasound, shows potential in assessing blood oxygenation in breast nodules. However, its effectiveness in distinguishing between malignant and benign nodules remains insufficiently explored. PURPOSE: This study aims to develop nomogram models for predicting the benign or malignant nature of breast nodules using PAI. METHOD: A prospective cohort study enrolled 369 breast nodules, subjecting them to PAI and ultrasound examination. The training and testing cohorts were randomly divided into two cohorts in a ratio of 3:1. Based on the source of the variables, three models were developed, Model 1: photoacoustic-BIRADS+BMI + blood oxygenation, Model 2: BIRADS+Shape+Intranodal blood (Doppler) + BMI, Model 3: photoacoustic-BIRADS+BIRADS+ Shape+Intranodal blood (Doppler) + BMI + blood oxygenation. Risk factors were identified through logistic regression, resulting in the creation of three predictive models. These models were evaluated using calibration curves, subject receiver operating characteristic (ROC), and decision curve analysis. RESULTS: The area under the ROC curve for the training cohort was 0.91 (95% confidence interval, 95% CI: 0.88-0.95), 0.92 (95% CI: 0.89-0.95), and 0.97 (95% CI: 0.96-0.99) for Models 1-3, and the ROC curve for the testing cohort was 0.95 (95% CI: 0.91-0.98), 0.89 (95% CI: 0.83-0.96), and 0.97 (95% CI: 0.95-0.99) for Models 1-3. CONCLUSIONS: The calibration curves demonstrate that the model's predictions agree with the actual values. Decision curve analysis suggests a good clinical application.

Ultrafast four-dimensional imaging of cardiac mechanical wave propagation with sparse optoacoustic sensing.

Propagation of electromechanical waves in excitable heart muscles follows complex spatiotemporal patterns holding the key to understanding life-threatening arrhythmias and other cardiac conditions. Accurate volumetric mapping of cardiac wave propagation is currently hampered by fast heart motion, particularly in small model organisms. Here we demonstrate that ultrafast four-dimensional imaging of cardiac mechanical wave propagation in entire beating murine heart can be accomplished by sparse optoacoustic sensing with high contrast, ∼115-µm spatial and submillisecond temporal resolution. We extract accurate dispersion and phase velocity maps of the cardiac waves and reveal vortex-like patterns associated with mechanical phase singularities that occur during arrhythmic events induced via burst ventricular electric stimulation. The newly introduced cardiac mapping approach is a bold step toward deciphering the complex mechanisms underlying cardiac arrhythmias and enabling precise therapeutic interventions.

Frequency-Resolved High-Frequency Broadband Measurement of Acoustic Longitudinal Waves by Laser-Based Excitation and Detection.

Optoacoustics is a metrology widely used for material characterisation. In this study, a measurement setup for the selective determination of the frequency-resolved phase velocities and attenuations of longitudinal waves over a wide frequency range (3-55 MHz) is presented. The ultrasonic waves in this setup were excited by a pulsed laser within an absorption layer in the thermoelastic regime and directed through a layer of water onto a sample. The acoustic waves were detected using a self-built adaptive interferometer with a photorefractive crystal. The instrument transmits compression waves only, is low-contact, non-destructive, and has a sample-independent excitation. The limitations of the approach were studied both by simulation and experiments to determine how the frequency range and precision can be improved. It was shown that measurements are possible for all investigated materials (silicon, silicone, aluminium, and water) and that the relative error for the phase velocity is less than 0.2%.

Merged Molecular Switches Excel as Optoacoustic Dyes: Azobenzene-Cyanines Are Loud and Photostable NIR Imaging Agents.

Optoacoustic (or photoacoustic) imaging promises micron-resolution noninvasive bioimaging with much deeper penetration (>cm) than fluorescence. However, optoacoustic imaging of enzyme activity would require loud, photostable, NIR-absorbing molecular contrast agents: which remain unknown. Most organic molecular contrast agents are repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging, as consequences of their slow S1→S0 electronic relaxation. We now report that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches to cyanine dyes to give ultrafast relaxation (<10 ps, >100-fold faster). Without even adapting instrument settings, these azohemicyanines display outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (here: >3-fold even at time zero). We show why this simple but unexplored design strategy can still offer stronger performance in the future, and can also increase the spatial resolution and the quantitative linearity of photoacoustic response over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on problems facing optoacoustic agents, this practical strategy will help to unleash the full potential of optoacoustic imaging in fundamental studies and translational uses.

