Optical imaging modalities are non-ionizing methods with significant potential for non-invasive, portable, and cost-effective medical diagnostics and treatments. The design of critical parameters of an optical imaging system depends on a thorough understanding of optical properties of the biological tissue within the purposed application. Integrating sphere technique combined with inverse adding doubling algorithm has been widely used for determination of biological tissue ex vivo. It has been studied for tissues typically with a large sample size and over a spectral range of 400 nm to 1100 nm. The aim of this study is to develop a methodology for calculating optical absorption and reduced scattering of small size biological tissues from reflectance and transmittance measurements at a wide spectral range of 400 to 1800 nm. We developed a small sample adaptor kit to allow integrating sphere measurements of samples with small sizes using a commercial device. We proposed a two-tier IAD algorithm to mitigate the profound cross-talk effect in reduced scattering using IAD. We evaluated the two-tier IAD with both simulated data by Monte Carlo Simulation and data obtained from phantom experiments. We also investigated the accuracy the proposed work flow of using small sample kit and condensed incident light beam. We found that the small sample measurements despite with condense beam size led to overestimated absorption coefficient across the whole wavelength range while the spectrum shape well preserved. Our proposed method of a two-tier IAD and small sample kit could be a useful and reliable tool to characterise optical properties of biological tissue ex vivo particularly when only small size samples are available.
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