Effective fluence and dose at skin depth of daylight and lamp sources for PpIX-based photodynamic therapy.

SIGNIFICANCE: Skin-based photodynamic therapy (PDT) is used for the clinical treatment of actinic keratosis (AKs) and other skin lesions with continued expansion into the standard of care. Due to the spectral dependency of photosensitizer activation and skin optical fluence, there is a need for more accurate methods to estimate the delivered dose at depth from different PDT light sources and treatment regimens. AIM: Develop radiometric methods for calculating photosensitizer-effective fluence and dose at depth and determine differences between red-lamp, blue-lamp, and daylight-based PDT treatments. METHODS: Radiometric measurements of FDA-approved PDT lamp sources, outdoor daylight, and indoor daylight were performed for clinically relevant AK treatments. The protoporphyrin IX (PpIX) equivalent irradiance, fluence, and dose for each light source were calculated from the PpIX absorption spectrum and a 7-layer skin fluence model. The effective fluence and dose at depth was estimated by combining the spectral attenuation predicted at each wavelength and depth with the source fluence at each wavelength. RESULTS: The red-lamp source had the highest illuminance (112,000 lumen/m2), but lowest PpIX-effective irradiance (9.6 W/m2), and highest effective fluence at depth (10.8 W/m2 at 500 µm). In contrast, the blue light source had the lowest illuminance (2300 lumen/m2), but highest PpIX effective irradiance (37.0 W/m2), and ultimately the lowest effective fluence at depth (0.18 W/cm2 at 500 µm). The daylight source had values of (outdoor | indoor) illuminance of (49,200 | 37,800 lumen/m2), effective irradiance of (19.2 | 10.7 W/m2), and effective fluence of (1.50 | 1.08 W/m2 at 500 µm). The effective fluence and dose at depth facilitated the comparison of treatment regimens, for example, calculating an equivalent dose for a 2 hr indoor daylight treatment and a 10 min red-light treatment for the 300-1000 µm depth range. CONCLUSIONS: The consideration of PpIX-effective fluence at varying depths is necessary to provide adequate comparisons of the delivered dose from PDT light sources. Methods for calculating radiometric fluence and delivered dose at depth were introduced, with open source MATLAB code, to help overcome the limitations of commonly used photometric and irradiance-based reporting.

3D-Printed Tumor Phantoms for Assessment of In Vivo Fluorescence Imaging Analysis Methods.

PURPOSE: Interventional fluorescence imaging is increasingly being utilized to quantify cancer biomarkers in both clinical and preclinical models, yet absolute quantification is complicated by many factors. The use of optical phantoms has been suggested by multiple professional organizations for quantitative performance assessment of fluorescence guidance imaging systems. This concept can be further extended to provide standardized tools to compare and assess image analysis metrics. PROCEDURES: 3D-printed fluorescence phantoms based on solid tumor models were developed with representative bio-mimicking optical properties. Phantoms were produced with discrete tumors embedded with an NIR fluorophore of fixed concentration and either zero or 3% non-specific fluorophore in the surrounding material. These phantoms were first imaged by two fluorescence imaging systems using two methods of image segmentation, and four assessment metrics were calculated to demonstrate variability in the quantitative assessment of system performance. The same analysis techniques were then applied to one tumor model with decreasing tumor fluorophore concentrations. RESULTS: These anatomical phantom models demonstrate the ability to use 3D printing to manufacture anthropomorphic shapes with a wide range of reduced scattering (µs': 0.24-1.06 mm-1) and absorption (µa: 0.005-0.14 mm-1) properties. The phantom imaging and analysis highlight variability in the measured sensitivity metrics associated with tumor visualization. CONCLUSIONS: 3D printing techniques provide a platform for demonstrating complex biological models that introduce real-world complexities for quantifying fluorescence image data. Controlled iterative development of these phantom designs can be used as a tool to advance the field and provide context for consensus-building beyond performance assessment of fluorescence imaging platforms, and extend support for standardizing how quantitative metrics are extracted from imaging data and reported in literature.

Smartphone-based dual radiometric fluorescence and white-light imager for quantification of protoporphyrin IX in skin.

