Submitted on November 22, 2004
Accepted on March 8, 2005
Hemoglobin Oxygen Saturation Measurements Using Resonance Raman Intravital Microscopy
Ivo P Torres Filho1*, James Terner2, Roland N Pittman3, Leonardo G Somera4, and Kevin R Ward3
1 Anesthesiology, Virginia Commonwealth University, Richmond, VA, USA; Physiology, Virginia Commonwealth University, Richmond, VA, USA; Emergency Medicine, Virginia Commonwealth University, Richmond, VA, USA
2 Chemistry, Virginia Commonwealth University, Richmond, VA, USA
3 Physiology, Virginia Commonwealth University, Richmond, VA, USA; Emergency Medicine, Virginia Commonwealth University, Richmond, VA, USA
4 Anesthesiology, Virginia Commonwealth University, Richmond, VA, USA
* To whom correspondence should be addressed. E-mail: itorres{at}vcu.edu.
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 the 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 of 15-30 µm in diameter. Raman spectra were obtained in backscattering geometry using a microscope coupled to a spectrometer and a cooled detector. Calibration was performed in vitro using glass capillaries containing blood at several Hb concentrations, equilibrated at various oxygen tensions. HbO2Sat was estimated using the Raman band intensities at 1360 cm-1 and 1375 cm-1. Glass capillary pathlength 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 using 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.
