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The short- and long-term effects of probe contact pressure on in vivo diffuse reflectance and fluorescence spectroscopy were investigated using an animal model. Elevation in probe contact pressure induced major profile alterations in the diffuse reflectance spectra between 400 and 650 nm, and led to significant intensity increases in the fluorescence spectra. The pressure threshold that was required to induce statistically significant spectral alterations was dependent upon the type of tissue. The observed spectral alterations may be attributed to decreases in local blood volume, blood oxygenation, and tissue metabolism, resulting from high probe contact pressure.
©2008 Optical Society of America
1. Introduction
Interactions between light and tissue are governed by the morphological, biochemical, and physiological characteristics of tissue. Based upon this principle, many studies have been conducted over the past two decades to explore the feasibility of developing optical diagnostic tools for cancer and tissue-injury detection, and many successful results have been achieved to date [1–11]. In the majority of the reported studies on optical diagnosis, a fiber-optic probe is used in conjunction with a spectroscopy system to remotely acquire optical spectra. During a spectral acquisition procedure, the fiber-optic probe is kept in contact with the tissue site to be investigated, in order to avoid spectral artifacts induced by mismatches in refractive index and inconsistent illumination-detection geometry [12, 13]. The contact nature of a fiber-optic probe inevitably introduces a certain amount of pressure (probe contact pressure) on the local tissue, which, in turn, could affect the intrinsic characteristics of the tissue.
The pressure effects on tissue optics and in vivo optical spectroscopy have been reported in a few studies [14–19]. For example, Chan, et al., reported decreased reflectance and increased transmittance from in vitro tissue under compression [14]. In an in vivo human study, Nath, et al., and Rivoire, et al., found that probe contact pressure had little to no effect on fluorescence spectra measured from cervical tissue [17, 18]. Shim, et al., showed that varying the pressure of the probe tip on tissue does not induce significant alterations in the Raman spectra acquired from human gastrointestinal tissues in vivo [19]. While some insights have been gained through these studies, a systematic evaluation of the effects of probe contact pressure on in vivo diffuse reflectance and fluorescence spectroscopy remains lacking. With this in mind, the objectives of the currently reported study were (1) to quantify the short- and long-term effects of various probe contact pressures on diffuse reflectance and fluorescence spectra in vivo; and (2) to determine the pressure threshold required to induce such effects using an animal study. The heart and the liver were selected as the target organs, because of the research interests of our group.
2. Material and methods
2.1 Animal preparation
The study protocol described below was reviewed and approved by the Institutional Animal Care and Use Committee at Florida International University. Six male Sprague-Dawley rats were purchased from the Charles River Laboratory (Wilmington, MA). The weights of these rats varied between 250 and 400 grams. Prior to each experiment, one rat was anesthetized using pentobarbital (50 mg/Kg, IM). Once sedated, a tracheotomy procedure was performed and a respirator connected to the rat through a trachea tube. The rat’s breathing rate and volume of air intake were controlled at 30 breaths per minute and 5 cc, respectively. A steady supply of 2–3% isoflorane/oxygen mixture was provided to the rat, so as to maintain an adequate level of anesthesia throughout the study. Upon completing the initial anesthetic and surgical routine, a mid-line sternotomy was performed to expose the heart and the liver of the rat, so that the pressure effect study could be conducted. At the end of each experiment, each rat was euthanized by injecting saturated sodium chloride solution directly into the heart.
2.2 Instrumentation
A fiber-optic spectroscopy system was used in this study to acquire in vivo diffuse reflectance and fluorescence spectra from the heart and the liver in a continuous fashion (Fig. 1). Two light sources were employed in this spectroscopy system: (1) a tungsten-halogen light (LS-1, Ocean Optics, Dunedin, FL) for diffuse reflectance spectroscopy; and (2) a 337-nm nitrogen laser (VLS-337, Spectra-Physics, Mountain View, CA) for fluorescence spectroscopy. A portable spectrometer with a detection range of 250 to 932 nm and spectral resolution of 5 nm (USB 2000, Ocean Optics, Dunedin, FL) was used to record reflected light. Two 380-nm, long-pass filters were installed at the entrance port of the spectrometer, to remove 337 nm excitation in the fluorescence spectra. The spectrometer and light sources were controlled by a means of a portable computer.
