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Phantom-based performance test methods are critically needed to support development and clinical translation of emerging photoacoustic microscopy (PAM) devices. While phantoms have been recently developed for macroscopic photoacoustic imaging systems, there is an unmet need for well-characterized tissue-mimicking materials (TMMs) and phantoms suitable for evaluating PAM systems. Our objective was to develop and characterize a suitable dermis-mimicking TMM based on polyacrylamide hydrogels and demonstrate its utility for constructing image quality phantoms. TMM formulations were optically characterized over 400–1100 nm using integrating sphere spectrophotometry and acoustically characterized using a pulse through-transmission method over 8–24 MHz with highly confident extrapolation throughout the usable band of the PAM system. This TMM was used to construct a spatial resolution phantom containing gold nanoparticle point targets and a penetration depth phantom containing slanted tungsten filaments and blood-filled tubes. These phantoms were used to characterize performance of a custom-built PAM system. The TMM was found to be broadly tunable and specific formulations were identified to mimic human dermis at an optical wavelength of 570 nm and acoustic frequencies of 10–50 MHz. Imaging results showed that tungsten filaments yielded 1.1–4.2 times greater apparent maximum imaging depth than blood-filled tubes, which may overestimate real-world performance for vascular imaging applications. Nanoparticles were detectable only to depths of 120–200 µm, which may be due to the relatively weaker absorption of single nanoparticles vs. larger targets containing high concentration of hemoglobin. The developed TMMs and phantoms are useful tools to support PAM device characterization and optimization, streamline regulatory decision-making, and accelerate clinical translation.

Fluorescence imaging techniques such as fluorescein angiography and fundus autofluorescence are often used to diagnose retinal pathologies; however, there are currently no standardized test methods for evaluating device performance. Here we present microstructured fluorescent phantoms fabricated using a submicron-scale three-dimensional printing technology, direct laser writing (DLW). We employ an in situ DLW technique to print 10 µm diameter microfluidic channels that support perfusions of fluorescent dyes. We then demonstrate how broadband photoresist fluorescence can be exploited to generate resolution targets and biomimetic models of retinal vasculature using standard DLW processes. The results indicate that these approaches show significant promise for generating better performance evaluation tools for fluorescence microscopy and imaging devices.

Cerebral oximetry based on near-infrared spectroscopy represents a unique noninvasive tool for real-time surgical monitoring, yet studies have shown a significant discrepancy in accuracy among commercial systems. Towards the establishment of a standardized method for performance testing, we have studied a solid phantom approach – based on a 3D-printed cerebrovascular module (CVM) incorporating an array of 148 cylindrical channels – that has several advantages over liquid phantoms. Development and characterization of a CVM prototype are described, including high-resolution imaging and spectrophotometry measurements. The CVM was filled with whole bovine blood tuned over an oxygen saturation range of 30-90% and molded-silicone layers simulating extracerebral tissues were used to evaluate penetration depth. Saturation measurement accuracy was assessed in two commercially-available clinical cerebral oximeters. For one oximeter, both neonatal and pediatric sensors showed a high degree of precision, whereas accuracy was strongly dependent on saturation level and extracerebral geometry. The second oximeter showed worse precision, yet greater robustness to variations in extracerebral layers. These results indicate that 3D-printed channel array phantoms represent a promising new approach for standardized testing of clinical oximeters.

Innovative biophotonic modalities such as photoacoustic imaging (PAI) have the potential to provide enhanced sensitivity and molecule-specific detection when used with nanoparticles. However, high peak irradiance levels generated by pulsed lasers can lead to modification of plasmonic nanoparticles. Thus, there is an outstanding need to develop practical methods to effectively predict the onset nanoparticle photomodification as well as a need to better understand the process during PAI. To address this need, we studied pulsed laser damage of gold nanorods (GNRs) using turbid phantoms and a multi-spectral near-infrared PAI system, comparing results with spectrophotometric measurements of non-scattering samples. Transmission electron microscopy and Monte Carlo modeling were also performed to elucidate damage processes. In the phantoms, shifts in PAI-detected spectra indicative of GNR damage were initiated at exposure levels one-third of that seen in non-scattering samples, due to turbidity-induced enhancement of subsurface fluence. For exposures approaching established safety limits, damage was detected at depths of up to 12.5 mm. Typically, GNR damage occurred rapidly, over the course of a few laser pulses. This work advances the development of test methods and numerical models as tools for assessment of nanoparticle damage and its implications, and highlights the importance of considering GNR damage in development of PAI products, even for exposures well below laser safety limits.

Multispectral photoacoustic oximetry imaging (MPOI) is an emerging hybrid modality that enables the spatial mapping of blood oxygen saturation (SO_2) to depths of several centimeters. To facilitate MPOI device development and clinical translation, well-validated performance test methods and improved quantitative understanding of physical processes and best practices are needed. We developed a breast-mimicking blood flow phantom with tunable SO_2 and used this phantom to evaluate a custom MPOI system. Results provide quantitative evaluation of the impact of phantom medium properties (Intralipid versus polyvinyl chloride plastisol) and device design parameters (different transducers) on SO_2 measurement accuracy, especially depth-dependent performance degradation due to fluence artifacts. This approach may guide development of standardized test methods for evaluating MPOI devices.

Emerging three-dimensional (3D) printing technology enables the fabrication of optically realistic and morphologically complex tissue-simulating phantoms for the development and evaluation of novel optical imaging products. In this study, we assess the potential to print image-defined neurovascular phantoms with patent channels for contrast-enhanced near-infrared fluorescence (NIRF) imaging. An anatomical map defined from clinical magnetic resonance imaging (MRI) was segmented and processed into files suitable for printing a forebrain vessel network in rectangular and curved-surface biomimetic phantoms. Methods for effectively cleaning samples with complex vasculature were determined. A final set of phantoms were imaged with a custom NIRF system at 785 nm excitation using two NIRF contrast agents. In addition to demonstrating the strong potential of 3D printing for creating highly realistic, patient-specific biophotonic phantoms, our work provides insight into optimal methods for accomplishing this goal and elucidates current limitations of this approach.