Effect of mechanical optical clearing on near-infrared spectroscopy.

Near-infrared Spectroscopy (NIRS) is a broadly utilized technology with many emerging applications including clinical diagnostics, sports medicine, and functional neuroimaging, to name a few. For functional brain imaging NIR light is delivered at multiple wavelengths through the scalp and skull to the brain to enable spatial oximetry measurements. Dynamic changes in brain oxygenation are highly correlated with neural stimulation, activation, and function. Unfortunately, NIRS is currently limited by its low spatial resolution, shallow penetration depth, and, perhaps most importantly, signal corruption due to light interactions with superficial non-target tissues such as scalp and skull. In response to these issues, we have combined the non-invasive and rapidly reversible method of mechanical tissue optical clearing (MOC) with a commercially available NIRS system. MOC utilizes a compressive loading force on tissue, causing the lateral displacement of blood and water, while simultaneously thinning the tissue. A MOC-NIRS Breath Hold Test displayed a ∼3.5-fold decrease in the time-averaged standard deviation between channels, consequentially promoting greater channel agreement. A Skin Pinch Test was implemented to negate brain and muscle activity from affecting the recorded signal. These results displayed a 2.5-3.0 fold increase in raw signal amplitude. Existing NIRS instrumentation has been further integrated within a custom helmet device to provide a uniform force distribution across the NIRS sensor array. These results showed a gradual decrease in time-averaged standard deviation among channels with an increase in applied pressure. Through these experiments, and the development of the MOC-NIRS helmet device, MOC appears to provide enhancement of NIRS technology beyond its current limitations.

Melanometry for objective evaluation of skin pigmentation in pulse oximetry studies.

Pulse oximetry enables real-time, noninvasive monitoring of arterial blood oxygen levels. However, results can vary with skin color, thus detecting disparities during clinical validation studies requires an accurate measure of skin pigmentation. Recent clinical studies have used subjective methods such as self-reported color, race/ethnicity to categorize skin. Melanometers based on optical reflectance may offer a more effective, objective approach to assess pigmentation. Here, we review melanometry approaches and assess evidence supporting their use as clinical research tools. We compare performance data, including repeatability, robustness to confounders, and compare devices to each other, to subjective methods, and high-quality references. Finally, we propose best practices for evaluating melanometers and discuss alternate optical approaches that may improve accuracy. Whilst evidence indicates that melanometers can provide superior performance to subjective approaches, we encourage additional research and standardization efforts, as these are needed to ensure consistent and reliable results in clinical studies.

Tissue mimicking materials and finger phantom design for pulse oximetry.

Pulse oximetry represents a ubiquitous clinical application of optics in modern medicine. Recent studies have raised concerns regarding the potential impact of confounders, such as variable skin pigmentation and perfusion, on blood oxygen saturation measurement accuracy in pulse oximeters. Tissue-mimicking phantom testing offers a low-cost, well-controlled solution for characterizing device performance and studying potential error sources, which may thus reduce the need for costly in vivo trials. The purpose of this study was to develop realistic phantom-based test methods for pulse oximetry. Material optical and mechanical properties were reviewed, selected, and tuned for optimal biological relevance, e.g., oxygenated tissue absorption and scattering, strength, elasticity, hardness, and other parameters representing the human finger's geometry and composition, such as blood vessel size and distribution, and perfusion. Relevant anatomical and physiological properties are summarized and implemented toward the creation of a preliminary finger phantom. To create a preliminary finger phantom, we synthesized a high-compliance silicone matrix with scatterers for embedding flexible tubing and investigated the addition of these scatterers to novel 3D printing resins for optical property control without altering mechanical stability, streamlining the production of phantoms with biologically relevant characteristics. Phantom utility was demonstrated by applying dynamic, pressure waveforms to produce tube volume change and resultant photoplethysmography (PPG) signals. 3D printed phantoms achieved more biologically relevant conditions compared to molded phantoms. These preliminary results indicate that the phantoms show strong potential to be developed into tools for evaluating pulse oximetry performance. Gaps, recommendations, and strategies are presented for continued phantom development.

Imaging and characterization of bioengineered blood vessels within a bioreactor using free-space and catheter-based OCT.

