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INNOVATIVE METHODOLOGY

Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy

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

Submitted 22 November 2004 ; accepted in final form 8 March 2005

A system is described for in vivo noninvasive measurements of hemoglobin oxygen saturation (HbO₂Sat) at the microscopic level. The spectroscopic basis for the application is resonant Raman enhancement of Hb in the violet/ultraviolet region, allowing simultaneous identification of oxy- and deoxyhemoglobin with the same excitation wavelength. The heme vibrational bands are well known, but the technique has never been used to determine microvascular HbO₂Sat in vivo. A diode laser light (power: 0.3 mW) was focused onto sample areas 15–30 µm in diameter. Raman spectra were obtained in backscattering geometry by using a microscope coupled to a spectrometer and a cooled detector. Calibration was performed in vitro by using glass capillaries containing blood at several Hb concentrations, equilibrated at various oxygen tensions. HbO₂Sat was estimated using the Raman band intensities at 1,360 and 1,375 cm^–1. Glass capillary path length and Hb concentration had no effect on HbO₂Sat estimated from Raman spectra. In vivo observations were made in blood flowing in microvessels of the rat mesentery. The Hb Raman peaks observed in oxygenated and deoxygenated blood were consistent with earlier Raman studies that used Hb solutions and isolated cells. The method allowed HbO₂Sat determinations in the whole range of arterioles, venules, and capillaries. Tissue transillumination allowed diameter and erythrocyte velocity measurements in the same vessels. Raman microspectroscopy offers distinct advantages over other currently used techniques by providing noninvasive and reliable in vivo determinations of HbO₂Sat in thin tissues as well as in solid organs and tissues, which are unsuitable for techniques requiring transillumination.

resonance Raman spectroscopy; microcirculation; rat

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