Significance: The quantification of protoporphyrin IX (PpIX) in skin can be used to study photodynamic therapy (PDT) treatments, understand porphyrin mechanisms, and enhance preoperative mapping of non-melanoma skin cancers. Aim: We aim to develop a smartphone-based imager for performing simultaneous radiometric fluorescence (FL) and white light (WL) imaging to study the baseline levels, accumulation, and photobleaching of PpIX in skin. Approach: A smartphone-based dual FL and WL imager (sDUO) is introduced alongside new radiometric calibration methods for providing SI-units of measurements in both pre-clinical and clinical settings. These radiometric measurements and corresponding PpIX concentration estimations are applied to clinical measurements to understand mechanistic differences between PDT treatments, accumulation differences between normal tissue and actinic keratosis lesions, and the correlation of photosensitizer concentrations to treatment outcomes. Results: The sDUO alongside the developed methods provided radiometric FL measurements (nW/cm2) with a demonstrated sub nanomolar PpIX sensitivity in 1% intralipid phantoms. Patients undergoing PDT treatment of actinic keratosis (AK) lesions were imaged, capturing the increase and subsequent decrease in FL associated with the incubation and irradiation timepoints of lamp-based PDT. Furthermore, the clinical measurements showed mechanistic differences in new daylight-based treatment modalities alongside the selective accumulation of PpIX within AK lesions. The use of the radiometric calibration enabled the reporting of detected PpIX FL in units of nW/cm2 with the use of liquid phantom measurements allowing for the estimation of in-vivo molar concentrations of skin PpIX. Conclusions: The phantom, pre-clinical, and clinical measurements demonstrated the capability of the sDUO to provide quantitative measurements of PpIX FL. The results demonstrate the use of the sDUO for the quantification of PpIX accumulation and photobleaching in a clinical setting, with implications for improving the diagnosis and treatment of various skin conditions.

Equivalent efficacy of indoor daylight and lamp-based 5-aminolevulinic acid photodynamic therapy for treatment of actinic keratosis.

Background: Photodynamic therapy (PDT) is widely used as a treatment for actinic keratoses (AK), with new sunlight-based regimens proposed as alternatives to lamp-based treatments. Prescribing indoor daylight activation could help address the seasonal temperature, clinical supervision, and access variability associated with outdoor treatments. Objective: To compare the AK lesion clearance efficacy of indoor daylight PDT treatment (30 min of 5-aminolevulinic acid (ALA) pre-incubation, followed by 2 h of indoor sunlight) versus a lamp-based PDT treatment (30 min of ALA preincubation, followed by 10 min of red light). Methods: A prospective clinical trial was conducted with 41 patients. Topical 10% ALA was applied to the entire treatment site (face, forehead, scalp). Patients were assigned to either the lamp-based or indoor daylight treatment. Actinic keratosis lesion counts were determined by clinical examination and recorded for pre-treatment, 1-month, and 6-month follow-up visits. Results: There was no statistical difference in the efficacy of AK lesion clearance between the red-lamp (1-month clearance = 57 ± 17%, 6-month clearance = 57 ± 20%) and indoor daylight treatment (1-month clearance = 61 ± 19%, 6-month clearance = 67 ± 20%). A 95% confidence interval of the difference of the means was measured between -4.4% and 13.4% for 1-month, and -2.2% and +23.6% for 6-month timepoints when comparing the indoor daylight to the red-lamp treatment, with a priori interval of equivalence of ±20%. Limitations: Ensuring an equivalent dose between the indoor and lamp treatment cohorts limited randomisation since it required performing indoor daylight treatments only during sunny days. Conclusion: Indoor-daylight PDT provided equivalent AK treatment efficacy to a lamp-based regimen while overcoming temperature limitations and UV-block sunscreen issues associated with outdoor sunlight treatments in the winter. Clinical trial registration: Clinicaltrials.gov listing: NCT03805737.

Ultracompact fluorescence smartphone attachment using built-in optics for protoporphyrin-IX quantification in skin.

Smartphone-based fluorescence imaging systems have the potential to provide convenient quantitative image guidance at the point of care. However, common approaches have required the addition of complex optical attachments, which reduce translation potential. In this study, a simple clip-on attachment appropriate for fluorescence imaging of protoporphyrin-IX (PpIX) in skin was designed using the built-in light source and ultrawide camera sensor of a smartphone. Software control for image acquisition and quantitative analysis was developed using the 10-bit video capability of the phone. Optical performance was characterized using PpIX in liquid tissue phantoms and endogenously produced PpIX in mice and human skin. The proposed system achieves a very compact form factor (<30 cm3) and can be readily fabricated using widely available low-cost materials. The limit of detection of PpIX in optical phantoms was <10 nM, with good signal linearity from 10 to 1000 nM (R2 >0.99). Both murine and human skin imaging verified that in vivo PpIX fluorescence was detected within 1 hour of applying aminolevulinic acid (ALA) gel. This ultracompact handheld system for quantification of PpIX in skin is well-suited for dermatology clinical workflows. Due to its simplicity and form factor, the proposed system can be readily adapted for use with other smartphone devices and fluorescence imaging applications. Hardware design and software for the system is made freely available on GitHub (https://github.com/optmed/CompactFluorescenceCam).

3D printing fluorescent material with tunable optical properties.

