As photoacoustic imaging (PAI) begins to mature and undergo clinical translation, there is a need for well-validated, standardized performance test methods to support device development, quality control, and regulatory evaluation. Despite recent progress, current PAI phantoms may not adequately replicate tissue light and sound transport over the full range of optical wavelengths and acoustic frequencies employed by reported PAI devices. Here we introduce polyacrylamide (PAA) hydrogel as a candidate material for fabricating stable phantoms with well-characterized optical and acoustic properties that are biologically relevant over a broad range of system design parameters. We evaluated suitability of PAA phantoms for conducting image quality assessment of three PAI systems with substantially different operating parameters including two commercial systems and a custom system. Imaging results indicated that appropriately tuned PAA phantoms are useful tools for assessing and comparing PAI system image quality. These phantoms may also facilitate future standardization of performance test methodology.

Photoacoustic mesoscopy visualises vascular architecture at high-resolution up to ~3 mm depth. Despite promise in preclinical and clinical imaging studies, with applications in oncology and dermatology, the accuracy and precision of photoacoustic mesoscopy is not well established. Here, we evaluate a commercial photoacoustic mesoscopy system for imaging vascular structures. Typical artefact types are first highlighted and limitations due to non-isotropic illumination and detection are evaluated with respect to rotation, angularity, and depth of the target. Then, using tailored phantoms and mouse models, we investigate system precision, showing coefficients of variation (COV) between repeated scans [short term (1 h): COV= 1.2%; long term (25 days): COV= 9.6%], from target repositioning (without: COV=1.2%, with: COV=4.1%), or from varying in vivo user experience (experienced: COV=15.9%, unexperienced: COV=20.2%). Our findings show robustness of the technique, but also underscore general challenges of limited-view photoacoustic systems in accurately imaging vessel-like structures, thereby guiding users when interpreting biologically-relevant information.

Pulsed laser diodes may offer a smaller, less expensive alternative to conventional optoacoustic laser sources; however they do not provide pulse rates faster than a few tens of kHz and emit at wavelengths only within the near-infrared region. We investigated whether continuous wave (CW) laser diodes, which are available in visible and near-infrared regions, can be good optoacoustic light sources when overdriven with a peak current >40-fold higher than the CW absolute maximum. We found that overdriven CW diodes provided ∼10 ns pulses of ∼200 nJ/pulse and repetition rates higher than 600 kHz without being damaged, outperforming many pulsed laser diodes. Using this system, we obtained images of phantoms and mouse ear and human arm in vivo, confirming their use in optoacoustic imaging and sensing.

Optoacoustic imaging is a rapidly expanding field for the diagnosis, characterization, and treatment evaluation of cancer. However, the availability of tumor specific exogenous contrast agents is still limited. Here, we report on a small targeted contrast agent for optoacoustic imaging using a black hole quencher® (BHQ) dye. The sonophore BHQ-1 exhibited strong, concentration-dependent, optoacoustic signals in phantoms, demonstrating its ideal suitability for optoacoustic imaging. After labeling BHQ-1 with cyclic RGD-peptide, BHQ-1-cRGD specifically bound to αvβ3-integrin expressing glioblastoma cell spheroids in vitro. The excellent optoacoustic properties of BHQ-1-cRGD could furthermore be proven in vivo. Together with this emerging imaging modality, our sonophore labeled small peptide probe offers new possibilities for non-invasive detection of molecular structures with high resolution in vivo and furthers the specificity of optoacoustic imaging. Ultimately, the discovery of tailor-made sonophores might offer new avenues for various molecular optoacoustic imaging applications, similar to what we see with fluorescence imaging.

This work presents spatially compounded plane wave imaging using a laser-induced ultrasound source. The plane wave source consisted of a 30 μm thick film of carbon black-doped PDMS cured on a 100 μm thick polyester substrate and presented a rectangular aperture of 40 × 3 mm. It was placed in front of a linear ultrasound array, passing through the imaging plane allowing overlap of the detection plane and the insonification plane. Illumination was provided by an array of optical fibre bundles placed above the imaging plane, at an angle. We will first present the general imaging set up and instrumentation used, after which details are given on the fabrication of the transmitter itself and on the objects that were imaged. Comparing laser-induced and conventional ultrasound images of wire phantoms shows the point-spread-function to be, in general, slightly better laterally in the conventional case but more homogeneous throughout the imaging plane with the laser-induced source. Imaging of a tissue-mimicking phantom shows a 55% improvement in contrast between a tumour and the background when using laser-induced ultrasound, as compared to the conventional case.

