Near-infrared spectroscopy (NIRS) is an established technique for measuring tissue oxygen saturation (StO2), which is of high clinical value. For tissues that have layered structures, it is challenging but clinically relevant to obtain StO2 of the different layers, e.g. brain and scalp. For this aim, we present a new method of data analysis for time-domain NIRS (TD-NIRS) and a new two-layered blood-lipid phantom. The new analysis method enables accurate determination of even large changes of the absorption coefficient (Δµa) in multiple layers. By adding Δµa to the baseline µa, this method provides absolute µa and hence StO2 in multiple layers. The method utilizes (i) changes in statistical moments of the distributions of times of flight of photons (DTOFs), (ii) an analytical solution of the diffusion equation for an N-layered medium, (iii) and the Levenberg-Marquardt algorithm (LMA) to determine Δµa in multiple layers from the changes in moments. The method is suitable for NIRS tissue oximetry (relying on µa) as well as functional NIRS (fNIRS) applications (relying on Δµa). Experiments were conducted on a new phantom, which enabled us to simulate dynamic StO2 changes in two layers for the first time. Two separate compartments, which mimic superficial and deep layers, hold blood-lipid mixtures that can be deoxygenated (using yeast) and oxygenated (by bubbling oxygen) independently. Simultaneous NIRS measurements can be performed on the two-layered medium (variable superficial layer thickness, L), the deep (homogeneous), and/or the superficial (homogeneous). In two experiments involving ink, we increased the nominal µa in one of two compartments from 0.05 to 0.25 cm-1, L set to 14.5 mm. In three experiments involving blood (L set to 12, 15, or 17 mm), we used a protocol consisting of six deoxygenation cycles. A state-of-the-art multi-wavelength TD-NIRS system measured simultaneously on the two-layered medium, as well as on the deep compartment for a reference. The new method accurately determined µa (and hence StO2) in both compartments. The method is a significant progress in overcoming the contamination from the superficial layer, which is beneficial for NIRS and fNIRS applications, and may improve the determination of StO2 in the brain from measurements on the head. The advanced phantom may assist in the ongoing effort towards more realistic standardized performance tests in NIRS tissue oximetry. Data and MATLAB codes used in this study were made publicly available.
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