Tunable Interparticle Connectivity in Gold Nanosphere Assemblies for Efficient Photoacoustic Conversion.

Manipulating matter at the nanometer scale to create desired plasmonic nanostructures holds great promise in the field of biomedical photoacoustic (PA) imaging. We demonstrate a strategy for regulating PA signal generation from anisotropic nano-sized assemblies of gold nanospheres (Au NSs) by adjusting the inter-particle connectivity between neighboring Au NSs. The inter-particle connectivity is controlled by modulating the diameter and inter-particle spacing of Au NSs in the nanoassemblies. The results indicate that nanoassemblies with semi-connectivity, i.e., assemblies with a finite inter-particle spacing shorter than the theoretical limit of repulsion between nearby Au NSs, exhibit 3.4-fold and 2.4-fold higher PA signals compared to nanoassemblies with no connectivity and full connectivity, respectively. Furthermore, due to the reduced diffusion of Au atoms, the semi-connectivity Au nanoassemblies demonstrate high photodamage threshold and, therefore, excellent photostability at fluences above the current American National Standards Institute limits. The exceptional photostability of the semi-connectivity nanoassemblies highlights their potential to surpass conventional plasmonic contrast agents for continuing PA imaging. Collectively, our findings indicate that semi-connected nanostructures are a promising option for reliable, high-contrast PA imaging applications over multiple imaging sessions due to their strong PA signals and enhanced photostability.

Effect of pulsed laser parameters on photoacoustic flow cytometry efficiency in vitro and in vivo.

Photoacoustic flow cytometry is one of the most effective approaches to detect "alien" objects in the bloodstream, including circulating tumor cells, blood clots, parasites, and emboli. However, the possibility of detecting high-amplitude signals from these objects against the background of blood depends on the parameters of the laser pulse. So, the dependencies of photoacoustic signals amplitude and number on laser pulse energy (5-150 µJ), pulse length (1, 2, 5 ns), and pulse repetition rate (2, 5, 10 kHz) for the melanoma cells were investigated. First, the PA responses of a melanoma cell suspension in vitro were measured to directly assess the efficiency of converting laser light into an acoustic signal. After it, the same dependence with the developed murine model based on constant rate melanoma cell injection into the animal blood flow was tested. Both in vivo and in vitro experiments show that signal generation efficiency increases with laser pulse energy above 15 µJ. Shorter pulses, especially 1 ns, provide more efficient signal generation as well as higher pulse rates. A higher pulse rate also provides more efficient signal generation, but also leads to overheating of the skin. The results show the limits where the photoacoustic flow cytometry system can be effectively used for the detection of circulating tumor cells in undiluted blood both for in vitro experiments and for in vivo murine models.

Feasibility of photoacoustic-guided ultrasound treatment for port wine stains.

BACKGROUND AND OBJECTIVES: Port wine birthmark, also known as port wine stain (PWS) is a skin discoloration characterized by red/purple patches caused by vascular malformation. PWS is typically treated by using lasers to destroy abnormal blood vessels. The laser heating facilitates selective photothermolysis of the vessels and attenuates quickly in the tissue due to high optical scattering. Therefore, residual abnormal capillaries deep in the tissue survive and often lead to the resurgence of PWS. Ultrasound (US) has also been proposed to treat PWS, however, it is nonselective with respect to the vasculature but penetrates deeper into the tissue. We aim to study the feasibility of a hybrid PWS treatment modality combining the advantages of both modalities. MATERIALS AND METHODS: In this manuscript, we propose a photoacoustic (PA) guided US focusing methodology for PWS treatment which combines the optical contrast-based selectivity with US penetration to focus the US energy onto the vasculature. The PA signals collected by the transducers, when time-reversed, amplified, and transmitted, converge onto the PWS, thus minimally affecting the neighboring tissue. We performed two- and three-dimensional simulations that mimic realistic transducers and medium properties in this proof of concept study. RESULTS: The time-reversed PA signals when transmitted from the transducers converged onto the vasculature, as expected, thus reducing the heating of the neighboring tissue. We observed that while the US focus is indeed affected due to experimental factors such as limited-view, large detector separation and finite detection bandwidth, and so forth, the US did focus completely or partially onto the vasculature demonstrating the feasibility of the proposed methodology. CONCLUSION: The results demonstrate the potential of the proposed methodology for PWS treatment. This treatment method can destroy the deeper capillaries while minimally heating the neighboring tissue, thus reducing the chances of the resurgence of PWS and as well as cosmetic scarring.