BACKGROUND AND OBJECTIVE: Regenerative medicine involves the bioengineering of a functional tissue or organ by seeding living cells on a biodegradable scaffold cultured in a bioreactor. A major barrier to creating functional tissues, however, has been the inability to monitor the dynamic and complex process of scaffold maturation in real time, making control and optimization extremely difficult. Current methods to assess maturation of bioengineered constructs, such as histology or organ bath physiology, are sample-destructive. Optical coherence tomography (OCT) has recently emerged as a key modality for structural assessment of native blood vessels as well as engineered vessel mimics. The objective of this study was to monitor and assess in real time the development of a bioengineered blood vessel using a novel approach of combining both free-space and catheter-based OCT imaging in a new quartz-walled bioreactor. Development of the blood vessel was characterized by changes in thickness and scattering coefficient over a 30-day period. MATERIALS AND METHODS: We constructed a novel blood vessel bioreactor utilizing a rotating cylindrical quartz cuvette permitting free-space OCT imaging of an installed vessel's outer surface. A vascular endoscopic OCT catheter was used to image the lumen of the vessels. The quartz cuvette permits 360 degree, free-space OCT imaging of the blood vessel. Bioengineered blood vessels were fabricated using biodegradable polymers (15% PCL/collagen, ∼300 µm thick) and seeded with CH3 10t1/2 mesenchymal stem cells. A swept-source OCT imaging system comprised of a 20 kHz tunable laser (Santec HSL2000) with 1,300 nm central wavelength and 110 nm FWHM bandwidth was used to assess the vessels. OCT images were obtained at days 1, 4, 7, 14, 21, and 30. Free-space (exterior surface) OCT images were co-registered with endoscopic OCT images to determine the vessel wall thickness. DAPI-stained histological sections, acquired at same time point, were evaluated to quantify wall thickness and cellular infiltration. Non-linear curve fitting of free-space OCT data to the extended Huygen-Fresnel model was performed to determine optical scattering properties. RESULTS: Vessel wall thickness increased from 435 ± 15 µm to 610 ± 27 µm and Vessel scattering coefficient increased from 3.73 ± 0.32 cm⁻¹ to 5.74 ± 0.06 cm⁻¹ over 30 days. Histological studies showed cell migration from the scaffold surface toward the lumen and cell proliferation over the same time course. The imaging procedure did not have any significant impact on scaffold dimensions, cell migration, or cell proliferation. CONCLUSIONS: This study suggests that combination of free-space and catheter-based OCT for blood vessel imaging provides accurate structural information of the developing blood vessel. We determined that free-space OCT images could be co-registered with catheter-based OCT images to monitor structural features such as wall thickness or delamination of the developing tissue-engineered blood vessel within a bioreactor. Structural parameters and optical properties obtained from OCT imaging correlate with histological sections of the blood vessel and could potentially be used as markers to non-invasively and non-destructively assess regeneration of engineered tissues in real time.

Phantoms for evaluating the impact of skin pigmentation on photoacoustic imaging and oximetry performance.

Recent reports have raised concerns of potential racial disparities in performance of optical oximetry technologies. To investigate how variable epidermal melanin content affects performance of photoacoustic imaging (PAI) devices, we developed plastisol phantoms combining swappable skin-mimicking layers with a breast phantom containing either India ink or blood adjusted to 50-100% SO2 using sodium dithionite. Increasing skin pigmentation decreased maximum imaging depth by up to 25%, enhanced image clutter, and increased root-mean-square error in SO2 from 8.0 to 17.6% due to signal attenuation and spectral coloring effects. This phantom tool can aid in evaluating PAI device robustness to ensure high performance in all patients.

A review of a strategic roadmapping exercise to advance clinical translation of photoacoustic imaging: From current barriers to future adoption.

Photoacoustic imaging (PAI), also referred to as optoacoustic imaging, has shown promise in early-stage clinical trials in a range of applications from inflammatory diseases to cancer. While the first PAI systems have recently received regulatory approvals, successful adoption of PAI technology into healthcare systems for clinical decision making must still overcome a range of barriers, from education and training to data acquisition and interpretation. The International Photoacoustic Standardisation Consortium (IPASC) undertook an community exercise in 2022 to identify and understand these barriers, then develop a roadmap of strategic plans to address them. Here, we outline the nature and scope of the barriers that were identified, along with short-, medium- and long-term community efforts required to overcome them, both within and beyond the IPASC group.

