One of the chief applications of diffuse optical spectroscopy is the measurement of chromophore concentrations in biological tissue, which requires measurements of tissue absorption. To achieve absolute absorption measurements, two chief confounds must be accounted for: instrumental contributions and tissue scattering. To account for instrumental contributions, a preliminary calibration on a phantom of known optical properties is typically done. The need for a calibration is eliminated by self-calibrating or dual-slope techniques using specially designed probe geometries. A technique that is capable of measuring tissue scattering is frequency domain near-infrared spectroscopy. However, it is typically not implemented for a spectrum of wavelengths due to instrumental complexity. Here we present a technique that combines self-calibrating frequency-domain at two wavelengths, to account for tissue scattering, and dual-slope continuous-wave broadband diffuse reflectance spectroscopy to achieve spectral measurements of absolute absorption between 600 nm and 1064 nm without any need for calibration. We apply this technique to two human tissues in vivo to determine concentrations of oxy-hemoglobin, deoxy-hemoglobin, lipids, and water. We found that the quality of the spectral fits may be significantly improved by the inclusion of a wavelength-independent background absorption. This leads to a discussion on the origin of this background absorption, and on the meaning of the chromophore concentrations that are recovered from spectral analysis. Current work is seeking to further understand and possibly correct for this apparent background absorption.
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