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The benefits of contrast-enhancing imaging probes have become apparent over the past decade. However, there is a gap in the literature when it comes to the assessment of the phototoxic potential of imaging probes and systems emitting visible and/or near-infrared radiation. The primary mechanism of fluorescent agent phototoxicity is thought to involve the production of reactive molecular species (RMS), yet little has been published on the best practices for safety evaluation of RMS production levels for clinical products. We have proposed methods involving a cell-free assay to quantify singlet oxygen [(SO) a known RMS] generation of imaging probes, and performed testing of Indocyanine Green (ICG), Proflavine, Methylene Blue, IR700 and IR800 at clinically relevant concentrations and radiant exposures. Results indicated that SO production from IR800 and ICG were more than two orders of magnitude below that of the known SO generator Rose Bengal. Methylene Blue and IR700 produced much higher SO levels than ICG and IR800. These results were in good agreement with data from the literature. While agents that exhibit spectral overlap with the assay may be more prone to errors, our tests for one of these agents (Proflavine) appeared robust. Overall, our results indicate that this methodology shows promise for assessing the phototoxic potential of fluorophores due to SO production.

Detailed overview of 510(k) cleared imaging devices by the FDA.

Considerable advances in cancer-specific optical imaging have improved the precision of tumor resection. In comparison to traditional imaging modalities, this technology is unique in its ability to provide real-time feedback to the operating surgeon. Given the significant clinical implications of optical imaging, there is an urgent need to standardize surgical navigation tools and contrast agents to facilitate swift regulatory approval. Because fluorescence-enhanced surgery requires a combination of both device and drug, each may be developed in conjunction, or separately, which are important considerations in the approval process. This report is the result of a one-day meeting held on May 4, 2016 with officials from the National Cancer Institute, the FDA, members of the American Society of Image-Guided Surgery, and members of the World Molecular Imaging Society, which discussed consensus methods for FDA-directed human testing and approval of investigational optical imaging devices as well as contrast agents for surgical applications. The goal of this workshop was to discuss FDA approval requirements and the expectations for approval of these novel drugs and devices, packaged separately or in combination, within the context of optical surgical navigation. In addition, the workshop acted to provide clarity to the research community on data collection and trial design. Reported here are the specific discussion items and recommendations from this critical and timely meeting. Cancer Res; 77(9); 2197–206. ©2017 AACR.

Significance: Raman spectroscopy has emerged as a promising technique for a variety of biomedical applications. The unique ability to provide molecular specific information offers insight to the underlying biochemical changes that result in disease states such as cancer. However, one of the hurdles to successful clinical translation is a lack of international standards for calibration and performance assessment of modern Raman systems used to interrogate biological tissue. Aim: To facilitate progress in the clinical translation of Raman-based devices and assist the scientific community in reaching a consensus regarding best practices for performance testing. Approach: We reviewed the current literature and available standards documents to identify methods commonly used for bench testing of Raman devices (e.g., relative intensity correction, wavenumber calibration, noise, resolution, and sensitivity). Additionally, a novel 3D-printed turbid phantom was used to assess depth sensitivity. These approaches were implemented on three fiberoptic-probe-based Raman systems with different technical specifications. Results: While traditional approaches demonstrated fundamental differences due to detectors, spectrometers, and data processing routines, results from the turbid phantom illustrated the impact of illumination-collection geometry on measurement quality. Conclusions: Specifications alone are necessary but not sufficient to predict in vivo performance, highlighting the need for phantom-based test methods in the standardized evaluation of Raman devices.

The editorial introduces the JBO Special Section on Tissue Phantoms to Advance Biomedical Optical Systems.

A lack of accepted standards and standardized phantoms suitable for the technical validation of biophotonic instrumentation hinders the reliability and reproducibility of its experimental outputs. In this Perspective, we discuss general criteria for the design of tissue-mimicking biophotonic phantoms, and use these criteria and state-of-the-art developments to critically review the literature on phantom materials and on the fabrication of phantoms. By focusing on representative examples of standardization in diffuse optical imaging and spectroscopy, fluorescence-guided surgery and photoacoustic imaging, we identify unmet needs in the development of phantoms and a set of criteria (leveraging characterization, collaboration, communication and commitment) for the standardization of biophotonic instrumentation.