Effects of mechanical indentation on diffuse reflectance spectra, light transmission, and intrinsic optical properties in ex vivo porcine skin.

BACKGROUND AND OBJECTIVES: Mechanical indentation has been shown to increase light transmission through turbid tissue. In this study, we investigated the effects of localized indentation on the optical properties of ex vivo porcine skin specimens by dynamically monitoring diffuse reflectance spectra, light transmission, and applied load while controlling tissue thickness. STUDY DESIGN/METHODS: A custom-built diffuse reflectance spectroscopy (DRS) system was used to capture diffuse reflectance spectra from tissue specimens undergoing indentation. The DRS probe was designed to perform both optical sensing and tissue indentation. A mechanical load frame was used to dynamically control probe displacement and resultant specimen thickness change while recording applied load. Diffuse reflectance spectra, as well as light transmission at 630 nm, were recorded during stress relaxation tests where tissue specimens were displaced to and held at a final thickness. Tissue optical properties were extracted from reflectance spectra using a previously established look-up table (LUT) approach. RESULTS: Indentation increased light transmission through tissue during linear displacement, and continued to increase transmission during subsequent stress relaxation at constant tissue thickness. The magnitude of relative transmission increases was shown to be a function of bulk tissue compressive strain (relative thickness change). Reduced scattering coefficients calculated from the LUT at 630 nm decreased during stress relaxation, with the relative decrease in scattering also depending strongly on tissue compressive strain. Reduced scattering coefficients decreased by 12.0 ± 4.7% at 0.44 ± 0.022 compressive strain, and reduced by 35.6 ± 1.3% at 0.71 ± 0.01 compressive strain. CONCLUSION: DRS can be used to capture transient changes in intrinsic tissue optical properties during mechanical loading. Mechanical indentation modifies tissue optical properties and may be harnessed as a minimally-invasive optical clearing technique to improve optical diagnostics and therapeutics.

Photoacoustic imaging phantoms for assessment of object detectability and boundary buildup artifacts.

Standardized phantoms and test methods are needed to accelerate clinical translation of emerging photoacoustic imaging (PAI) devices. Evaluating object detectability in PAI is challenging due to variations in target morphology and artifacts including boundary buildup. Here we introduce breast fat and parenchyma tissue-mimicking materials based on emulsions of silicone oil and ethylene glycol in polyacrylamide hydrogel. 3D-printed molds were used to fabricate solid target inclusions that produced more filled-in appearance than traditional PAI phantoms. Phantoms were used to assess understudied image quality characteristics (IQCs) of three PAI systems. Object detectability was characterized vs. target diameter, absorption coefficient, and depth. Boundary buildup was quantified by target core/boundary ratio, which was higher in transducers with lower center frequency. Target diameter measurement accuracy was also size-dependent and improved with increasing transducer frequency. These phantoms enable evaluation of multiple key IQCs and may support development of comprehensive standardized test methods for PAI devices.

Tissue-mimicking phantoms for performance evaluation of photoacoustic microscopy systems.

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.

Evaluation of the robustness of cerebral oximetry to variations in skin pigmentation using a tissue-simulating phantom.

Clinical studies have demonstrated that epidermal pigmentation level can affect cerebral oximetry measurements. To evaluate the robustness of these devices, we have developed a phantom-based test method that includes an epidermis-simulating layer with several melanin concentrations and a 3D-printed cerebrovascular module. Measurements were performed with neonatal, pediatric and adult sensors from two commercial oximeters, where neonatal probes had shorter source-detector separation distances. Referenced blood oxygenation levels ranged from 30 to 90%. Cerebral oximeter outputs exhibited a consistent decrease in saturation level with simulated melanin content; this effect was greatest at low saturation levels, producing a change of up to 15%. Dependence on pigmentation was strongest in a neonatal sensor, possibly due to its high reflectivity. Overall, our findings indicate that a modular channel-array phantom approach can provide a practical tool for assessing the impact of skin pigmentation on cerebral oximeter performance and that modifications to algorithms and/or instrumentation may be needed to mitigate pigmentation bias.

The IPASC data format: A consensus data format for photoacoustic imaging.

