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INNOVATIVE METHODOLOGY

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

Departments of ¹Anesthesiology, ²Chemistry, ³Physiology and Biophysics, ⁴Emergency Medicine, ⁵Biomedical Engineering, and ⁶Biochemistry, Virginia Commonwealth University Reanimation Engineering Shock Center, Virginia Commonwealth University, Richmond, Virginia

Submitted 10 January 2008 ; accepted in final form 24 March 2008

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 ₄), as well as from the ₁₉ and ₁₀ 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 ₄, ₁₀, and ₁₉ 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.

intravital microscopy; resonance Raman spectroscopy; microcirculation; rat