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Am J Physiol Heart Circ Physiol 289: H488-H495, 2005. First published March 11, 2005; doi:10.1152/ajpheart.01171.2004
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INNOVATIVE METHODOLOGY

Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy

Ivo P. Torres Filho,1,3,4 James Terner,2 Roland N. Pittman,3,4 Leonardo G. Somera, III,1 and Kevin R. Ward3,4

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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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

INTRAVASCULAR MEASUREMENTS of hemoglobin (Hb) oxygen saturation (HbO2Sat) in the microcirculation have been obtained primarily by means of spectrophotometry of the Hb molecule (29). The procedure is based on measurements of light absorption at an isosbestic wavelength of the Hb absorption spectrum and at a wavelength where there is a maximum difference between oxy- and deoxyhemoglobin (28). This method can be implemented noninvasively and yields reliable measurements when the secondary light from other portions of the tissue can be accounted for or effectively shielded. The technique has been circumscribed primarily to the measurement of HbO2Sat in relatively thin tissues in which transillumination is possible (15, 17). In addition, the procedure becomes substantially more complex if HbO2Sat needs to be determined in capillaries as well as in larger microvessels such as arterioles and venules (14, 16). Consequently, it is not possible to determine HbO2Sat in all microvessels using the same setup.

Raman spectroscopy can be used to uniquely identify molecules and has a wide range of in vivo applications (2, 8, 9, 22). In terms of HbO2Sat determination, this method has the advantage that the oxygenation information is related only to the specific interaction of the photons with the Hb molecules, generating a unique Raman spectrum (6, 7, 12, 36, 40, 41). The vibrational bands of heme are well known (1, 5), and previous Raman investigations of Hb have been carried out on isolated and purified Hb or in isolated red blood cells (27, 33, 34). However, the technique has not yet been used to determine HbO2Sat in microvessels in vivo. We adapted this technology to the measurement of HbO2Sat in flowing blood of single microvessels of living tissues, in a configuration that provides the measurement of other microvascular parameters, such as diameter and blood flow, using other noninvasive optical techniques.

The principles of the technique have been described in detail elsewhere (22, 37, 47), and only a brief explanation is given here. Raman spectroscopy, or Raman scattering, is based on the well-documented Raman effect: an inelastic phenomenon in which the scattered photon is shifted in frequency from the incident photon as it either gains energy from or loses energy to a particular vibrational mode of the molecule. Therefore, Raman spectroscopy is a form of vibrational spectroscopy in which the energy transitions arise from molecular vibrations. Because these vibrations involve identifiable functional groups, when the energies of these transitions are plotted as a spectrum, they can be used to uniquely identify a molecule. The inelastic interaction of a primary light quantum with a molecule in its vibrational ground state produces the Stokes Raman spectrum. If a molecule is not in its vibrational ground state, the interaction of the primary light produces the anti-Stokes Raman spectrum. A Raman spectrum is a plot of the intensity of Raman scattered radiation as a function of its frequency difference from the incident radiation. This difference is called the Raman shift (usually expressed in relative wavenumber units, cm–1):

where {lambda}inc and {lambda}scatt are the incident and Raman scattered light wavelengths (in cm), respectively. Because it is a difference value, the Raman shift is independent of the frequency of the incident radiation. Typically, only the Stokes region is used (the anti-Stokes spectrum is identical in pattern but much less intense). Other exciting light quanta are elastically scattered to give Rayleigh scattering (same {lambda}inc) of unscattered energy. The intensity of the Rayleigh line is several orders of magnitude higher that that of the Raman lines and may dominate the Raman spectrum of a weak Raman scatterer at low energies.