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. Funding: FDA Office of Minority Health and Health Equity and FDA Perinatal Health Center of Excellence

Recent advances in plasmonic nanoparticle synthesis have enabled extremely high per-particle surface-enhanced Raman scattering (SERS) efficiencies. This has led to the development of SERS tags for in vivo applications (e.g. tumor targeting and detection), providing high sensitivity and fingerprint-like molecular specificity. While the SERS enhancement factor is a major contributor to SERS tag performance, in practice the throughput and excitation-collection geometry of the optical system can significantly impact detectability. Test methods to objectively quantify SERS particle performance under realistic conditions are necessary to facilitate clinical translation. Towards this goal, we have developed 3D-printed phantoms with tunable, biologically-relevant optical properties. Phantoms were designed to include 1 mm-diameter channels at different depths, which can be filled with SERS tag solutions. The effects of channel depth and particle concentration on the detectability of three different SERS tags were evaluated using 785 nm laser excitation at the maximum permissible exposure for skin. Two of these tags were commercially available, featuring gold nanorods as the SERS particle, while the third tag was prepared in-house using silver-coated gold nanostars. Our findings revealed that the measured SERS intensity of tags in solution is not always a reliable predictor of detectability when applied in a turbid medium such as tissue. The phantoms developed in this work can be used to assess the suitability of specific SERS tags and instruments for their intended clinical applications and provide a means of optimizing new SERS device-tag combination products.

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.

Significance: Raman spectroscopy has emerged as a promising technique for a variety of biomedical applications. The unique ability to provide molecular specific information offers insight to the underlying biochemical changes that result in disease states such as cancer. However, one of the hurdles to successful clinical translation is a lack of international standards for calibration and performance assessment of modern Raman systems used to interrogate biological tissue. Aim: To facilitate progress in the clinical translation of Raman-based devices and assist the scientific community in reaching a consensus regarding best practices for performance testing. Approach: We reviewed the current literature and available standards documents to identify methods commonly used for bench testing of Raman devices (e.g., relative intensity correction, wavenumber calibration, noise, resolution, and sensitivity). Additionally, a novel 3D-printed turbid phantom was used to assess depth sensitivity. These approaches were implemented on three fiberoptic-probe-based Raman systems with different technical specifications. Results: While traditional approaches demonstrated fundamental differences due to detectors, spectrometers, and data processing routines, results from the turbid phantom illustrated the impact of illumination-collection geometry on measurement quality. Conclusions: Specifications alone are necessary but not sufficient to predict in vivo performance, highlighting the need for phantom-based test methods in the standardized evaluation of Raman devices.

Infrared thermographs (IRTs) have been used for fever screening during infectious disease epidemics. However, their performance is inconsistent in literature, due to wide quality/implementation variations. We overview standards and FDA guidance for IRT performance evaluation, implementation, and regulation policies. Additionally, we present results from a large-scale clinical study of fever-screening IRTs and discuss impact of consensus guidelines and facial measurement location on performance. We found that: high-quality IRTs implemented according to international standards can help to accurately measure temperature; current standards can be improved to further enhance IRT performance. Overall, fever screening is only one element in infectious disease detection.

Photoacoustic imaging (PAI) and photoacoustic microscopy (PAM) continue to undergo advancement, but standardized performance test methods are needed to facilitate development and translation. Most tissue-mimicking materials (TMMs) have not been adequately characterized at high acoustic frequencies relevant to PAM systems (>20 MHz). We characterized acoustic properties of various polyacrylamide TMM formulations over 10-60 MHz using a pulse-echo method, while optical properties were characterized over 400-1000 nm. We evaluated performance of a custom PAM system using phantoms containing gold nanoparticles. Polyacrylamide had highly tunable acoustic properties similar to human skin, and performance tests provided key insights into PAM system performance.

Recent advances in optical imaging have the potential to significantly improve patient outcomes. Among the most promising approaches under development is contrast-enhanced fluorescence imaging, which can be performed with molecular targeting to enhance tumor visualization. One key issue that has not been rigorously addressed is the potential phototoxicity of these fluorophores. In this study, we have used a commercial cell-free assay to qualify the singlet oxygen production rate in approved (ICG, Methylene Blue) and unapproved (IRDye700, IRDye800) fluorophores. Results shed light on the relative phototoxicity of each agent and will help to establish standardized methods for photochemical safety testing.

