Effects of calibration factors and intensity dependent non-linearity on functional photoacoustic microscopy

Functional photoacoustic microscopy is a valuable tool in quantifying hemoglobin oxygenation within single vessels. In several functional studies with this tool, quantitative sO₂ measurements were taken both in vitro and in vivo. Although in vitro measurements of sO₂ showed high agreement with expected values from premade samples, in practice, in vivo measurements were less accurate. The reported values of 70%-100% sO2 in the arteries present large deviations from the expected range of 95-100%. Several factors, such as fluence wavelength dependence, optical wavelength range, and transducer central frequency have been suggested and investigated in order to understand these discrepancies. Despite additional knowledge of systematic errors arising from such factors, measuring the absolute value of sO₂ in vivo remains a challenge. All previous studies assumed linear dependence of the photoacoustic signal on absorption and used the linear least squares model. However, several factors, such as wavelength calibration errors, photodiode-wavelength dependence, and intensity dependent non-linearity, all of which may have a significant effect on the final calculation, have not been investigated. Here we evaluate both in vitro and in vivo the effects on sO ₂ measurements of photodiode wavelength dependence, laser wavelength accuracy, and intensity dependent absorption of oxygenated and deoxygenated hemoglobin. We show that these factors may contribute significantly to the deviations in sO₂ calculations in vivo.