Raman spectroscopy is an attractive optical technique because it provides direct access to the state of Hb. Excitation within the {alpha}, {beta}, and Soret bands of Hb results in enhancement of numerous Raman peaks between 100 and 1,700 cm–1 whose relative intensities show a dramatic dependence on the excitation wavelength (35). Resonance Raman (RR) scattering from Hb is a selective enhancement of the porphyrin vibrations in the Raman spectrum when the frequency of the exciting light falls within the electronic absorption band of the molecule (40). Excitation within the Soret band of heme proteins provides strong enhancement in the 1,350–1,380 cm–1 range, also known as {nu}4 (1, 38). This polarized Raman peak appears to be sensitive to the electron density in the heme ring and shifts to higher frequency upon oxidation of ferrous derivatives to ferric derivatives. Therefore, Raman scattering provides information about the oxygenation state, as well as the oxidation and spin state, of the heme irons (39). Several authors have reported RR scattering of laser radiation from vibrational modes in the heme group of oxy- and deoxyhemoglobin in aqueous solution (5, 6). Because RR scattering from Hb occurs only at its prosthetic group, it is possible to investigate exclusively vibrations of the heme groups of Hb without interference by scattering of the surrounding globin or other parts of the red blood cell or of the suspending medium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Raman Intravital Microscope

A commercial upright microscope (Axioplan Imaging 2; Zeiss) was adapted for RR spectroscopy by using laser excitation. The main optical elements were similar to those used in regular microscopy, and the system was designed to provide noninvasive measurement of geometric and hemodynamic parameters of the microcirculation (Fig. 1). The microscope was equipped with transillumination and epi-illumination optics, including dry [Zeiss x10, numerical aperture (NA) 0.46; x20, NA 0.60] and immersion objectives (Zeiss x20 water, NA 0.45; x40 water, NA 0.80; x40 oil, NA 1.30). All Raman spectra reported were obtained using the x40 water immersion objective.



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  		Fig. 1. Schematic diagram of the system used to obtain resonance Raman spectra and hemodynamic parameters in the microcirculation. The area under study can be observed using simultaneous trans- and epi-illumination, if desired. The head of the velocimeter system placed just before the charge-coupled device (CCD) video camera allows online red blood cell velocity measurements. Geometric measurements can be made using the images generated by the video camera. The filter wheel is also controlled by computer 2. The data-acquisition system also receives input from pressure transducers connected to the animal. HbO2Sat, Hb O2 saturation.

 
The 406.7-nm excitation was provided by an external cavity (Littrow configuration), semiconductor tunable continuous wave laser (Sacher TEC 100; Sacher Lasertechnik, Marburg, Germany) connected to a controller (MLD-1000; Sacher Lasertechnik). The laser beam was directed via a mirror onto the epi-illumination optical path of the microscope. The plasma emission lines were removed from the laser beam with a band-pass filter and by adjusting the diameter of two pinholes placed in the optical path. The laser intensity entering the microscope was controlled by a set of neutral density filters mounted in a vertical filter holder and by adjusting the laser current using the controller. A 2-mm mirror in the microscope then reflected the laser light down onto the preparation. Backscattered light from the sample was collected and collimated by the objective and directed back into the main part of the instrument along the same path as the incident laser beam. A high-quality long-pass filter was used to attenuate the Rayleigh line while providing good transmission of the Raman scattered light in the desired range (>420 nm). The light was projected into the entrance slit (set at an aperture of 10–20 µm in this study) of the spectrometer (SpectraPro; Acton Research, Acton, MA) via a mechanical adapter. A lens then recollimated the light, which was directed by a mirrored prism onto the diffraction grating (2,400 grooves/mm). The diffracted light was directed by the second mirrored face of the prism to a 150-mm focal length lens that focused the light onto the detector. The spectral responses of different detectors were compared, and a backilluminated charge-coupled device (CCD) detector (Spec10:400B; Princeton Instruments) was selected to maximize quantum efficiency at the desired wavelength range (410–450 nm). The resolution of the detector (1,340 x 400 pixels) was also selected to maximize the acquisition of RR spectra, because relatively large pixels degrade the spectral resolution. The detector was Peltier-cooled to either –60 or –90°C to reduce thermal noise. The cooling temperature did not affect the ability to detect the RR Hb peaks in vivo. The detector was connected to a controller interfaced to a personal computer, where spectra were stored and analyzed.

