3D printed vascular phantoms for high-resolution biophotonic image quality assessment via direct laser writing.

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.

Infrared Thermography for Measuring Elevated Body Temperature: Clinical Accuracy, Calibration, and Evaluation.

Infrared thermographs (IRTs) implemented according to standardized best practices have shown strong potential for detecting elevated body temperatures (EBT), which may be useful in clinical settings and during infectious disease epidemics. However, optimal IRT calibration methods have not been established and the clinical performance of these devices relative to the more common non-contact infrared thermometers (NCITs) remains unclear. In addition to confirming the findings of our preliminary analysis of clinical study results, the primary intent of this study was to compare methods for IRT calibration and identify best practices for assessing the performance of IRTs intended to detect EBT. A key secondary aim was to compare IRT clinical accuracy to that of NCITs. We performed a clinical thermographic imaging study of more than 1000 subjects, acquiring temperature data from several facial locations that, along with reference oral temperatures, were used to calibrate two IRT systems based on seven different regression methods. Oral temperatures imputed from facial data were used to evaluate IRT clinical accuracy based on metrics such as clinical bias (Δcb), repeatability, root-mean-square difference, and sensitivity/specificity. We proposed several calibration approaches designed to account for the non-uniform data density across the temperature range and a constant offset approach tended to show better ability to detect EBT. As in our prior study, inner canthi or full-face maximum temperatures provided the highest clinical accuracy. With an optimal calibration approach, these methods achieved a Δcb between ±0.03 °C with standard deviation (σΔcb) less than 0.3 °C, and sensitivity/specificity between 84% and 94%. Results of forehead-center measurements with NCITs or IRTs indicated reduced performance. An analysis of the complete clinical data set confirms the essential findings of our preliminary evaluation, with minor differences. Our findings provide novel insights into methods and metrics for the clinical accuracy assessment of IRTs. Furthermore, our results indicate that calibration approaches providing the highest clinical accuracy in the 37-38.5 °C range may be most effective for measuring EBT. While device performance depends on many factors, IRTs can provide superior performance to NCITs.

3D-printed phantoms for characterizing SERS nanoparticle detectability in turbid media.

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.

Free-Form Deformation Approach for Registration of Visible and Infrared Facial Images in Fever Screening.

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.

Solid hemoglobin-polymer phantoms for evaluation of biophotonic systems.

Stable tissue phantoms that incorporate the spectral absorption properties of hemoglobin would benefit a wide range of biophotonic technologies. Toward this end, we have developed and validated a novel polymer material incorporating hemoglobin. Our solid hemoglobin-polymer (SHP) material is fabricated by mixing liquid silicone base with a hemoglobin solution, followed by sonication and low temperature curing. The optical properties of samples were determined over 450-1000 nm using the inverse adding-doubling method and the Beer-Lambert law. Measurements indicated SHP optical stability over four months. Near-infrared spectroscopy and hyperspectral imaging measurements of SHP samples were performed to demonstrate the utility of this approach. SHP materials have the potential to improve tissue-simulating phantoms used for development, evaluation, and standardization of optical devices for oximetry and other applications.

Three-dimensional printing of tissue phantoms for biophotonic imaging.

We have investigated the potential of tissue phantoms fabricated with thermosoftening- and photopolymerization-based three-dimensional (3D) printers for use in evaluation of biophotonic imaging systems. The optical properties of printed polymer samples were measured and compared to biological tissues. Phantoms with subsurface channels as small as 0.2 mm in diameter were fabricated and imaged with microscopy, x-ray microtomography, and optical coherence tomography to characterize morphology. These phantoms were then implemented to evaluate the penetration depth of a hyperspectral reflectance imaging system used in conjunction with a near-infrared contrast agent. Results indicated that 3D printing may provide a suitable platform for performance testing in biophotonics, although subsurface imaging is critical to mitigate printer-to-printer variability in matrix homogeneity and feature microstructure.

Vascular contrast in narrow-band and white light imaging.

Narrow-band imaging (NBI) is a spectrally selective reflectance imaging technique that is used clinically for enhancing visualization of superficial vasculature and has shown promise for applications such as early endoscopic detection of gastrointestinal neoplasia. We have studied the effect of vessel geometry and illumination wavelength on vascular contrast using idealized geometries in order to more quantitatively understand NBI and broadband or white light imaging of mucosal tissue. Simulations were performed using a three-dimensional, voxel-based Monte Carlo model incorporating discrete vessels. In all cases, either 415 or 540 nm illumination produced higher contrast than white light, yet white light did not always produce the lowest contrast. White light produced the lowest contrast for small vessels and intermediate contrast for large vessels (diameter≥100 µm) at deep regions (vessel depth≥200 µm). The results show that 415 nm illuminations provided superior contrast for smaller vessels at shallow depths while 540 nm provided superior contrast for larger vessels in deep regions. Besides 540 nm, our studies also indicate the potential of other wavelengths to achieve high contrast of large vessels at deep regions. Simulation results indicate the importance of three key mechanisms in determining spectral variations in contrast: intravascular hemoglobin (Hb) absorption in the vessel of interest, diffuse Hb absorption from collateral vasculature, and bulk tissue scattering. Measurements of NBI contrast in turbid phantoms incorporating 0.1-mm-diameter hemoglobin-filled capillary tubes indicated good agreement with modeling results. These results provide quantitative insights into light-tissue interactions and the effect of device and tissue properties on NBI performance.