Cerebral saccular aneurysms represent a life-threatening condition that typically requires surgical intervention, often accomplished with a clip ligation approach across the aneurysm neck. Fluorescent angiography (FA) with indocyanine green (ICG) - a technique commonly used in neurosurgery – can be utilized to ensure that the neck of the aneurysm is fully ligated, while the adjacent vessels remain patent after clip placement. However, there is currently a lack of standardized performance test methods for surgical microscopes with FA capability. We have developed a 3D-printed, biomimetic aneurysm phantom with the potential to facilitate development and evaluation of these critical surgical instruments. Digital models of the Circle of Willis vasculature, including the multimodal imaging-based detailed anatomical (MIDA) model of the head and neck and a public domain model of a basilar tip aneurysm, were combined to generate a modular aneurysm phantom system. This system includes a basilar artery aneurysm and posterior communicator artery aneurysm phantoms. Non-fluorescent phantom components representing surrounding tissue were derived from the MIDA model. A stereolithography printer was used to create a solid vascular phantom from a custom turbid photopolymer doped with ICGsimulating dye. Feature sizes of printed components were found to be within 2.5% of digital models. Using a custom fluorescence imaging system, we were able to clearly visualize vessels and aneurysms amongst non-fluorescent background structures in the model. The methods for fabrication of a biomimetic neurovascular phantom incorporating realistic pathology have the potential to facilitate development, standardized bench testing and clinical training for intraoperative FA 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.

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.

Significance: Infrared thermographs (IRTs) have been used for fever screening during infectious disease epidemics, including severe acute respiratory syndrome, Ebola virus disease, and coronavirus disease 2019 (COVID-19). Although IRTs have significant potential for human body temperature measurement, the literature indicates inconsistent diagnostic performance, possibly due to wide variations in implemented methodology. A standardized method for IRT fever screening was recently published, but there is a lack of clinical data demonstrating its impact on IRT performance. Aim: Perform a clinical study to assess the diagnostic effectiveness of standardized IRT-based fever screening and evaluate the effect of facial measurement location. Approach: We performed a clinical study of 596 subjects. Temperatures from 17 facial locations were extracted from thermal images and compared with oral thermometry. Statistical analyses included calculation of receiver operating characteristic (ROC) curves and area under the curve (AUC) values for detection of febrile subjects. Results: Pearson correlation coefficients for IRT-based and reference (oral) temperatures were found to vary strongly with measurement location. Approaches based on maximum temperatures in either inner canthi or full-face regions indicated stronger discrimination ability than maximum forehead temperature (AUC values of 0.95 to 0.97 versus 0.86 to 0.87, respectively) and other specific facial locations. These values are markedly better than the vast majority of results found in prior human studies of IRT-based fever screening. Conclusion: Our findings provide clinical confirmation of the utility of consensus approaches for fever screening, including the use of inner canthi temperatures, while also indicating that full-face maximum temperatures may provide an effective alternate approach.

Fluorescence imaging for surgical guidance is a proven modality that allows for visualization of fluorescent markers in numerous biological imaging applications. As the field continues to develop there is an urgent need for fluorescence-imaging standards that enable system characterization and performance monitoring. Here, we present a long-term stable standard that mimics indocynine green (ICG) spectral behavior and is capable of probing the sensitivity of commercially available imaging systems. The standard is composed of three different tests: a varying concentration sensitivity test (0.1nM-3000nM), a tissue-equivalent-depth sensitivity test (0.2mm-7mm), and a 1951 USFA fluorescence resolution test. The fluorescence standard platform developed can incorporate 3-D printing with tissue-equivalent layers, enabling a wide variety of anatomical-specific designs. The manufacturing capabilities developed allow for incorporation of different fluorophores for applications beyond ICG and NIR.

We have optimized our prior phantom-based test method for cerebral oximetry performance using a new 3D-printed cerebrovascular module (CVM). In addition, we have and added a surrogate for a thin perfused scalp layer to evaluate perfusion- related confounding factors. The new CVM’s optical properties better represented biological tissue and also incorporated a water-mimicking dye. The modular phantom also included biologically relevant scalp/skull and cerebrospinal fluid (CSF) layers. Performance testing of two commercially available clinical oximeters with the modified CVM over a range of oxygen saturation levels illustrates the utility of our solid phantom-based approach for standardized cerebral oximeter performance assessment.