Calibration

Wave-number calibration. To precisely assign a wave number to each individual detector channel, we used two types of external sources: calibration lamps and reference compounds whose Raman peak positions are known. Emission lines of low-pressure argon-mercury and mercury calibration lamps (Oriel Instruments, Stratford, CT), as well as benzene and toluene, were used for calibration. Calibration points distributed over the whole spectral region of interest were fitted by a polynomial (WinSpec software; Roper Scientific, Trenton, NJ). The wavelength of the incident laser light was determined at the beginning of each experiment to transform the wavelength calibration into a wave-number calibration. The final spectral resolution was 1.54 cm–1 (0.03 nm) in the range of interest (1,300–1,400 cm–1). The power of the incident laser at the x40 microscope objective focal point also was determined (PD300-TP Power Meter; Ophir Optronics, Wilmington, MA) and kept at 0.3 mW.

In vitro measurements. Calibration of the HbO2Sat apparatus was performed in vitro with rat Hb solutions, as well as with rat and pig blood, equilibrated with known oxygen tensions. Prolonged equilibration of blood with pure nitrogen was used to obtain measurements under anaerobic conditions (13). A separate group of Sprague-Dawley rats and pigs was used for collection of blood. Blood was withdrawn under sterile conditions from the exposed carotid artery of anesthetized animals into sterile syringes containing heparin. The blood was used either undiluted or diluted with normal saline to different Hb concentrations. Hb solutions were prepared by rupturing red blood cells, followed by ultracentrifugation. Total Hb concentration, methemoglobin, and HbO2Sat was measured with a CO oximeter adjusted for the animal's Hb absorption spectra (OSM3; Radiometer, Copenhagen, Denmark). An additional 0.06-ml sample was used for hematocrit determination. The tested Hb concentration of the samples used for the Raman measurements ranged from 1 to 14 g/dl, and hematocrits ranged from 0 to 45%. Raman spectroscopy of sample solutions was investigated using glass capillary tubes with different path lengths (50–800 µm). Various laser excitation areas were tested, ranging from a minimum spot size of 15 µm to a maximum of 150 µm in diameter.

In vivo measurements. This study was approved in advance by the Institutional Animal Care and Use Committee of Virginia Commonwealth University Health System and conformed to the Public Health Service Policy on Human Care and Use of Laboratory Animals (August 2002) and the American Physiological Society's "Guiding Principles in the Care and Use of Animals." In vivo experiments were performed in anesthetized male Sprague-Dawley rats (250–450 g body wt) spontaneously breathing room air or 100% oxygen. Anesthesia was provided as a mixture of ketamine (70 mg/kg ip; Fort Dodge Animal Health, Fort Dodge, IA) and acepromazine (3 mg/kg ip; Vedco, St. Joseph, MO), followed by constant intravenous infusion (0.24–0.36 mg·kg–1·min–1) of alfaxalone-alfadolone acetate (Saffan; Schering-Plough Animal Health, Welwyn Garden City, United Kingdom). The rats had acute cannulas implanted into the femoral (or carotid) artery and the jugular vein to measure blood pressure and to collect blood samples. The rat mesentery was prepared for microvascular studies as previously described (42, 43, 45). Briefly, via a midline laparotomy, an ileal loop and the associated mesentery were carefully exteriorized. The ileum and mesentery were placed on top of a temperature-controlled Plexiglas viewing platform (21) without excessive stretch. Any exposed gut was covered with swabs soaked in saline. After exposure, the preparation was covered with a thin plastic film (Saran Wrap; Dow Corning, Midland, MI) to minimize both desiccation of the tissue and gas exchange with the atmosphere. We have not observed any obvious deleterious effects (e.g., altered microvascular patency, platelet aggregation, or increased numbers of leukocytes) of Saran Wrap on these preparations or changes in the Raman spectra.