In vitro evaluation of fluorescence glucose biosensor response.

Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor.

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.

AAPM Task Group Report 311: Guidance for performance evaluation of fluorescence-guided surgery systems.

The last decade has seen a large growth in fluorescence-guided surgery (FGS) imaging and interventions. With the increasing number of clinical specialties implementing FGS, the range of systems with radically different physical designs, image processing approaches, and performance requirements is expanding. This variety of systems makes it nearly impossible to specify uniform performance goals, yet at the same time, utilization of different devices in new clinical procedures and trials indicates some need for common knowledge bases and a quality assessment paradigm to ensure that effective translation and use occurs. It is feasible to identify key fundamental image quality characteristics and corresponding objective test methods that should be determined such that there are consistent conventions across a variety of FGS devices. This report outlines test methods, tissue simulating phantoms and suggested guidelines, as well as personnel needs and professional knowledge bases that can be established. This report frames the issues with guidance and feedback from related societies and agencies having vested interest in the outcome, coming from an independent scientific group formed from academics and international federal agencies for the establishment of these professional guidelines.

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.

Biomimetic tissue phantoms for neurosurgical near-infrared fluorescence imaging.

Significance: Neurosurgical fluorescence imaging is a well-established clinical approach with a growing range of indications for use. However, this technology lacks effective phantom-based tools for development, performance testing, and clinician training. Aim: Our primary aim was to develop and evaluate 3D-printed phantoms capable of optically and morphologically simulating neurovasculature under fluorescence angiography. Approach: Volumetric digital maps of the circle of Willis with basilar and posterior communicator artery aneurysms, along with surrounding cerebral tissue, were generated. Phantoms were fabricated with a stereolithography printer using custom photopolymer composites, then visualized under white light and near-infrared fluorescence imaging. Results: Feature sizes of printed components were found to be within 13% of digital models. Phantoms exhibited realistic optical properties and convincingly recapitulated fluorescence angiography scenes. Conclusions: Methods identified in this study can facilitate the development of realistic phantoms as powerful new tools for fluorescence imaging.

Test method for evaluating the photocytotoxic potential of fluorescence imaging products.

Various fluorescence imaging agents are currently under clinical studies. Despite significant benefits, phototoxicity is a barrier to the clinical translation of fluorophores. Current regulatory guidelines on medication-based phototoxicity focus on skin effects during sun exposure. However, with systemic and local administration of fluorophores and targeted illumination, there is now possibility of photochemical damage to deeper tissues during intraoperative imaging procedures. Hence, independent knowledge regarding phototoxicity is required to facilitate the development of fluorescence imaging products. Previously, we studied a cell-free assay for initial screening of reactive molecular species generation from fluorophores. The current work addresses a safety test method based on cell viability as an adjunct and a comparator with the cell-free assay. Our goal is to modify and implement an approach based on the in vitro 3T3 neutral red uptake assay of the Organization for Economic Co-Operation and Development Test Guideline 432 (OECD TG432) to evaluate the photocytotoxicity of clinically relevant fluorophores. These included indocyanine green (ICG), proflavine, methylene blue (MB), and IRDye800, as well as control photosensitizers, benzoporphyrin derivative (BPD) and rose bengal (RB). We performed measurements at agent concentrations and illumination parameters used for clinic imaging. Our results aligned with prior studies, indicating photocytotoxicity in RB and BPD and an absence of reactivity for ICG and IRDye800. DNA interactive agents, proflavine and MB, exhibited drug/light dose-response curves like photosensitizers. This study provides evidence and insights into practices useful for testing the photochemical safety of fluorescence imaging products.

Special Section Guest Editorial: Tissue Phantoms to Advance Biomedical Optical Systems.

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

Evaluation of standardized performance test methods for biomedical Raman spectroscopy (Erratum).

The erratum corrects an error in Fig. 4 of the published article.

Criteria for the design of tissue-mimicking phantoms for the standardization of biophotonic instrumentation.

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.

Quantifying the Photochemical Damage Potential of Contrast-Enhanced Fluorescence Imaging Products: Singlet Oxygen Production.

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.

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.

Evaluation of standardized performance test methods for biomedical Raman spectroscopy.

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.