Significance: Expanded use of fluorescence-guided surgery with devices approved for use with indocyanine green (ICG) has led to a range of commercial systems available. There is a compelling need to be able to independently characterize system performance and allow for cross-system comparisons. Aim: The goal of this work is to expand on previous proposed fluorescence imaging standard designs to develop a long-term stable phantom that spectrally matches ICG characteristics and utilizes 3D printing technology for incorporating tissue-equivalent materials. Approach: A batch of test targets was created to assess ICG concentration sensitivity in the 0.3- to 1000-nM range, tissue-equivalent depth sensitivity down to 6 mm, and spatial resolution with a USAF test chart. Comparisons were completed with a range of systems that have significantly different imaging capabilities and applications, including the Li-Cor® Odyssey, Li-Cor® Pearl, PerkinElmer® Solaris, and Stryker® Spy Elite. Results: Imaging of the ICG-matching phantoms with all four commercially available systems showed the ability to benchmark system performance and allow for cross-system comparisons. The fluorescence tests were able to assess differences in the detectable concentrations of ICG with sensitivity differences >10× for preclinical and clinical systems. Furthermore, the tests successfully assessed system differences in the depth-signal decay rate, as well as resolution performance and image artifacts. The manufacturing variations, photostability, and mechanical design of the tests showed promise in providing long-term stable standards for fluorescence imaging. Conclusions: The presented ICG-matching phantom provides a major step toward standardizing performance characterization and cross-system comparisons for devices approved for use with ICG. The developed hybrid manufacturing platform can incorporate long-term stable fluorescing agents with 3D printed tissue-equivalent material. Further, long-term testing of the phantom and refinements to the manufacturing process are necessary for future implementation as a widely adopted fluorescence imaging standard.

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.

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.

Purpose Near-infrared fluorescence (NIRF) imaging using exogenous contrast has gained much attention as a technique for enhancing visualization of vasculature using untargeted agents, as well as for the detection and localization of cancer with targeted agents. In order to address the emerging need for standardization of NIRF imaging technologies, it is necessary to identify the best practices suitable for objective, quantitative testing of key image quality characteristics. Toward the development of a battery of test methods that are rigorous yet applicable to a wide variety of devices, we have evaluated techniques for phantom design, measurement, and calculation of specific performance metrics. Methods Using a NIRF imaging system for indocyanine green imaging, providing excitation at 780 nm and detection above 830 nm, we explored methods to evaluate uniformity, field of view, spectral crosstalk, spatial resolution, depth of field, sensitivity, linearity, and penetration depth. These measurements were performed using fluorophore-doped multiwell plate and high turbidity planar phantoms, as well as a 3D-printed multichannel phantom and a USAF 1951 resolution target. Results and conclusions Based on a wide range of approaches described in medical and fluorescence imaging literature, we have developed and demonstrated a cohesive battery of test methods for evaluation of fluorescence image quality in wide-field imagers. We also propose a number of key metrics that can facilitate direct, quantitative comparison of device performance. These methods have the potential to facilitate more uniform evaluation and inter-comparison of clinical and preclinical imaging systems than is typically achieved, with the long-term goal of establishing international standards for fluorescence image quality assessment.

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.

Considerable advances in cancer-specific optical imaging have improved the precision of tumor resection. In comparison to traditional imaging modalities, this technology is unique in its ability to provide real-time feedback to the operating surgeon. Given the significant clinical implications of optical imaging, there is an urgent need to standardize surgical navigation tools and contrast agents to facilitate swift regulatory approval. Because fluorescence-enhanced surgery requires a combination of both device and drug, each may be developed in conjunction, or separately, which are important considerations in the approval process. This report is the result of a one-day meeting held on May 4, 2016 with officials from the National Cancer Institute, the FDA, members of the American Society of Image-Guided Surgery, and members of the World Molecular Imaging Society, which discussed consensus methods for FDA-directed human testing and approval of investigational optical imaging devices as well as contrast agents for surgical applications. The goal of this workshop was to discuss FDA approval requirements and the expectations for approval of these novel drugs and devices, packaged separately or in combination, within the context of optical surgical navigation. In addition, the workshop acted to provide clarity to the research community on data collection and trial design. Reported here are the specific discussion items and recommendations from this critical and timely meeting. Cancer Res; 77(9); 2197–206. ©2017 AACR.