Short-wavelength optoacoustic spectroscopy based on water muting.

Infrared (IR) optoacoustic spectroscopy can separate a multitude of molecules based on their absorption spectra. However, the technique is limited when measuring target molecules in aqueous solution by strong water absorption at IR wavelengths, which reduces detection sensitivity. Based on the dependence of optoacoustic signal on the temperature of the probed medium, we introduce cooled IR optoacoustic spectroscopy (CIROAS) to mute water contributions in optoacoustic spectroscopy. We showcase that spectral measurements of proteins, lipids, and glucose in the short-wavelength IR region, performed at 4 °C, lead to marked sensitivity improvements over conventional optoacoustic or IR spectroscopy. We elaborate on the dependence of optoacoustic signals on water temperature and demonstrate polarity changes in the recorded signal at temperatures below 4 °C. We further elucidate the dependence of the optoacoustic signal and the muting temperature on sample concentration and demonstrate that changes in these dependences enable quantification of the solute concentration. We discuss how CIROAS may enhance abilities for molecular sensing in the IR.

Quartz-Enhanced Photoacoustic Spectroscopy Assisted by Partial Least-Squares Regression for Multi-Gas Measurements.

We report on the use of quartz-enhanced photoacoustic spectroscopy (QEPAS) for multi-gas detection. Photoacoustic (PA) spectra of mixtures of water (H2O), ammonia (NH3), and methane (CH4) were measured in the mid-infrared (MIR) wavelength range using a mid-infrared (MIR) optical parametric oscillator (OPO) light source. Highly overlapping absorption spectra are a common challenge for gas spectroscopy. To mitigate this, we used a partial least-squares regression (PLS) method to estimate the mixing ratio and concentrations of the individual gasses. The concentration range explored in the analysis varies from a few parts per million (ppm) to thousands of ppm. Spectra obtained from HITRAN and experimental single-molecule reference spectra of each of the molecular species were acquired and used as training data sets. These spectra were used to generate simulated spectra of the gas mixtures (linear combinations of the reference spectra). Here, in this proof-of-concept experiment, we demonstrate that after an absolute calibration of the QEPAS cell, the PLS analyses could be used to determine concentrations of single molecular species with a relative accuracy within a few % for mixtures of H2O, NH3, and CH4 and with an absolute sensitivity of approximately 300 (±50) ppm/V, 50 (±5) ppm/V, and 5 (±2) ppm/V for water, ammonia, and methane, respectively. This demonstrates that QEPAS assisted by PLS is a powerful approach to estimate concentrations of individual gas components with considerable spectral overlap, which is a typical scenario for real-life adoptions and applications.

Predicting optoacoustic spectral behaviour of human erythrocytes, stomatocytes and echinocytes using a modified Green's function method.

Optoacoustic (OA) spectral properties of various sources mimicking normal and pathological red blood cells (RBCs) have been studied. The shapes of normal RBC and cells suffering from stomatocytosis (denoted by ST) were generated using mathematical models. However, the shape corresponding to the cells affected by echinocytosis (referred to as ET) was constructed by uniformly distributing half prolate spheroids on a central spherical object. The OA field emitted by an acoustically inhomogeneous source was calculated for a wide acoustic frequency bandwidth (1-1500 MHz with an increment 5 MHz) by solving the time-independent wave equation employing a modified Green's function approach. The OA spectra averaged over 200 orientations for normal RBC and STs demonstrate similar features (one minimum occurring nearly at 906 MHz). The same graphs for ETs are remarkably different from that of normal RBC and exhibit better match with that of a spherical RBC (first minimum appearing at around 425 MHz). The spectral features of ETs above 425 MHz may enable us to differentiate diseased cells (echinocytosis) from normal RBCs.

Piezoelectric and Opto-Acoustic Material Properties of Bone.

In this chapter, recent piezoelectric and opto-acoustic studies on bone are introduced. The former are certainly related to ultrasound since piezoelectricity is one of the electro-mechanical properties. The latter are divided into two parts: Photo Acoustics (PA) and Brillouin Scattering (BS). PA is the energy conversion from light to ultrasound while Brillouin scattering is the interaction between phonons and photons. These studies seem very different; however, they are all studies on the ultrasonic material characterization of bone. Another common aspect of these studies is that they are generally targeting the material characterization of bone extracellular matrix. These studies have started later than the conventional ultrasonic bone studies and are expected to provide different characteristics of bone in the micrometer scale area.

