Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation

Abstract

The resonant Raman enhancement of hemoglobin (Hb) in the Q band region allows simultaneous identification of oxy- and deoxy-Hb. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular Hb oxygen saturation (So2) in vivo. We implemented a system for in vivo noninvasive measurements of So2. A laser light was focused onto areas of 15–30 μm in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering geometry. Calibration was performed in vitro using blood at several Hb concentrations, equilibrated at various oxygen tensions. So2 was estimated by measuring the intensity of Raman signals (peaks) in the 1,355- to 1,380-cm−1 range (oxidation state marker band ν4), as well as from the ν19 and ν10 bands (1,500- to 1,650-cm−1 range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary pathlength and Hb concentration did not affect So2 estimations from Raman spectra. The Hb Raman peaks observed in blood were consistent with earlier Raman studies using Hb solutions and isolated cells. The correlation between Raman-based So2 estimations and So2 measured by CO-oximetry was highly significant for ν4, ν10, and ν19 bands. The method allowed So2 determinations in all microvessel types, while diameter and erythrocyte velocity could be measured in the same vessels. Raman microspectroscopy has advantages over other techniques by providing noninvasive and reliable in vivo So2 determinations in thin tissues, as well as in solid organs and tissues in which transillumination is not possible.