Pulsed photothermal radiometry (PPTR) allows noninvasive determination of laser-induced temperature depth profiles in optically scattering layered structures. The obtained profiles provide information on spatial distribution of selected chromophores such as melanin and hemoglobin in human skin. We apply the described approach to study time evolution of incidental bruises (hematomas) in human subjects. By combining numerical simulations of laser energy deposition in bruised skin with objective fitting of the predicted and measured PPTR signals, we can quantitatively characterize the key processes involved in bruise evolution (i.e., hemoglobin mass diffusion and biochemical decomposition). Simultaneous analysis of PPTR signals obtained at various times post injury provides an insight into the variations of these parameters during the bruise healing process. The presented methodology and results advance our understanding of the bruise evolution and represent an important step toward development of an objective technique for age determination of traumatic bruises in forensic medicine.

Diffusion approximation (DA) of the radiative transport equation allows derivation of enclosed solutions for diffuse reflectance from multi-layer scattering structures, such as human skin. Although the DA is known to be inadequate near tissue boundaries and light sources, analytical tractability makes such solutions very attractive for use in noninvasive characterization of biological organs based on measured diffuse reflectance spectra (DRS). For the presented three-layer model of human skin, which enables a good match with DRS in visible spectral range measured with an integrating sphere, the DA solutions systematically overshoot numerically simulated DRS (using Monte Carlo approach) by 1-2 percentage points. However, using the former in inverse analysis of the latter can result in much larger artifacts, most notably overestimations of the melanin and blood contents by up to 15%, which must be considered when analyzing experimental DRS. Despite such systematic errors, the described approach allows simple and robust monitoring of physiological changes in human skin, as demonstrated in tests involving temporary obstruction of blood circulation and seasonal variations due to extensive sun exposure.

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Diffuse reflectance spectra (DRS) of biological samples are commonly measured using an integrating sphere (IS). To account for the incident light spectrum, measurement begins by placing a highly reflective white standard against the IS sample opening and collecting the reflected light. After replacing the white standard with the test sample of interest, DRS of the latter is determined as the ratio of the two values at each involved wavelength. However, such a substitution may alter the fluence rate inside the IS. This leads to distortion of measured DRS, which is known as single-beam substitution error (SBSE). Barring the use of more complex experimental setups, the literature states that only approximate corrections of the SBSE are possible, e.g., by using look-up tables generated with calibrated low-reflectivity standards. We present a practical method for elimination of SBSE when using IS equipped with an additional reference port. Two additional measurements performed at this port enable a rigorous elimination of SBSE. Our experimental characterization of SBSE is replicated by theoretical derivation. This offers an alternative possibility of computational removal of SBSE based on advance characterization of a specific DRS setup. The influence of SBSE on quantitative analysis of DRS is illustrated in one application example.