Optical spectroscopy is being used increasingly in medical applications to noninvasively investigate tissues below the skin. In order to assure adequate sampling of tissues underlying the skin, photon penetration depth must be known. Photon penetration in tissues has been studied with near-infrared (NIR) light, but experimental study of visible light propagation in tissue has been limited. In this study, a micro-motion system coupled with a reflectance spectroscopy system was used to determine the penetration depth of visible-range and NIR photons (535-800 nm) in phantoms composed of Intralipid and hemoglobin. An absorbing target was placed at intervals of 0.1mm along a 15mm line perpendicular to and bisecting the line between the ends of the source and detector optical fiber bundles. Comparisons between detected light intensities at different target positions were used to determine the most probable photon path depths at 576 nm and at 760 nm. Scattering coefficients, hemoglobin concentrations, and source-detector separations were varied to evaluate their effects on the penetration depth of photons. Results from phantoms containing Intralipid only showed that the most-probable penetration depth at 576 nm was comparable to that at 760 nm. Larger sourcedetector separations resulted in deeper photon penetration depths for both spectral regions. Changes in scattering over a 4-fold range did not affect the photon path depth appreciably. In the presence of hemoglobin with a source-detector separation of 13 mm, the most probable depth of photon penetration in the visible range was greater than 2.5 mm, and was within 1 mm of the most probable depth of photon penetration in the NIR. This study demonstrates the feasibility of using the visible and NIR regions in transcutaneous reflectance spectroscopy.
Myoglobin is an important intracellular oxygen transport molecule in muscle. Oxygen binding to myoglobin can be determined spectroscopically due to differences in absorption of oxymyoglobin and deoxymyoglobin. Myoglobin oxygenation can be used as a measure of intracellular oxygen tension in muscle. We sought to determine the effects of differences in temperature and pH on myoglobin absorption spectra in the near-infrared spectral region. Transmission spectra were taken of pure solutions of oxymyoglobin and deoxymyoglobin at 10°, 20°, 30°, and 40°C at pH values of 6.0, 7.0, and 8.0 (n=4). In second derivative spectra at 40°C, the deoxymyoglobin peak near 760 nm was shifted by 0.9-1.2 nm toward longer wavelengths relative to 10°C at constant pH. Differences in pH did not result in statistically significant shifts in this peak at constant temperature. Estimations of myoglobin saturation from myoglobin spectra with intermediate saturations were obtained by least squares (LS) and partial least squares (PLS) analyses. Both algorithms estimate myoglobin saturation with small root mean square errors (<1e-6) when component spectra and calibration set spectra are at the same temperature as test spectra (n=100). However, when spectra at 20°C or 40°C were used as component spectra in LS with test spectra at 30°C (all at pH 7.0), errors were 0.8% and 1.4%, respectively. PLS analysis of 30°C test spectra using 20°C or 40°C calibration set spectra yielded errors of 1.6% and 1.5%, respectively. When the PLS analysis is endpoint corrected, these errors become vanishingly small. These results demonstrate that peak shifts due to temperature are potential sources of error if calibration and test spectra differ by 10°C. These errors can be minimized by appropriate spectral analytic methods.