Photoacoustic imaging (PAI) is an emerging modality that has shown promise for improving patient management in a range of applications. Unfortunately, the current lack of uniformity in PAI data formats compromises inter-user data exchange and comparison, which impedes: technological progress; effective research collaboration; and efforts to deliver multi-centre clinical trials. To overcome this challenge, the International Photoacoustic Standardisation Consortium (IPASC) has established a data format with a defined consensus metadata structure and developed an open-source software application programming interface (API) to enable conversion from proprietary file formats into the IPASC format. The format is based on Hierarchical Data Format 5 (HDF5) and designed to store photoacoustic raw time series data. Internal quality control mechanisms are included to ensure completeness and consistency of the converted data. By unifying the variety of proprietary data and metadata definitions into a consensus format, IPASC hopes to facilitate the exchange and comparison of PAI data.

Mechanical tissue optical clearing technique increases imaging resolution and contrast through ex vivo porcine skin.

BACKGROUND AND OBJECTIVES: Mechanical tissue optical clearing permits light delivery deeper into turbid tissue, which may improve current optical diagnostics and laser-based therapeutic techniques. We investigated the effects of localized compression on brightfield imaging through ex vivo porcine skin by evaluating resolution and contrast of a target positioned beneath native, mechanically compressed, or chemically cleared specimens. We also evaluated the effects of indentation on dynamic tissue thickness and light transmission. STUDY DESIGN/METHODS: A 5 mm diameter, hemispherically tipped, manual load transducer was used to compress specimens using 2-44 N for 60 seconds. Chemically cleared specimens were immersed for 1 hour in glycerol or dimethyl sulfoxide. A USAF 1951 resolution target was positioned beneath specimens and imaged using brightfield microscopy. Resolution and contrast of target features were analyzed. In separate experiments, a mechanical test instrument was used to compress and hold specimens at a final thickness while measuring applied load and light transmission. RESULTS: Image intensity profiles showed that while uncompressed skin did not allow resolution of any target, localized compression allowed maximum resolution up to a line width of 173 ± 21 µm. Mechanical clearing achieved up to four times higher maximum resolution and 2-3 times higher contrast sensitivity than chemical immersion. Resolving capability was highly correlated with compressive tissue strain. Light transmission increased during tissue compression, but also increased while holding final thickness constant. CONCLUSION: Localized compression is an effective technique for increasing resolution and contrast of target features through tissue and may improve light-based diagnostics. Thickness reduction and other mechanisms appear to contribute to this effect.

Review of consensus test methods in medical imaging and current practices in photoacoustic image quality assessment.

SIGNIFICANCE: Photoacoustic imaging (PAI) is a powerful emerging technology with broad clinical applications, but consensus test methods are needed to standardize performance evaluation and accelerate translation. AIM: To review consensus image quality test methods for mature imaging modalities [ultrasound, magnetic resonance imaging (MRI), x-ray CT, and x-ray mammography], identify best practices in phantom design and testing procedures, and compare against current practices in PAI phantom testing. APPROACH: We reviewed scientific papers, international standards, clinical accreditation guidelines, and professional society recommendations describing medical image quality test methods. Observations are organized by image quality characteristics (IQCs), including spatial resolution, geometric accuracy, imaging depth, uniformity, sensitivity, low-contrast detectability, and artifacts. RESULTS: Consensus documents typically prescribed phantom geometry and material property requirements, as well as specific data acquisition and analysis protocols to optimize test consistency and reproducibility. While these documents considered a wide array of IQCs, reported PAI phantom testing focused heavily on in-plane resolution, depth of visualization, and sensitivity. Understudied IQCs that merit further consideration include out-of-plane resolution, geometric accuracy, uniformity, low-contrast detectability, and co-registration accuracy. CONCLUSIONS: Available medical image quality standards provide a blueprint for establishing consensus best practices for photoacoustic image quality assessment and thus hastening PAI technology advancement, translation, and clinical adoption.

Polyacrylamide hydrogel phantoms for performance evaluation of multispectral photoacoustic imaging systems.

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.

Evaluation of Fluence Correction Algorithms in Multispectral Photoacoustic Imaging.

