Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy (original) (raw)
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Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation
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The resonant Raman enhancement of hemoglobin (Hb) in the Q-band region allows simultaneous identification of oxy-and deoxyhemoglobin. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular hemoglobin oxygen saturation (SO 2 ) in vivo. We implemented a system for in vivo noninvasive measurements of SO 2 . 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. SO 2 was estimated by measuring the intensity of Raman signals (peaks) in the 1355-1380 cm -1 range (oxidation state marker band 4 ), as well as from the 19 and 10 bands (1500-1650 cm -1 range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary pathlength and Hb concentration did not affect SO 2 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 SO 2 estimations and SO 2 measured by CO-oximetry was highly significant for 4, 10 and 19 bands. The method allowed SO 2 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 SO 2 determinations in thin tissues, as well as in solid organs and tissues where transillumination is not possible.
2008
First published March 27, 2008; doi:10.1152/japplphysiol.00025.2008.— 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 m in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering ge-ometry. 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-cm1 range (oxidation state marker band 4), as well as from the 19 and 10 bands (1,500- to 1,650-cm1 range). In vivo observations were made in microvessels of anesthetized rats. Glass
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Raman Spectroscopy in Clinical Investigations
e-mail vh kart ha malic manipal edii Vhsiracl Raman speciioscopy has been successfully applied in seveial areas of biology and medicine, including diagnosis of malignancy The .ippliiaiinii'. of Surface Enhanced Raman speciioscopy and imcro-Kaman have improved to the e?cieml of studying single molecule dynamies and .dliilai iMOLhcmisiiy icspcciively Rimiaii spectroscopy studies cairied out in our laboratory on oral cancer, osteoradionecrosis, radiation induced il,nil.ILLS in mouse models arc piescnlcd and discussed We have recorded Raman speciia of normal and malignant oial tissues and the obtained spectra uiu analysed using statistical (PCA) methods An ob)cciivc diagnosis method with high sensitivity and s]ieciricity based on Muhalanohis distance and >|Kiii.iI icNidual IS developed foi oral inalignaiuy The study of radiation induced damage in mouse brain and muscle tissue suggests that ladiaiion .Kinaicil chemical cascade is similar to those in stress, but it persists foi longei periods Radiulioii treatment on bone leads to immediate structural Juni'is III the mmcial part of the bone K nuords Raman spcclioscojiy, SERS, oral cancer, PC.'A analysis, radiation induced damage. ORN bone r v r s Nos 7K ^0 Am. S7 M) H|. S7 b4 .le I. Iiifroductiori riic discovery ofRiurian elTcd in the year 1928 dcinonslraled iliai (lie analysis of inelastieally scattered light from the simplest mnlLci-ilc H ,0, can provide unique finger print of molecular siiiiciurc 11, 2|. In the last 75 years, popularity and versatility of Riiiium scatlering spectroscopy have increased in many ways iiul a diverse fai)iily ol Raman-based techniques has been ilcvciopcd. More and more sensitive experimental approaches ^'Miiiiuic to be developed to explore the molecular mechanisms ''I u>mplcx biological phenomena. Raman spectroscopy has also 'ven idenlilied as a reliable diagnostic technique [3-5). A larger luimhcr of biological molecules can be probed by using Raman ^"'V tioscopy. Several studies show the potential of near-infrared kiiMian spectroscopy for the detection ol cancer and pre-cancer 'll 1 itro/in vivo, as a new tool [3-5]. Resonance Raman scattering selectively increases the uicring signal from the ground stale vibration modes that arc ^'4i|)led U) excited vibronic levels (6J. This large enhancement Raman scattering cross section of specific molecular ^'hiation modes, offers great advantages over non-resonancê ^"'(spondmg AutKor Raman scattering. Research findings show that UVRR spectroscopy can be used to characterize normal and diseased colon tissue by selectively enhancing spectra of aromatic amino acids, and parameterizing their contribution to the colon spectrum That means, UV RR spectroscopy can provide complete biochemical characterization of the tissue under study as well as it can describe the pathological change [6|. Micro-Raman spectroscopy is a powerful tool for study of the structural variations in samples of sizes down to sub microns [7, 8]. In the Raman microanalysis, a laser beam is focused onto a very small area with a microscope objective and Raman scattered light from the area is collected by the same objective, dispersed by a monochiomalor and spectra recorded. Raman microscopy has potential utility in structural studies in situ. Recent advances in lasers, detectors, and spectrograph and filter technologies have made it possible to detect even very weak Raman signals from a single living cell [7, 8]. Ultrasensitive Raman detection based on surface enhanced Raman scattering is now well established [9, 10]. Surfaceenhanced Raman spectroscopy (SERS) is a phenomenon resulting in strongly increased Raman signals of molecules that ©20031ACS