Comprehensive characterization of biomedical imaging systems require phantoms that are easy to fabricate and can mimic human tissue. Additionally, with the arrival of engineered tissues, it is key to develop phantoms that can mimic bioengineered samples. In ultrasound and photoacoustic imaging, water-soluble phantom materials such as gelatin undergo rapid degradation while polymer-based materials such as polyvinyl alcohol are not conducive for generating bioengineered tissues that can incorporate cells. Here we propose silk protein-based hydrogels as an ultrasound and photoacoustic phantom material that has potential to provide a 3D environment for long-term sustainable cell growth. Common acoustic, optical, and biomechanical properties such as ultrasound attenuation, reduced scattering coefficient, and Young's modulus were measured. The results indicate that silk acoustically mimics many tissue types while exhibiting similar reduced optical scattering in the wavelength range of 400-1200 nm. Furthermore, silk-based materials can be stored long-term with no change in acoustic and optical properties, and hence can be utilized to assess the performance of ultrasound and photoacoustic systems.

Thermal strain imaging (TSI) is a widely investigated ultrasound (US) thermometry technique that is based on the temperature-dependent change in speed of sound. However, a major challenge of TSI is a calibration process to account for material-dependent thermal strain. In this study, we leverage nanoparticle (NP)-mediated photoacoustic (PA) thermometry to calibrate thermal strain and guide US thermal imaging. By controlling the molecular composition of the sub-micrometer layer surrounding the NPs, PA thermometry becomes independent of the thermal characteristics of the overall background tissue where the NPs reside. Thus accurate temperature measurements are obtainable from sparse NP-mediated PA signals. These measurements are used to guide TSI, allowing US thermometry to produce an expanded temperature map over the entire region of interest without prior knowledge of tissue composition. Our feasibility study in tissue-mimicking phantoms demonstrates the potential to improve TSI by integrating a PA-based calibration method that complements and guides US thermometry.

Combined ultrasound and photoacoustic (USPA) imaging has attracted several pre-clinical and clinical applications due to its ability to simultaneously display structural, functional, and molecular information of deep biological tissue in real time. However, the depth and wavelength dependent optical attenuation and the unknown optical and acoustic heterogeneities limit the USPA imaging performance in deep tissue regions. Novel instrumentation, image reconstruction, and artificial intelligence (AI) methods are currently being investigated to overcome these limitations and improve the USPA image quality. Effective implementation of these approaches requires a reliable USPA simulation tool capable of generating US based anatomical and PA based molecular contrasts of deep biological tissue. Here, we developed a hybrid USPA simulation platform by integrating finite element models of light (NIRFast) and ultrasound (k-Wave) propagations for co-simulation of B-mode US and PA images. The platform allows optimization of different design parameters for USPA devices, such as the aperture size and frequency of both light and ultrasound detector arrays. For designing tissue-realistic digital phantoms, a dictionary-based function has been added to k-Wave to generate various levels of ultrasound speckle contrast. The feasibility of modeling US imaging combined with optical fluence dependent multispectral PA imaging is demonstrated using homogeneous as well as heterogeneous tissue phantoms mimicking human organs (e.g., prostate and finger). In addition, we also demonstrate the potential of the simulation platform to generate large scale application-specific training and test datasets for AI enhanced USPA imaging. The complete USPA simulation codes together with the supplementary user guides have been posted to an open-source repository (https://github.com/KothapalliLabPSU/US-PA_simulation_codes).

Recent advances in high power, pulsed, light emitting diodes (LEDs) have shown potential as fast, robust and relatively inexpensive excitation sources for photoacoustic imaging (PAI), yet systematic characterization of performance for biomedical imaging is still lacking. We report here technical and biological validation studies of a commercial dual-wavelength LED-based PAI and ultrasound system. Phantoms and small animals were used to assess temporal precision. In phantom studies, we found high temporal stability of the LED-based PAI system, with no significant drift in performance observed during 6 h of operation or over 30 days of repeated measurements. In vivo dual-wavelength imaging was able to map the dynamics of changes in blood oxygenation during oxygen-enhanced imaging and reveal the kinetics of indocyanine green contrast agent inflow after intravenous administration (Tmax∼6 min). Taken together, these studies indicate that LED-based excitation could be promising for future application in functional and molecular PAI.

Multimode fibres (MMFs) are becoming increasingly attractive in optical endoscopy as they promise to enable unparallelled miniaturisation, spatial resolution and cost. However, high-speed imaging with wavefront shaping has been challenging. Here, we report the development of a video-rate dual-modal photoacoustic (PA) and fluorescence microscopy probe with a high-speed digital micromirror device (DMD) towards forward-viewing endomicroscopy. Optimal DMD patterns were obtained using a real-valued intensity transmission matrix algorithm to raster-scan a 1.5  μ m-diameter focused beam at the distal fibre tip for imaging. The PA imaging speed and spatial resolution were varied from  ∼ 2 to 57 frames per second and from 1.7 to 3  μ m, respectively. Further, high-fidelity PA images of carbon fibres and mouse red blood cells were acquired at unprecedented speed. The capability of dual-modal imaging was demonstrated with phantoms. We anticipate that with further miniaturisation of the ultrasound detector, this probe could be integrated into medical needles to guide minimally invasive procedures.

