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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fluorescence, both intrinsic and exogenously induced, is being used for diagnosis of abnormal tissue. Excitation wavelengths used by these methods range from 320 to 450 nm. The presence of absorbing or fluorescing drugs is rarely taken into account by practitioners of fluorescence diagnosis and has the potential to yield false-positive or false-negative results. Our aim is to quantify this potential by (1) comparing the quantum yield of fluoroquinolone antibiotics to those of known tissue fluorophores and (2) taking into account drug concentrations in the tissue during treatment. Quantum yields are determined relative to a working standard of Rhodamine 6G in ethanol. The working standard was calibrated against a fluorescein standard. We concentrated our initial efforts on (1) the fluoroquinolone antibiotics, ciprofloxacin, norfloxacin and ofloxacin and (2) the intrinsic tissue fluorophores, NADH, FAD and protoporphyrin IX. When ciprofloxacin, norfloxacin and ofloxacin were excited at wavelengths 310–390 nm, emission occurred from 350 to 650 nm with quantum yields ranging from 0.03 to 0.3. Quantum yields for intrinsic fluorophores excited at their peak absorption wavelengths were 0.02 (NADH, 340 nm), 0.035 (FAD, 450 nm) and 0.087 (protoporphyrin IX, 408 nm). A review of the literature shows that these fluoroquinolones have a large volume of distribution and can be found in high concentrations in almost every organ during a treatment regimen. The product of the drug tissue concentration and quantum yield, which we term the fluorescence effective concentration, is such that it is likely these fluoroquinolones will interfere during fluorescence diagnosis techniques. [ABSTRACT FROM AUTHOR]