Each animal was positioned over the stage of the microscope, and the mesenteric tissue image was projected onto a television camera (DC-330; DAGE-MTI, Michigan City, IN) connected to a videotape recorder (AG-7350; Panasonic, Osaka, Japan), a video timer, and a video monitor (SSM 175A; Sony, Tokyo, Japan). Each field was recorded under transmitted light before and after each HbO2Sat determination. Vessel diameter was measured from the digitized video images at a resolution of 0.12 µm/pixel. Red blood cell velocity was determined online using an optical Doppler velocimeter (Texas A&M University, College Station, TX). This is a well-established method that has been used successfully in several previous studies (4, 13). The method gives an average red blood cell velocity that can be digitally displayed on a meter and allows for calculation of microvessel blood flow. The temperature of the area under study was periodically measured using a thermocouple (YSI, Dayton, OH). Total Hb concentration, methemoglobin, and HbO2Sat were measured as described above in blood samples obtained from the femoral artery.

To obtain a range of HbO2Sat levels, we evaluated arterioles, capillaries, and venules in situ under control conditions and after the rat was subjected to stepwise hemorrhage until death. Hemorrhagic hypotension was induced by withdrawing blood from the carotid artery using a heparinized syringe. Blood pressure was simultaneously recorded using a data acquisition system (Biopac Systems, Goleta, CA). The only method for microvascular HbO2Sat measurements that could be used as an in vivo reference in our preparation was the microspectrophotometric Hb absorption method (also known as the 3-wavelength method) of Pittman and Duling (30, 31). Therefore, we compared the values of HbO2Sat obtained using RR spectroscopy with those obtained using the three-wavelength method in arterioles and venules. A digital video-based version of this method using a high-resolution digital charge-coupled device camera (CoolSnap cf; Roper Scientific, Trenton, NJ) was implemented to calculate HbO2Sat using standard digital video image analysis. Wavelengths in the green region of the spectrum were used for microvessels up to 100 µm in diameter. Raman spectra and digital images were captured in the dark to eliminate noise caused by stray light. For each microvessel, one to two Raman spectra and four snap shots (for 3-wavelength evaluation) were recorded sequentially but in random order. During HbO2Sat measurements, the tissue was exposed to only one light source at a time. The tested laser excitation spot sizes were in the range of 10–100 µm in diameter, but the RR data reported were obtained using an epi-illuminated area of ~30 µm in diameter (relative to the actual microscopic field) that was placed in the interstitium (close to the vessel wall) or in the center of the vessel under study.

Analysis

Each RR spectrum was the cumulative signal of one to two exposures, each one 20–30 s in duration. Each spectrum was processed for removal of cosmic ray spikes and normalized to the same baseline level by subtracting each spectrum from the average intensity obtained from ranges of the spectrum where no Raman peaks were present. No further processing of the RR spectra was performed, and the RR spectra are presented without smoothing.

As a first step in the estimation of the HbO2Sat, the peak ratio (PR) of the intensities of the RR bands for oxygenated (Ioxy) and deoxygenated (Ideoxy) hemoglobin was computed using the formula:

Ideoxy and Ioxy were calculated using three different approaches: 1) absolute intensity at 1,360 cm–1 (Ideoxy) and at 1,375 cm–1 (Ioxy); 2) average intensity from 1,355 to 1,365 cm–1 (for Ideoxy) and from 1,370 to 1,380 cm–1 (for Ioxy); and 3) integral area over each of the two ranges: 1,355–1,365 cm–1 and 1,370–1,380 cm–1. These spectral ranges were chosen so as to maximize signal-to-noise ratio and to minimize the contribution of the overlap between the two Hb Raman bands. Similar approaches (using ranges) have been employed to estimate tissue water content from Raman signal intensities in the human lens (24), cornea (3), and skin (10).

HbO2Sat was then estimated (in %) using the PR, according to the formula:

The coefficients a (1.280 ± 0.018) and b (–6.390 ± 0.756) were obtained from calibration experiments in which HbO2Sat was independently measured using a well-established device, a CO oximeter adjusted for the animal's Hb absorption spectra (OSM3; Radiometer).