Approximately 19 % of breast cancer patients undergoing breast conserving surgery (BCS) must return for a secondary surgery due to incomplete tumour removal. Our previous work demonstrated that the lower lipid content, characteristic of tumour tissue, was observed as regions of hypo-intense photoacoustic (PA) contrast. The goal of this work was to evaluate feasibility of a low-frequency, hand-held PA imaging probe for surgical margin assessment based on lipid content differences. Here, we describe (i) the design of a prototype hand-held PA imaging probe, (ii) the effect of limited-bandwidth on image contrast, (iii) accuracy towards hypo-intense contrast detection, (iv) the limited-view characteristics of the single sensor design, and (iv) early imaging results of an ex-vivo breast cancer specimen. The probe incorporates a single polyvinylidene fluoride acoustic sensor, a 1-to-4 optical fibre bundle and a polycarbonate axicon lens for light delivery. Imaging results on phantoms designed to mimic positive margins demonstrated the ability to detect gaps in optical absorption as small as 1 mm in width. Compared to images from a near full-view PAI system, the hand-held PAI probe had higher signal to noise ratio but suffered from negativity image artifacts. Lumpectomy specimen imaging showed that strong signals can be obtained from the fatty tissue. Taken together, the results show this imaging approach with a hand-held probe has potential for detection of residual breast cancer tissue during BCS; however, more work is needed to reduce the size of the probe to fit within the surgical cavity.

We report a new approach to preparing phantoms using 3D printing. This device supports plastic tubing containing the contrast agent and is immersed in a solution with absorption or scattering properties that mimic tissue. Up to 12 tubing samples could be placed in the device with sample-to-sample spacing as low as 0.3 mm and at a constant distance from the transducer (±0.16 mm), which is critical in validating photoacoustic contrast agents. We also studied different types of tubing and found that tubing with a larger outside diameter has more inherent signal. Both 40% India Ink and lipids in the immersion media modulated the signal. Finally, we created a depth phantom and found that signal decayed following a linear relationship (R2 = 0.997) with respect to distance from the focal point. We include computer-assisted drafting code the community can use to print this phantom or customized versions of this phantom.

In ultrasound imaging, fully-developed speckle arises from the spatiotemporal superposition of pressure waves backscattered by randomly distributed scatterers. Speckle appearance is affected by the imaging system characteristics (lateral and axial resolution) and the random-like nature of the underlying tissue structure. In this work, we examine speckle formation in acoustic-resolution photoacoustic (PA) imaging using simulations and experiments. Numerical and physical phantoms were constructed to demonstrate that PA speckle carries information related to unresolved absorber structure in a manner similar to ultrasound speckle and unresolved scattering structures. A fractal-based model of the tumor vasculature was used to study PA speckle from unresolved cylindrical vessels. We show that speckle characteristics and the frequency content of PA signals can be used to monitor changes in average vessel size, linked to tumor growth. Experimental validation on murine tumors demonstrates that PA speckle can be utilized to characterize the unresolved vasculature in acoustic-resolution photoacoustic imaging.

The assessment of tissue chromophores at a volumetric scale is vital for an improved diagnosis and treatment of a large number of diseases. Spectral photoacoustic imaging (sPAI) co-registered with high-resolution ultrasound (US) is an innovative technology that has a great potential for clinical translation as it can assess the volumetric distribution of the tissue components. Conventionally, to detect and separate the chromophores from sPAI, an input of the expected tissue absorption spectra is required. However, in pathological conditions, the prediction of the absorption spectra is difficult as it can change with respect to the physiological state. Besides, this conventional approach can also be hampered due to spectral coloring, which is a prominent distortion effect that induces spectral changes at depth. Here, we are proposing a novel data-driven framework that can overcome all these limitations and provide an improved assessment of the tissue chromophores. We have developed a superpixel spectral unmixing (SPAX) approach that can detect the most and less prominent absorber spectra and their volumetric distribution without any user interactions. Within the SPAX framework, we have also implemented an advanced spectral coloring compensation approach by utilizing US image segmentation and Monte Carlo simulations, based on a predefined library of optical properties. The framework has been tested on tissue-mimicking phantoms and also on healthy animals. The obtained results show enhanced specificity and sensitivity for the detection of tissue chromophores. To our knowledge, this is a unique framework that accounts for the spectral coloring and provides automated detection of tissue spectral signatures at a volumetric scale, which can open many possibilities for translational research.