Gold-implanted plasmonic quartz plate as a launch pad for laser-driven photoacoustic microfluidic pumps.

Enabled initially by the development of microelectromechanical systems, current microfluidic pumps still require advanced microfabrication techniques to create a variety of fluid-driving mechanisms. Here we report a generation of micropumps that involve no moving parts and microstructures. This micropump is based on a principle of photoacoustic laser streaming and is simply made of an Au-implanted plasmonic quartz plate. Under a pulsed laser excitation, any point on the plate can generate a directional long-lasting ultrasound wave which drives the fluid via acoustic streaming. Manipulating and programming laser beams can easily create a single pump, a moving pump, and multiple pumps. The underlying pumping mechanism of photoacoustic streaming is verified by high-speed imaging of the fluid motion after a single laser pulse. As many light-absorbing materials have been identified for efficient photoacoustic generation, photoacoustic micropumps can have diversity in their implementation. These laser-driven fabrication-free micropumps open up a generation of pumping technology and opportunities for easy integration and versatile microfluidic applications.

Improving PMUT Receive Sensitivity via DC Bias and Piezoelectric Composition.

The receive sensitivity of lead zirconate titanate (PZT) piezoelectric micromachined ultrasound transducers (PMUTs) was improved by applying a DC bias during operation. The PMUT receive sensitivity is governed by the voltage piezoelectric coefficient, h31,f. With applied DC biases (up to 15 V) on a 2 µm PbZr0.52Ti0.48O3 film, e31,f increased 1.6 times, permittivity decreased by a factor of 0.6, and the voltage coefficient increased by ~2.5 times. For released PMUT devices, the ultrasound receive sensitivity improved by 2.5 times and the photoacoustic signal improved 1.9 times with 15 V applied DC bias. B-mode photoacoustic imaging experiments showed that with DC bias, the PMUT received clearer photoacoustic signals from pencil leads at 4.3 cm, compared to 3.7 cm without DC bias.

Chlorophyll fluorometry in evaluating photosynthetic performance: key limitations, possibilities, perspectives and alternatives.

Non-destructive methods for the assessment of photosynthetic parameters of plants are widely applied to evaluate rapidly the photosynthetic performance, plant health, and shifts in plant productivity induced by environmental and cultivation conditions. Most of these methods are based on measurements of chlorophyll fluorescence kinetics, particularly on pulse modulation (PAM) fluorometry. In this paper, fluorescence methods are critically discussed in regard to some their possibilities and limitations inherent to vascular plants and microalgae. Attention is paid to the potential errors related to the underestimation of thylakoidal cyclic electron transport and anoxygenic photosynthesis. PAM-methods are also observed considering the color-addressed measurements. Photoacoustic methods are discussed as an alternative and supplement to fluorometry. Novel Fourier modifications of PAM-fluorometry and photoacoustics are noted as tools allowing simultaneous application of a dual or multi frequency measuring light for one sample.

Design Strategies of Photoacoustic Molecular Probes.

Photoacoustic (PA) probes have been developed very quickly and applied in broad areas in recent years. Most of them are constructed based on organic dyes with intrinsic near-infrared (NIR) absorption properties. To increase PA contrast and improve imaging resolution and the sensitivity of detection, various methods for the design of PA probes have been developed. This minireview mainly focuses on the development and design strategies of activatable small-molecule PA probes in four aspects: reaction-cleavage, metal ion chelation, photoswitch, and protonation-deprotonation. It highlights some key points of designing PA probes corresponding to their properties and applications. The challenges and perspectives for small-molecule PA probes are also discussed.

Optoacoustic imaging of the skin.

Optoacoustic (OA, photoacoustic) imaging capitalizes on the synergistic combination of light excitation and ultrasound detection to empower biological and clinical investigations with rich optical contrast while effectively bridging the gap between micro and macroscopic imaging realms. State-of-the-art OA embodiments consistently provide images at micron-scale resolution through superficial tissue layers by means of focused illumination that can be smoothly exchanged for acoustic-resolution images at diffuse light depths of several millimetres to centimetres via ultrasound beamforming or tomographic reconstruction. Taken together, this unique multi-scale imaging capacity opens unprecedented capabilities for high-resolution in vivo interrogations of the skin at scalable depths. Moreover, diverse anatomical and functional information is retrieved via dynamic mapping of endogenous chromophores such as haemoglobin, melanin, lipids, collagen, water and others. This, along with the use of non-ionizing radiation, facilitates a clinical translation of the OA modalities. We review recent progress in OA imaging of the skin in preclinical and clinical studies exploiting the rich contrast provided by endogenous substances in tissues. The imaging capabilities of existing approaches are discussed in the context of initial translational studies on skin cancer, inflammatory skin diseases, wounds and other conditions.