The spectra were stored and analyzed as described above with commercially available software (WinSpec software; Roper Scientific) and with customized software specifically developed to streamline processing of Raman spectra and estimate HbO2Sat.

Statistics

Values are reported as means ± SE. Differences among the HbO2Sat values estimated under different conditions (different laser spot sizes, different glass capillary path lengths) were analyzed using analysis of variance. For correlation analysis, linear least-squares regressions were performed, and significance of the correlation coefficients was tested. The statistical tests were performed using commercial computer software (OriginLab Origin 7 and Microsoft Excel 2002). All P values correspond to two-tailed tests with significance set at 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In Vitro Measurements

Raman spectra were obtained with rat Hb solutions, as well as with rat and pig blood, equilibrated with known O2 tensions. When the HbO2Sat (measured by the OSM3 oximeter) was <1% or >98%, a single RR peak was observed. For intermediate levels of HbO2Sat, the spectra showed two clearly defined Hb peaks in the 1,350–1,400 cm–1 range. Each RR peak was always in the same position within the spectrum (±2 cm–1) independent of the source of Hb. Figure 2 shows RR spectra of rat blood in vitro. With the HbO2Sat kept constant, similar Raman spectra were obtained from solutions with the use of glass capillary tubes with different path lengths (50–800 µm) and using different laser excitation areas (15–150 µm).



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  		Fig. 2. Resonance Raman spectra obtained in vitro with blood equilibrated with different oxygen tensions. Each spectrum is positioned in the HbO2Sat axis according to the HbO2Sat value independently measured using an oximeter. The spectra are presented without smoothing. Intensity is expressed in arbitrary units (A.U.).

 
In vitro calibration was performed using blood with different Hb concentrations (range: 5–13 g/dl) equilibrated with known O2 tensions. All approaches used to calculate Ideoxy and Ioxy (peak intensity, average intensity, and area) resulted in significant correlations (r > 0.9, n = 42, P < 0.01) between HbO2Sat values measured with the oximeter (the standard of reference) and HbO2Sat values estimated from Raman spectra. Figure 3 presents data for the PR of the intensities of the hemoglobin RR spectra measured at different HbO2Sat values (range: 5–99%, measured using an oximeter). Successive determinations of HbO2Sat in a given area of constant oxygen level provided results varying ±1–4%. The detection of all physiological HbO2Sat levels was possible. The fraction of methemoglobin was always <3%.



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  		Fig. 3. In vitro calibration of the resonance Raman intravital microscope system performed with blood at different dilutions with saline, resulting in different hemoglobin concentrations ([Hb]). Each point represents 1 measurement at a given Hb O2 saturation. Because no differences were found among different [Hb], data using all [Hb] were pooled for least-squares regression line calculation. Peak ratio (PR) values were measured from Raman spectra by using the peak intensities of oxy- and deoxyhemoglobin.

 
In Vivo Measurements

Raman emission was detected only when the laser excitation spot was positioned over a microvessel. The HbO2Sat values estimated from spectra obtained in vivo using different laser excitation sizes were not significantly different from each other (data not shown). In practice, the excitation spot used was 30 µm in diameter, which gave an adequate signal-to-noise ratio to measure HbO2Sat in the whole range of microvessels. We did not observe any adverse reactions of the animals to the laser radiation, either acutely or after 4–6 h of experimentation. Neither arteriolar vasoconstriction nor increased venular leukocyte adhesion was observed when the same tissue area was illuminated with the laser for uninterrupted periods of up to 5 min.

Intravascular HbO2Sat was estimated from RR spectra in mesenteric arterioles (n = 15) and venules (n = 25) from anesthetized rats breathing 100% O2 under normal conditions and after hemorrhage. Under control conditions, arteriolar (mean diameter: 20.3 ± 7.1 µm, range: 11–31 µm) and venular (mean diameter: 28.3 ± 9.8 µm, range: 19–60 µm) HbO2Sat averaged 83.4 ± 7.1 and 70.0 ± 12.8%, respectively. Figure 4 shows RR spectra obtained from a microvessel before and during hemorrhagic hypotension. Under these conditions, a substantial reduction in perfusion is observed throughout the mesenteric network. Similar recordings were obtained from venules located in the mesentery and in the serosal surface of the exposed ileum.