The 3D printing of fluorescent materials could help develop, validate, and translate imaging technologies, including systems for fluorescence-guided surgery. Despite advances in 3D printing techniques for optical targets, no comprehensive method has been demonstrated for the simultaneous incorporation of fluorophores and fine-tuning of absorption and scattering properties. Here, we introduce a photopolymer-based 3D printing method for manufacturing fluorescent material with tunable optical properties. The results demonstrate the ability to 3D print various individual fluorophores at reasonably high fluorescence yields, including IR-125, quantum dots, methylene blue, and rhodamine 590. Furthermore, tuning of the absorption and reduced scattering coefficients is demonstrated within the relevant mamalian soft tissue coefficient ranges of 0.005-0.05 mm-1 and 0.2-1.5 mm-1, respectively. Fabrication of fluorophore-doped biomimicking and complex geometric structures validated the ability to print feature sizes less than 200 µm. The presented methods and optical characterization techniques provide the foundation for the manufacturing of solid 3D printed fluorescent structures, with direct relevance to biomedical optics and the broad adoption of fast manufacturing methods in fluorescence imaging.

Indocyanine green matching phantom for fluorescence-guided surgery imaging system characterization and 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.

Evaluation of bone perfusion during open orthopedic surgery using quantitative dynamic contrast-enhanced fluorescence imaging.

In this study, an indocyanine green (ICG)-based dynamic contrast- enhanced fluorescence imaging (DCE-FI) technique was evaluated as a method to provide objective real-time data on bone perfusion using a porcine osteotomy model. DCE-FI with sequentially increasing injury to osseous blood supply was performed in 12 porcine tibias. There were measurable, reproducible and predictable changes to DCE-FI data across each condition have been observed on simple kinetic curve-derived variables as well variables derived from a novel bone-specific kinetic model. The best accuracy, sensitivity and specificity of 89%, 88% and 90%, have been achieved to effectively differentiate injured from normal/healthy bone.

Endosteal and periosteal blood flow quantified with dynamic contrast-enhanced fluorescence to guide open orthopaedic surgery.

Due to the lack of objectively measurable or quantifiable methods to assess the bone perfusion, the success of removing devitalized bone is based almost entirely on surgeon's experience and varies widely across surgeons and centers. In this study, an indocyanine green (ICG)-based dynamic contrast-enhanced fluorescence imaging (DCE-FI) has been developed to objectively assess bone perfusion and guide surgical debridement. A porcine trauma model (n = 6 pigs × 2 legs) with up to 5 conditions of severity in loss of flow in each, was imaged by a commercial fluorescence imaging system. By applying the bone-specific hybrid plug-compartment (HyPC) kinetic model to four-minute video sequences, the perfusion-related metrics, such as peak intensity, total bone blood flow (TBBF) and endosteal bone blood flow to TBBF fraction (EFF) were calculated. The results shown that the combination of TBBF and EFF can effectively differentiate injured from normal bone with the accuracy, sensitivity and specificity of 89%, 88% and 90%, respectively. Our subsequent first in human bone blood flow imaging study confirmed DCE-FI can be successfully translated into human orthopaedic trauma patients.

Smartphone fluorescence imager for quantitative dosimetry of protoporphyrin-IX-based photodynamic therapy in skin.

Significance: While clinical treatment of actinic keratosis by photodynamic therapy (PDT) is widely practiced, there is a well-known variability in response, primarily caused by heterogeneous accumulation of the photosensitizer protoporphyrin IX (PpIX) between patients and between lesions, but measurement of this is rarely done. Aim: Develop a smartphone-based fluorescence imager for simple quantitative photography of the lesions and their PpIX levels that can be used in a new clinical workflow to guide the reliability of aminolevulinic acid (ALA) application for improved lesion clearance. Approach: The smartphone fluorescence imager uses an iPhone and a custom iOS application for image acquisition, a 3D-printed base for measurement standardization, an emission filter for PpIX fluorescence isolation, and a 405-nm LED ring for optical excitation. System performance was tested to ensure measurement reproducibility and the ability to capture photosensitizer accumulation and photobleaching in pre-clinical and clinical settings. Results: PpIX fluorescence signal from tissue-simulating phantoms showed linear sensitivity in the 0.01 to 2.0 µM range. Murine studies with Ameluz® aminolevulinic acid (ALA) gel and initial human testing with Levulan® ALA cream verified that in-vivo imaging was feasible, including that PpIX production over 1 h is easily captured and that photobleaching from the light treatment could be quantified. Conclusions: The presented device is the first quantitative wide-field fluorescence imaging system for PDT dosimetry designed for clinical skin use and for maximal ease of translation into clinical workflow. The results lay the foundation for using the system in clinical studies to establish treatment thresholds for the individualization of PDT treatment.