Multi-scale optoacoustic molecular imaging of brain diseases.

The ability to non-invasively visualize endogenous chromophores and exogenous probes and sensors across the entire rodent brain with the high spatial and temporal resolution has empowered optoacoustic imaging modalities with unprecedented capacities for interrogating the brain under physiological and diseased conditions. This has rapidly transformed optoacoustic microscopy (OAM) and multi-spectral optoacoustic tomography (MSOT) into emerging research tools to study animal models of brain diseases. In this review, we describe the principles of optoacoustic imaging and showcase recent technical advances that enable high-resolution real-time brain observations in preclinical models. In addition, advanced molecular probe designs allow for efficient visualization of pathophysiological processes playing a central role in a variety of neurodegenerative diseases, brain tumors, and stroke. We describe outstanding challenges in optoacoustic imaging methodologies and propose a future outlook.

Acoustics at the nanoscale (nanoacoustics): A comprehensive literature review.: Part II: Nanoacoustics for biomedical imaging and therapy.

In the past decade, acoustics at the nanoscale (i.e., nanoacoustics) has evolved rapidly with continuous and substantial expansion of capabilities and refinement of techniques. Motivated by research innovations in the last decade, for the first time, recent advancements of acoustics-associated nanomaterials/nanostructures and nanodevices for different applications are outlined in this comprehensive review, which is written in two parts. As part II of this two-part review, this paper concentrates on nanoacoustics in biomedical imaging and therapy applications, including molecular ultrasound imaging, photoacoustic imaging, ultrasound-mediated drug delivery and therapy, and photoacoustic drug delivery and therapy. Firstly, the recent developments of nanosized ultrasound and photoacoustic contrast agents as well as their various imaging applications are examined. Secondly, different types of nanomaterials/nanostructures as nanocarriers for ultrasound and photoacoustic therapies are discussed. Finally, a discussion of challenges and future research directions are provided for nanoacoustics in medical imaging and therapy.

A Cost-Efficient Multiwavelength LED-Based System for Quantitative Photoacoustic Measurements.

The unique ability of photoacoustic (PA) sensing to provide optical absorption information of biomolecules deep inside turbid tissues with high sensitivity has recently enabled the development of various novel diagnostic systems for biomedical applications. In many cases, PA setups can be bulky, complex, and costly, as they typically require the integration of expensive Q-switched nanosecond lasers, and also presents limited wavelength availability. This article presents a compact, cost-efficient, multiwavelength PA sensing system for quantitative measurements, by utilizing two high-power LED sources emitting at central wavelengths of 444 and 628 nm, respectively, and a single-element ultrasonic transducer at 3.5 MHz for signal detection. We investigate the performance of LEDs in pulsed mode and explore the dependence of PA responses on absorber's concentration and applied energy fluence using tissue-mimicking phantoms demonstrating both optical absorption and scattering properties. Finally, we apply the developed system on the spectral unmixing of two absorbers contained at various relative concentrations in the phantoms, to provide accurate estimations with absolute deviations ranging between 0.4 and 12.3%. An upgraded version of the PA system may provide valuable in-vivo multiparametric measurements of important biomarkers, such as hemoglobin oxygenation, melanin concentration, local lipid content, and glucose levels.

Oxygen Saturation Imaging Using LED-Based Photoacoustic System.

Oxygen saturation imaging has potential in several preclinical and clinical applications. Dual-wavelength LED array-based photoacoustic oxygen saturation imaging can be an affordable solution in this case. For the translation of this technology, there is a need to improve its accuracy and validate it against ground truth methods. We propose a fluence compensated oxygen saturation imaging method, utilizing structural information from the ultrasound image, and prior knowledge of the optical properties of the tissue with a Monte-Carlo based light propagation model for the dual-wavelength LED array configuration. We then validate the proposed method with oximeter measurements in tissue-mimicking phantoms. Further, we demonstrate in vivo imaging on small animal and a human subject. We conclude that the proposed oxygen saturation imaging can be used to image tissue at a depth of 6-8 mm in both preclinical and clinical applications.