Yang, M.Wei, Y.Reineck, P.Wells, A.J.Ebendorff‐Heidepriem, H.Li, J.McLaughlin, R.A.2025-10-142025-10-142025Advanced Photonics Research, 2025; 6(8):2400209-1-2400209-122699-92932699-9293https://hdl.handle.net/2440/147786Fluorescence-guided surgery is an increasingly common technique in neurosurgery, where 5-aminolevulinic acid induces fluorescence in high-grade gliomas, aiding in tumor resection and improving surgical outcomes. Reliable detection of malignant tissue fluorescence depends critically upon the clinical imaging system. Factors such as nonuniform excitation light and the presence of non-fluorescent tissue layers over the tumor can reduce sensitivity. Characterizing imaging system performance in these scenarios is important to ensure clinical reliability. However, there are a lack of practical calibration standards available for this purpose. This study proposes a novel calibration standard to assist in characterizing a clinical fluorescence imaging system. The calibration standard uses multiple glass-based phantoms fabricated to mimic the optical properties of tissue. Silver nanoparticles mimic the absorption spectrum of hemoglobin; small air-filled cavities and crystals in the glass generate controlled levels of scattering; and samarium ions provide fluorescence to mimic malignant tissue. Single-layer and bilayer glass phantoms enable assessment of fluorescence across the field of view, including characterization of the sensitivity to detect fluorescence through layers of non-fluorescent glass, mimicking nonmalignant tissue. The glass-based phantoms demonstrate excellent photo-stability, homogeneity, and long-term shelf-life. Utility of this calibration standard is demonstrated with a commercial surgical fluorescence imaging system.en© 2025 The Author(s). Advanced Photonics Research published by Wiley- VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.bi-layer phantom; fluorescence; fluorescence-guided surgery; optical phantom; silver nanoparticlesJournal article10.1002/adpr.202400209739279Wei, Y. [0000-0002-7550-7397]Li, J. [0000-0001-8818-6070]McLaughlin, R.A. [0000-0001-6947-5061]