Absorption coefficient of oxygenated human lysed blood is evaluated with collimated transmission (SCT) to predict blood oxygen saturation using tabulated hemoglobin absorption spectra. We report on discrepancies in expected and observed oxygen levels.

Significance: For optical methods to accurately assess hemoglobin oxygen saturation in vivo, an independently verifiable tissue-like standard is required for validation. For this purpose, we propose three hemoglobin preparations and evaluate methods to characterize them.

Aim: To spectrally characterize three different hemoglobin preparations using multiple spectroscopic methods and to compare their absorption spectra to commonly used reference spectra.

Approach: Absorption spectra of three hemoglobin preparations in solution were characterized using spectroscopic collimated transmission: whole blood, lysed blood, and ferrous-stabilized hemoglobin. Tissue-mimicking phantoms composed of Intralipid, and the hemoglobin solutions were characterized using spatial frequency-domain spectroscopy (SFDS) and enhanced perfusion and oxygen saturation (EPOS) techniques while using yeast to deplete oxygen.

Results: All hemoglobin preparations exhibited similar absorption spectra when accounting for methemoglobin and scattering in their oxyhemoglobin and deoxyhemoglobin forms, respectively. However, systematic differences were observed in the fitting depending on the reference spectra used. For the tissue-mimicking phantoms, SFDS measurements at the surface of the phantom were affected by oxygen diffusion at the interface with air, associated with higher values than for the EPOS system.

Conclusions: We show the validity of different blood phantoms and what considerations need to be addressed in each case to utilize them equivalently.

Significance Spatial frequency domain imaging (SFDI) and spatial frequency domain spectroscopy (SFDS) are emerging tools to non-invasively assess tissues. However, the presence of aberrations can complicate processing and interpretation.

Aim This study develops a method to characterize optical aberrations when performing SFDI/S measurements. Additionally, we propose a post-processing method to compensate for these aberrations and recover arbitrary subsurface optical properties.

Approach Using a custom SFDS system, we extract absorption and scattering coefficients from a reference phantom at 0 to 15 mm distances from the ideal focus. In post-processing, we characterize aberrations in terms of errors in absorption and scattering relative to the expected in-focus values. We subsequently evaluate a compensation approach in multi-distance measurements of phantoms with different optical properties and in multi-layer phantom constructs to mimic subsurface targets.

Results Characterizing depth-specific aberrations revealed a strong power law such as wavelength dependence from ∼40 to ∼10 % error in both scattering and absorption. When applying the compensation method, scattering remained within 1.3% (root-mean-square) of the ideal values, independent of depth or top layer thickness, and absorption remained within 3.8%.

Conclusions We have developed a protocol that allows for instrument-specific characterization and compensation for the effects of defocus and chromatic aberrations on spatial frequency domain measurements.