Cerebral oximetry based on near-infrared spectroscopy (NIRS) has seen increasing clinical use for monitoring of premature infants as well as during neonatal, pediatric and adult cardiac surgery. One key confounding factor and a likely contributor to observed inconsistency amongst commercial NIRS oximeters is skin pigmentation. Clinical studies have shown negative bias in oxygen saturation (StO2) with increasing melanin content. In prior work, we developed a cerebral oximetry phantom comprised of a 3D-printed channel array module representing brain tissue and molded silicone layers simulating extracranial regions. The purpose of the current study was to develop and test epidermis-simulating layers that exhibit realistic human pigmentation properties. Initially, we performed spectroscopic characterization of potential melanin simulating agents – including coffee, India ink, synthetic melanin, and water-soluble nigrosin – in a polydimethylsiloxane (PDMS) substrate. We determined that the NIR absorption spectrum of water-soluble nigrosin most accurately matched human melanin. Layers of 0.1 mm thickness were fabricated with different nigrosin concentrations to simulate epidermis with light, moderate, and dark pigmentation. The brain module channels were filled with bovine blood in the 30-100% oxygenation range and measurements performed with neonatal/pediatric probes from commercially available cerebral oximeters. We found that StO2 reported by the oximeters decreased monotonically with increasing pigmentation level. The magnitude of this impact increased with decreasing StO2, producing a maximum change in saturation of approximately 8%. The consistency of our results with prior clinical findings provides preliminary evidence of the utility of our approach for assessing the impact of epidermal melanin in phantom-based performance testing.

Infrared thermography (IRT) – a non-contact, non-invasive technique – has been used for mass screenings to identify febrile individuals at transportation nodes (e.g., airports) during infectious disease pandemics such as SARS (Severe Acute Respiratory Syndrome), H1N1 virus, and Ebola outbreaks. Despite the potential of IRTs, the field lacks a well-established consensus methodology to ensure temperature measurement accuracy and reliability. This study aims to investigate the use of IRTs in a controlled setting to determine the effectiveness of IRT and the most reliable facial region for estimation of core temperature. We conducted a large clinical study, acquiring facial thermographs of 1,109 febrile and non-febrile subjects using Screening Thermographs (STs). Regression analyses between the reference oral temperature and different areas of the face, specifically the forehead and canthi, were carried out. The coefficients of determination of each regression were compared to determine how well facial and core body temperatures were correlated. Receiver operating characteristic (ROC) curves were constructed to compare the effectiveness of using different facial areas to identify febrile patients. Results show that the maximum temperature of the overall face has the best linear trend, followed by the maximum temperature at the inner canthus region. Both of these values show better correlations than forehead temperatures, which are commonly used as a target by non-contact infrared thermometers. For any chosen facial area, the maximum temperature collected always showed a stronger correlation than a specific point in that area. Results indicate that IRT performance is substantially approved when applying optimal measurement methodology.

Infrared thermographs (IRTs) have been implemented for mass fever screening in public areas such as airports during outbreaks of infectious disease pandemics such as Ebola virus disease, yet the approach has not been entirely successful. There has been increasing evidence in the literature that IRTs can provide greater accuracy in estimating core body temperature, if qualified systems are used and appropriate procedures are consistently applied. In this study, we addressed the issue of system qualification by implementing and evaluating a battery of test methods for objective and quantitative performance assessment of two commercial IRTs based on a recent international standard (IEC 80601-2-59). We evaluated stability and drift, image uniformity, minimum resolvable temperature difference, and measurement accuracy of the IRTs and illustrated how experimental and data processing procedures affect results. For instance, we demonstrated that offset temperature compensation, achieved using an external blackbody, is essential to meet the standard’s recommendations for temperature drift and stability. Additionally, we identified methods that can be implemented to optimize IRT evaluation. As an example, we identified a less burdensome approach to characterize image uniformity with a single image acquisition of a uniform blackbody. Overall, the insights into thermograph standardization and acquisition methods provided by this study may improve the utility of this technology and aid in comparing IRT performance, thus improving the potential for high quality disease pandemic countermeasures.