Multispectral photoacoustic imaging (MPAI) is a promising emerging diagnostic technology, but fluence artifacts can degrade device performance. Our goal was to develop well-validated phantom-based test methods for evaluating and comparing MPAI fluence correction algorithms, including a heuristic diffusion approximation, Monte Carlo simulations, and an algorithm we developed based on novel application of the diffusion dipole model (DDM). Phantoms simulated a range of breast-mimicking optical properties and contained channels filled with chromophore solutions (ink, hemoglobin, or copper sulfate) or connected to a previously developed blood flow circuit providing tunable oxygen saturation (SO2). The DDM algorithm achieved similar spectral recovery and SO2 measurement accuracy to Monte Carlo-based corrections with lower computational cost, potentially providing an accurate, real-time correction approach. Algorithms were sensitive to optical property uncertainty, but error was minimized by matching phantom albedo. The developed test methods may provide a foundation for standardized assessment of MPAI fluence correction algorithm performance.

Multidomain computational modeling of photoacoustic imaging: verification, validation, and image quality prediction.

As photoacoustic imaging (PAI) technology matures, computational modeling will increasingly represent a critical tool for facilitating clinical translation through predictive simulation of real-world performance under a wide range of device and biological conditions. While modeling currently offers a rapid, inexpensive tool for device development and prediction of fundamental image quality metrics (e.g., spatial resolution and contrast ratio), rigorous verification and validation will be required of models used to provide regulatory-grade data that effectively complements and/or replaces in vivo testing. To address methods for establishing model credibility, we developed an integrated computational model of PAI by coupling a previously developed three-dimensional Monte Carlo model of tissue light transport with a two-dimensional (2D) acoustic wave propagation model implemented in the well-known k-Wave toolbox. We then evaluated ability of the model to predict basic image quality metrics by applying standardized verification and validation principles for computational models. The model was verified against published simulation data and validated against phantom experiments using a custom PAI system. Furthermore, we used the model to conduct a parametric study of optical and acoustic design parameters. Results suggest that computationally economical 2D acoustic models can adequately predict spatial resolution, but metrics such as signal-to-noise ratio and penetration depth were difficult to replicate due to challenges in modeling strong clutter observed in experimental images. Parametric studies provided quantitative insight into complex relationships between transducer characteristics and image quality as well as optimal selection of optical beam geometry to ensure adequate image uniformity. Multidomain PAI simulation tools provide high-quality tools to aid device development and prediction of real-world performance, but further work is needed to improve model fidelity, especially in reproducing image noise and clutter.

Pulsed laser damage of gold nanorods in turbid media and its impact on multi-spectral photoacoustic imaging.

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.

Photoacoustic oximetry imaging performance evaluation using dynamic blood flow phantoms with tunable oxygen saturation.

Multispectral photoacoustic oximetry imaging (MPOI) is an emerging hybrid modality that enables the spatial mapping of blood oxygen saturation (SO2) 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 SO2 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 SO2 measurement accuracy, especially depth-dependent performance degradation due to fluence artifacts. This approach may guide development of standardized test methods for evaluating MPOI devices.

Experimental investigation of parameters influencing plasmonic nanoparticle-mediated bubble generation with nanosecond laser pulses.

Plasmonic nanoparticles (PNPs) continue to see increasing use in biophotonics for a variety of applications, including cancer detection and treatment. Several PNP-based approaches involve the generation of highly transient nanobubbles due to pulsed laser-induced vaporization and cavitation. While much effort has been devoted to elucidating the mechanisms behind bubble generation with spherical gold nano particles, the effects of particle shape on bubble generation thresholds are not well understood, especially in the nanosecond pulse regime. Our study aims to compare the bubble generation thresholds of gold nanospheres, gold nanorods, and silica-core gold nanoshells with different sizes, resonances, and surface coatings. Bubble generation is detected using a multimodality microscopy platform for simultaneous, nanosecond resolution pump-probe imaging, integrated scattering response, and acoustic transient detection. Nanoshells and large (40-nm width) nanorods were found to have the lowest thresholds for bubble generation, and in some cases they generated bubbles at radiant exposures below standard laser safety limits for skin exposure. This has important implications for both safety and performance of techniques employing pulsed lasers and PNPs.

Author Correction: Quantitative Evaluation of Nanosecond Pulsed Laser-Induced Photomodification of Plasmonic Gold Nanoparticles.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.