Effects of sample port size and sample thickness on optical properties quantification with a double integrating sphere system

Accurate quantification of tissue optical properties is essential for optical-based diagnostics and therapies. Conventional integrating sphere systems with one-inch sample ports often lead to insufficient sample coverage and measurement inaccuracies when evaluating small tissue specimens, such as those obtained during early carcinogenesis. This study investigates the influence of sample port size and sample thickness on the accuracy of measured optical properties. To evaluate these effects, we constructed a double integrating sphere system equipped with two interchangeable sample ports (half-inch and one-inch) and a collimated transmission setup. This system was employed to comprehensively measure optical phantoms (Intralipid and polystyrene microspheres) spanning a wide range of scattering properties and optical thicknesses, as well as ex vivo tissues, including mouse skin and porcine esophagus. The results revealed marked differences in optical properties measured with different port sizes, which became more pronounced with increasing optical thickness. Based on extensive phantom data, we developed an empirical correction model parameterized by optical thickness. This correction substantially improved the consistency of measurements across port sizes, reducing the average percentage deviation to below 8%, thereby demonstrating the robustness and practical utility of the approach. In summary, this study provides valuable guidance for the application of integrating sphere technology in accurately extracting optical properties from small tissue samples.