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The goal of quantitative optoacoustic tomography (qOAT) is to reconstruct a distribution of absolute chromophore concentrations and/or functional properties from measurements of the optically induced pressure (ultrasound signals) acquired at multiple excitation wavelengths. Estimating the distribution of hemoglobin, an endogenous OAT chromophore, is important because the oxygen saturation distribution of the blood vessels is a well-known indicator of aggressive growth of a cancerous tumor. In a number of studies, a spectral linear unmixing method has been applied to two-dimensional slices of tissue acquired with OAT at multiple wavelengths, leading to promising results at moderate penetration depths of ≤ 2 cm. In the three-dimensional (3D) OAT of the breast, such functional images cannot be accurately reconstructed via the spectral linear unmixing method due to unknown spatial distribution of the optical fluence in a relatively large size of the volume of interest (≥ 4 cm). Optical attenuation in biological tissue depends on the optical wavelength, and the optical fluence is exponentially attenuated with increasing imaging depth. Thus, the accuracy of the estimated distribution decreases with depth. To overcome this challenge, we investigated a spectral linear unmixing method with a simplified optical fluence normalization based on measurements of background absorbed optical energy in the breast. We compare estimates of blood oxygen saturations from two-wavelength clinical OAT breast images and demonstrate acceptable accuracy of ~10% while lack of compensation for the optical fluence distribution can lead to values outside the physiological range. We also quantitatively compare the accuracy of oxygen saturation estimates using numerical simulation of photon transport in realistic 3D OAT breast phantoms at dual wavelengths of 757 and 850 nm with inverse ratio of the optical absorption by deoxy- (Hb) and oxy-hemoglobin (HbO2) and three wavelengths of 757, 800, and 850 nm with inclusion of isosbestic point of the optical absorption in Hb/HbO2.
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