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  		Fig. 4. Examples of Raman spectra obtained in vivo before (control) and during hemorrhagic hypotension. The extravascular spectrum was collected by aiming the laser at the interstitial space next to the microvessel wall. All other spectra were obtained from an arteriole in the rat mesentery preparation. The mean arterial pressure (MAP) and the estimated HbO2Sat determined using Raman spectroscopy also are shown. Exposure, 20 s; laser excitation spot diameter, 30 µm; objective, x40, numerical aperture 0.80. Note that the strongest resonance Raman Hb peaks at 1,358 cm–1 (deoxyhemoglobin) and at 1,376 cm–1 (oxyhemoglobin) change in intensity as MAP (and microvessel flow) changes. Although all tracings start at the same baseline, each tracing has been offset upwardly to improve clarity and is presented without smoothing. Intensity is expressed in arbitrary units (a.u.).

 
When HbO2Sat values estimated from RR spectra were compared with HbO2Sat values measured using the three-wavelength method in the same microvessels (Fig. 5), a good correlation was found (r = 0.74, P < 0.001). The mean differences between HbO2Sat values estimated from RR spectra and HbO2Sat values measured using the three-wavelength method for arterioles and venules were 0.2 ± 11.5 and 0.4 ± 10.1%, respectively. The fraction of methemoglobin was always <2%.



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  		Fig. 5. In vivo values of HbO2Sat in arterioles and venules estimated from RR spectra and measured using the three-wavelength method in the same microvessels. The illustrated data are from a preparation of exteriorized mesentery of anesthetized rat. Each point represents a single HbO2Sat determination (Raman and 3-wavelength) in 1 microvessel. The least-squares regression line was calculated considering all measurements.

 
Because extravascular signals did not seem to interfere with RR signals from intravascular Hb, it was not critical to keep the laser excitation exclusively over the vessel under study. In other words, the same estimated HbO2Sat value was obtained, for instance, when a 30-µm-diameter laser spot was centered over a 15-µm-diameter vessel and when the laser spot was reduced to 15 µm. On several occasions, it was possible to observe the tissue (using transillumination) during the period of measurement, to check for movements of the preparation as potential sources of error. The position of the laser excitation spot was always checked before and after each measurement, and the measurement was repeated in case the two positions were different. The laser was the only light source to which the tissue was exposed during the HbO2Sat measurements reported using RR signals. However, we found that the use of transillumination concomitantly with the laser excitation and signal collection increased the background noise level but did not alter the RR hemoglobin peaks.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Raman Intravital Microscope

The results show, for the first time, the application of RR spectroscopy to evaluate dynamic changes in oxygenation of individual microvessels under physiological conditions. The system was implemented to allow the acquisition of RR spectra and other microcirculatory variables in vivo while systemic parameters (such as blood pressure) are simultaneously recorded. Although several Raman microscope systems are commercially available, they do not allow a complete evaluation of other physiological parameters in vivo. The system described is integrated and versatile, allowing digital imaging at a wide range of sensitivity levels and measurements of several microcirculatory parameters. The phosphorescence quenching method for PO2 determinations (44) can be added to the system.

We used a Raman excitation line within the Soret band, which causes a strong resonance enhancement. RR spectroscopy is suitable for the detection of strongly absorbing materials. Importantly, previous studies demonstrated that Hb spectra from red blood cells did not differ from purified Hb spectra (6). This is advantageous, because it allows the study of Hb in its natural environment, allowing chemical characteristics of descriptions obtained from the previous spectroscopic studies of the Hb molecule to be carried over to in vivo studies. In this regard, it is important to note that the hemoglobin RR peaks observed in flowing blood with the use of intravital microscopy are identical to those reported in previous in vitro studies (5).

