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The tissue fraction of red blood cells (RBCs) and their oxygenation and speed-resolved perfusion areestimated in absolute units by combining diffuse reflectance spectroscopy (DRS) and laser Doppler flowmetry(LDF). The DRS spectra (450 to 850 nm) are assessed at two source–detector separations (0.4 and 1.2 mm), allowingfor a relative calibration routine, whereas LDF spectra are assessed at 1.2mmin the same fiber-optic probe. Data areanalyzed using nonlinear optimization in an inverse Monte Carlo technique by applying an adaptive multilayeredtissue model based on geometrical, scattering, and absorbing properties, as well as RBC flow-speed information.Simulations of 250 tissue-like models including up to 2000 individual blood vessels were used to evaluatethe method. The absolute root mean square (RMS) deviation between estimated and true oxygenation was 4.1percentage units, whereas the relative RMS deviations for the RBC tissue fraction and perfusion were 19% and23%, respectively. Examples of in vivo measurements on forearm and foot during common provocations arepresented. The method offers several advantages such as simultaneous quantification of RBC tissue fractionand oxygenation and perfusion from the same, predictable, sampling volume. The perfusion estimate is speedresolved, absolute (% RBC × mm∕s), and more accurate due to the combination with DRS

Inverse Monte Carlo in a multilayered tissue model: merging diffuse reflectance spectroscopy and laser Doppler flowmetry