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.