Empirical data between 510 and 590 nm of diffuse reflected light from the pig heart in vivo have shown that myoglobin and cytochrome c absorption peaks with little apparent contribution of red blood cell (RBC) Hb. Monte Carlo simulations of photon migration in tissue were performed to compare the effects of myoglobin and cytochromes with those of blood Hb on photon pathlengths and diffuse reflectance of visible wavelengths (450–600 nm) from the pig heart in vivo. Wavelength dependence of the input parameters, including the transport-corrected scattering coefficients (1.1–1.2 mm−1) and the absorption coefficients of blood-free solubilized heart tissue (0.43–1.47 mm−1), as well as the absorption coefficients of Hb, were determined by an integrating sphere method and standard spectrophotometry, respectively. The Monte Carlo simulations indicate that in the 510- to 590-nm range the mean path length within the myocardium for diffusely reflected light varies from 1.4 to 1.2 mm, whereas their mean penetration depth within the epicardium is only 330–400 μm for blood-free heart tissue. Analysis shows that the blood Hb absorption extrema are only observable between 510 and 590 nm when RBC concentration in tissue is >0.5%. Blood within vessels much larger than capillaries does not contribute significantly to the spectral features, because virtually all light in this spectral range is absorbed during transit through large vessels (>100 μm). This analysis suggests that diffuse reflected light in the 510- to 590-nm region will show spectral features uniquely associated with myoglobin and cytochrome c oxygenation states within 400 μm of the surface of the heart in situ as long as the capillary RBC concentration remains <0.5%.