Near-infrared fluorescence (NIRF) angiography has been applied for intraoperative visualization of neurovascular circulation and pathologies such as aneurysms, as well as to enhance contrast in clinical imaging of retinal fundus microvasculature. The ability to quantitatively evaluate and compare the performance of NIRF imaging devices including contrast, resolution and linearity under biologically realistic, yet reproducible conditions would facilitate innovation and clinical translation of this technology. Towards development of methods that can fill this role, we have generated 3D-printed, image-defined tissue simulating phantoms at macro and micro scales. The macro-scale phantom was developed based on an MRI image volume of a human head. This map was segmented into white matter, gray matter and vessel regions and edited to provide a suitable file for 3D printing. The phantom was then printed with an Objet260 Connex3 printer using a material with a biologically relevant NIR scattering coefficient. The micro-scale phantom is based on a fundus camera image of a human retina. This phantom was printed using a Nanoscribe Photonic Professional GT printer with sub-micron resolution, but a maximum print volume of approximately 1 mm3. To demonstrate the neurovascular phantoms for NIRF imaging system, channels were injected with a solution of hemoglobin and Indocyanine Green and then imaged with CCD-based macro- and micro- NIRF imaging system. Overall, these approaches for fabricating biomimetic phantoms hold significant promise for evaluation of NIRF angiography devices image quality in a standardized, yet realistic manner.

The use of plasmonic nanoparticles (PNPs) in biophotonics has been steadily increasing, with a wide variety of emerging applications including detection and treatment of cancers. PNPs have unique, tunable optical properties arising from their surface plasmon resonance (SPR), and can be functionalized to achieve molecular targeting in vivo. Highly transient nanobubbles generated around PNPs due to pulsed laser-induced vaporization and cavitation may significantly impact diagnostic and therapeutic procedures. While much effort has been devoted to elucidating the mechanisms behind PNP nanobubble generation, there has not been a comprehensive study on the effect of nanoparticle size on nanobubble formation thresholds, and whether particle photodegradation occurs at these levels. Herein, we evaluate the melting and bubble generation thresholds of commercially available gold nanospheres in the 20-100 nm diameter range when exposed to nanosecond laser pulses. The 532-nm laser pulses are generated by a second-harmonic Q-switched Nd:YAG laser, providing output near the SPR of the gold nanospheres. Bubble generation is detected using a multimodality microscopy platform developed for simultaneous, nanosecond resolution pump-probe imaging, scattering response monitoring, and acoustic transient detection. PNP damage was further evaluated using transmission electron microscopy and spectrophotometry. The results of this study provide insights into damage thresholds as well as the concurrent and rapid optical, thermal, acoustic, and mechanical phenomena involved. This work will provide a foundation for improved understanding of a variety of PNPs and inform development of standard test methods for safety and efficacy evaluation of pulsed laser-PNP interactions in biophotonics.

Photoacoustic Imaging (PAI) is an emerging technology with strong potential for broad clinical applications from breast cancer detection to cerebral monitoring due to its ability to compute maps of blood oxygen saturation (SO 2 ) distribution in deep tissues using multispectral imaging. However, no well-validated consensus test methods currently exist for evaluating oximetry-specific performance characteristics of PAI devices. We have developed a phantombased flow system capable of rapid SO 2 adjustment to serve as a test bed for elucidation of factors impacting SO 2 measurement and quantitative characterization of device performance. The flow system is comprised of a peristaltic pump, membrane oxygenator, oxygen and nitrogen gas, and in-line oxygen, pH, and temperature sensors that enable real-time estimation of SO 2 reference values. Bovine blood was delivered through breast-relevant tissue phantoms containing vessel-mimicking fluid channels, which were imaged using a custom multispectral PAI system. Blood was periodically drawn for SO 2 measurement in a clinical-grade CO-oximeter. We used this flow phantom system to evaluate the impact of device parameters (e.g.,wavelength-dependent fluence corrections) and tissue parameters (e.g. fluid channel depth, blood SO 2 , spectral coloring artifacts) on oximetry measurement accuracy. Results elucidated key challenges in PAI oximetry and device design trade-offs, which subsequently allowed for optimization of system performance. This approach provides a robust benchtop test platform that can support PAI oximetry device optimization, performance validation, and clinical translation, and may inform future development of consensus test methods for performance assessment of photoacoustic oximetry imaging systems.