In Vitro Measurements

Raman spectra obtained with Hb solutions, as well as with blood, showed two clearly defined Hb peaks in the range of 1,350–1,400 cm–1. The position of these Hb RR bands in this range is within the values previously published (5). One peak is at maximum when Hb is fully deoxygenated, and the other one is at maximum when Hb is fully oxygenated. At intermediate oxygenation states, we have made the assumption that the peak heights are proportional to the concentrations of deoxyhemoglobin and oxyhemoglobin, respectively. The technique and the assumptions were tested in the ranges of 5–13 g/dl of Hb concentration and of 5–99% HbO2Sat. We have demonstrated empirically that the technique can be employed over a wide range of physiologically relevant Hb concentrations, microvessel diameters, and HbO2Sat (Figs. 3 and 5). In vitro calibration showed an excellent correlation between HbO2Sat values measured with the OSM3 oximeter and those estimated from RR spectra. The O2 saturation of a sample was related to the ratio of peak heights, thereby using one of the peaks as an internal reference for the sample. Therefore, HbO2Sat estimation was not affected by changes in overall intensity of the Raman signal.

Raman spectra were not affected by the path length of the glass capillary or different laser excitation areas (except for changes in overall Raman band intensity). Successive determinations of HbO2Sat (from RR signals) showed good reproducibility.

In Vivo Measurements

Previous Raman spectroscopic investigations were performed on single red blood cells (48–50). In some studies, the cells were immobilized by methanol fixation on a glass slide in air (27) and adsorbed on polylysine-coated glass surfaces (33). In addition, Raman spectra have been obtained from individual optically trapped cells by using different excitation lines (34). Those studies demonstrated the potential for Raman spectroscopy in the study of red blood cell disorders such as thalassemia, sickle cell disease, and malaria. Our approach was directed to an in vivo evaluation of blood oxygenation states in microvessels.

Intravascular HbO2Sat values measured in mesenteric arterioles and venules from anesthetized rats under normal conditions and after hemorrhage were comparable to those measured in the same species using the three-wavelength method. A good correlation was found when HbO2Sat values estimated from Raman spectra were compared with HbO2Sat values measured using the three-wavelength method in the same microvessels. An excitation laser spot of 30 µm in diameter gave an adequate signal-to-noise ratio to measure HbO2Sat in the whole range of microvessels in vivo. The measurement of all physiological HbO2Sat levels was possible.

Limitations and Applications

Previous studies on isolated red blood cells have shown that the type of glass coating and the laser irradiation had an important influence on the Raman spectra (33, 34). Cells may lose their intrinsic shape as result of substrate-induced effects and photo-induced effects when Raman spectroscopy is used (32, 34). However, these investigations pointed out that low accumulated photon dose and proper sample preparation allowed RR spectroscopic studies of single red blood cells. In our studies, these artifacts were further avoided by using very low laser energy (0.3 mW) and by studying flowing red blood cells in microvessels. To further minimize light-related tissue reactions, we restricted illumination to periods of <60 s. Our observations indicate that no photo-induced effects were present in the studied microvessels.

With the use of the current laser power (<0.5 mW at the preparation) and 30-s exposures, the minimum excitation spot diameter that provided reliable data was ~15 µm. Smaller sizes could be used in situations where longer collection times are possible to increase the signal-to-noise ratio. An improved signal-to-noise ratio also could be achieved by averaging a larger number of spectra. However, such a procedure would lead to a significant increase in the time of laser exposure of the tissue, increasing the possibility of photo damage (26). Another approach is the use of objectives with increased light-gathering power. Our preliminary measurements confirm that a superior lens (Zeiss x40, NA 1.3) delivers a considerably better RR signal. Other techniques of signal processing also would enhance the capabilities of the system. All in all, we anticipate that further refinements of the system will allow the acquisition times (and therefore the speed of HbO2Sat determinations) to be significantly reduced, possibly to a few seconds.

