Three-dimensional fluorescence laminar optical tomography (FLOT) can achieve resolutions of 100-200 µm and penetration depths of 2-3 mm. FLOT has been used in tissue engineering, neuroscience, as well as oncology. The limited dynamic range of the chargecoupled device-based system makes it difficult to image fluorescent samples with a large concentration difference, limits its penetration depth, and diminishes the quantitative accuracy of 3D reconstruction data. Here, incorporating the high-dynamic-range (HDR) method widely used in digital cameras, we present HDR-FLOT, increasing penetration depth and improving the ability to image fluorescent samples with a large concentration difference. The method was tested using an agar phantom and a B6 mouse for brain imaging in vivo.

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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.

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