Infrared (IR) modalities represent the only currently viable mass fever screening approaches for outbreaks of infectious disease pandemics such as Ebola virus disease and severe acute respiratory syndrome. Non-contact IR thermometers (NCITs) and IR thermographs (IRTs) have been used for fever screening in public areas such as airports. While NCITs remain a more popular choice than IRTs, there has been increasing evidences in the literature that IRTs can provide great accuracy in estimating body temperature if qualified systems are used and appropriate procedures are consistently applied. In this study, we addressed the issue of IRT qualification by implementing and evaluating a battery of test methods for objective, quantitative assessment of IRT performance based on a recent international standard (IEC 80601-2-59). We tested two commercial IRTs to evaluate their stability and drift, image uniformity, minimum resolvable temperature difference, and radiometric temperature laboratory accuracy. Based on these tests, we illustrated how experimental and data processing procedures could affect results, and suggested methods for clarifying and optimizing test methods. Overall, the insights into thermograph standardization and acquisition methods provided by this study may improve the utility of IR thermography and aid in comparing IRT performance, thus improving the potential for producing high quality disease pandemic countermeasures.

Hyperspectral reflectance imaging (HRI) is an emerging clinical tool for characterizing spatial and temporal variations in blood perfusion and oxygenation for applications such as burn assessment, wound healing, retinal exams and intraoperative tissue viability assessment. Since clinical HRI-based oximeters often use near-infrared (NIR) light, NIR-enabled mobile phones may provide a useful platform for future point-of-care devices. Furthermore, quantitative NIR imaging on mobile phones may dramatically increase the availability and accessibility of medical diagnostics for low-resource settings. We have evaluated the potential for phone-based NIR oximetry imaging and elucidated factors affecting performance using devices from two different manufacturers, as well as a scientific CCD. A broadband light source and liquid crystal tunable filter were used for imaging at 10 nm bands from 650 to 1000 nm. Spectral sensitivity measurements indicated that mobile phones with standard NIR blocking filters had minimal response beyond 700 nm, whereas one modified phone showed sensitivity to 800 nm and another to 1000 nm. Red pixel channels showed the greatest sensitivity up to 800 nm, whereas all channels provided essentially equivalent sensitivity at longer wavelengths. Referencing of blood oxygenation levels was performed with a CO-oximeter. HRI measurements were performed using cuvettes filled with hemoglobin solutions of different oxygen saturation levels. Good agreement between absorbance spectra measured with mobile phone and a CCD cameras were seen for wavelengths below 900 nm. Saturation estimates showed root-mean-squared-errors of 5.2% and 4.5% for the CCD and phone, respectively. Overall, this work provides strong evidence of the potential for mobile phones to provide quantitative spectral imaging in the NIR for applications such as oximetry, and generates practical insights into factors that impact performance as well as test methods for performance assessment.

Fever screening based on infrared (IR) thermographs (IRTs) is an approach that has been implemented during infectious disease pandemics, such as Ebola and Severe Acute Respiratory Syndrome. A recently published international standard indicates that regions medially adjacent to the inner canthi provide accurate estimates of core body temperature and are preferred sites for fever screening. Therefore, rapid, automated identification of the canthi regions within facial IR images may greatly facilitate rapid fever screening of asymptomatic travelers. However, it is more difficult to accurately identify the canthi regions from IR images than from visible images that are rich with exploitable features. In this study, we developed and evaluated techniques for multi-modality image registration (MMIR) of simultaneously captured visible and IR facial images for fever screening. We used free form deformation (FFD) models based on edge maps to improve registration accuracy after an affine transformation. Two widely used FFD models in medical image registration based on the Demons and cubic B-spline algorithms were qualitatively compared. The results showed that the Demons algorithm outperformed the cubic B-spline algorithm, likely due to overfitting of outliers by the latter method. The quantitative measure of registration accuracy, obtained through selected control point correspondence, was within 2.8 ± 1.2 mm, which enables accurate and automatic localization of canthi regions in the IR images for temperature measurement.