A potential problem with the use of intravital Raman spectroscopy is that RR signals generated in tissue compartments other than blood could interfere with the analysis of the Hb. This problem was approached by considering only the characteristic and strong Raman Hb bands. Therefore, it was easy to distinguish the spectrum of Hb from any other Hb-free compartment. In fact, intravascular areas filled with plasma studied in vivo never showed the Raman bands, whereas these bands could be observed when the excitation spot was placed over extravasated erythrocytes located in the fluid above avascular areas. The contribution from areas below the vessel under study is probably small, because the signal coming from those regions is strongly absorbed by the intravascular blood column. Considering the extinction coefficient of Hb at the excitation wavelength used in our experiments (406.7 nm), it is unlikely that the penetration exceeded the top 20–30 µm of a vessel (46). Moreover, because of the excitation wavelength and the high numerical aperture (0.80) of the objective used, the depth of field in our optical system was <2 µm, which resulted in a significant decrease in excitation and emission from regions outside the focal plane. However, in the current configuration some spatial averaging does occur, and the system may not allow the detection of small HbO2Sat gradients within a blood vessel. In addition, HbO2Sat should represent the temporal average during the measurement period, because 20–60 s are required to obtain a spectrum and the HbO2Sat in the sampled region could vary during this interval.

In vivo, the ability to determine HbO2Sat in given microvessels will depend on the amount of tissue present in the light path, and if this layer is too thick, the optical signal may be too weak to be detected by the system. However, because most preparations used in microvascular research rely on the direct visualization of the microvessels, the thickness of this layer is minimal, the corresponding absorption is minimal, and the technique should be applicable in most circumstances. In this regard, the methodology described extends the range of applicability of the RR spectroscopy technique and allows HbO2Sat determinations in vivo and in situ. RR spectroscopy may be used to study individual red blood cell behavior in living microvessels or overall tissue oxygenation when larger areas, comprising many microvessels, are evaluated. Therefore, the method allows oxygenation analysis at the tissue level or in a single red blood cell. In addition, this implementation allows the measurement of HbO2Sat in solid organs. The possibility of measuring important hemodynamic (e.g., microvascular blood flow) and oxygenation (PO2) variables also increases the ability to more accurately assess O2 transport in living systems.

In principle, RR spectroscopy can be used to determine HbO2Sat in any tissue surface, such as the human skin. Previous studies already have shown the suitability of Raman spectroscopy in human skin and eye (10, 11, 18–20, 23, 25). For transcutaneous measurements, both elastic and Raman scattering from the skin are potential concerns. Elastic scattering limits the photon penetration depth while increasing the Raman spot size. A consequence may be an increase in the error of estimating HbO2Sat. Thus understanding Raman scattering in complex environments, such as the blood-skin matrix, is critical for noninvasive, transcutaneous blood analysis using Raman spectroscopy. Implementing alternative collection geometries also may be important to allow measurements in very heterogeneous tissues. Further research is required to make this technique practical in clinical laboratories and to develop transcutaneous HbO2Sat-monitoring schemes.

In summary, resonance Raman spectroscopy was successfully employed to measure intravascular Hb O2 saturation in the flowing blood of microvessels noninvasively. In vivo comparisons between the Raman method and three-wavelength densitometry showed that the accuracy of the measurements at the lower extreme of saturation is questionable, suggesting that further refinement of the method may be necessary for measurements of Hb saturation levels in vivo below <10%. The system was implemented in the same optical system used for red blood cell velocity and microvessel diameter measurements, providing noninvasive measurements from all vessels in the microvascular network. This methodology can be used to study Hb O2 saturation in thin tissues (such as mesentery and small skeletal muscles), as well as in solid organs and tissues such as liver, brain, and tumors, which are unsuitable for techniques that require transillumination.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by Office of Naval Research Grants N00014-02-10344 and N00014-02-10642.


   ACKNOWLEDGMENTS
 
We thank R. Wayne Barbee, Brian Berger, and Luciana N. Torres for help during various phases of the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. P. Torres Filho, Dept. of Anesthesiology, Virginia Commonwealth Univ., 1101 E. Marshall St., Rm. B1-012, PO Box 980695, Richmond, VA 23298-0695 (E-mail: itorres{at}vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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