Excitation/emission light conduction was achieved using a fiber-optic probe that had been fabricated locally. The probe contained three optical fibers: two 200-µm core fibers for excitation, and one 400-µm core fiber for emission. All three fibers were enclosed in a stainless steel tube with a 1.3-mm outer diameter. To avoid inducing any tissue damage with the edge of the stainless steel tube, a black, 3-mm diameter, round bead was installed at the tip of the probe. The fiber-optic probe was held by a mechanical arm. During a spectral acquisition procedure, the optical probe was placed perpendicularly to the tissue surface and allowed to move freely only in the vertical plane. Due to gravity, the tip of the probe remained in contact with the tissue site being investigated.
2.3 Data acquisition
With the probe in contact with the tissue, fluorescence and diffuse reflectance spectra were acquired sequentially from the tissue at a rate of ~ 0.1 Hz for two minutes, which yielded a total of 12 spectra sets that were designated the normal control series. Since the probe itself induced sufficient contact pressure to alter the diffuse reflectance and fluorescence spectra from the liver tissue, the normal control series for the liver tissue was acquired with the tip of the probe barely touching the tissue surface (i.e., ‘0 Weight’ in Table 1). To change the probe contact pressure, a calibrated weight, either one, two or three grams, was added to the body of the probe, and the spectral acquisition was performed for two minutes (the pressure series). The duration of the data acquisition in the pressure series was limited to two minutes because no significant spectral alterations were observed beyond 90 seconds of recording. Upon removing the calibrated weight, spectral acquisition was performed for an additional two minutes at the original pressure level (the recovery series) to investigate any permanent effects, such as tissue damage, induced by the contact pressure of the probe. Here a one-second integration time was used, both for fluorescence and diffuse reflectance spectral acquisition. The resulting spectral S/N was greater than 5. A total of six unique sites (i.e., nsite = 6) were used for each studied pressure level.
2.4 Data analysis
All acquired spectra were smoothed using a Savitzky-Golay filter to eliminate high frequency noise originated from the spectrometer. The filtered spectra Iraw(λ) then were processed to remove those spectral alterations induced by the ambient light B(λ) and intrinsic instrumentation characteristics C(λ). That is,
where I(λ) represents a calibrated spectrum. The derivation of C(λ), also known as the calibration factor, can be found in a previous publication [20]. In brief, the calibration factors for fluorescence spectra, Cf(λ), compensated for the spectral alterations induced by the transmissitivities of the collection fiber and the laser light filters, as well as the responsivity of the spectrometer. Experimentally, Cf(λ) was obtained by comparing the true emission spectrum of a calibrated tungsten light (LS-1-CAL, Ocean Optics Inc, Dunedin, FL) with the one measured by means of the table-top spectroscopic system. The calibration factors for diffuse reflectance spectra, Crd(λ), accounted for all the instrumentation-induced spectral alterations stated above, as well as for those caused by the emissivity of the white light source (Ewhite(λ)). Here, Ewhite(λ) was obtained by measuring the emission spectrum of the white light source, using a calibrated spectrometer. The calibrated spectra was further downsampled to a 5 nm interval, and the spectral ranges limited to 380 nm – 750 nm and 400 nm – 900 nm for fluorescence spectra and diffuse reflectance spectra, respectively.
Table 1:. Weights and pressures applied by the optical probe (d = 1.3 mm) in the study.
Two spectral analysis procedures were conducted on the calibrated spectra, so as to identify and quantify those spectral alterations induced by probe contact pressure. The first procedure focused on detecting the changes in spectral profile and intensity induced by various probe contact pressures within the first 30 seconds of applying the pressure (i.e., short-term effects). In other words, the average of just the first three spectra acquired in a spectral series was used in the analysis. To minimize the effects of biological variations on the absolute quantities of spectral alterations, all average spectra were further normalized to the maximum spectral intensities of their corresponding normal controls. Identification of spectral alterations under various probe contact pressures was performed initially using empirical methods. The statistical significance (p<0.05) of the identified spectral alterations was determined using one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls (SNK) test for post-hoc analysis.
The second type of analysis focused on evaluating the time histories (i.e., long-term effects) of the spectral alterations identified above at various probe contact pressures. The full-spectral normalization (i.e., wavelength to wavelength) method was utilized in this analysis: the spectra recorded in a given pressure series were normalized to their corresponding average normal control. Trends in the selected spectral alterations were evaluated empirically, and their significance (p<0.01) was determined by comparing the start and end points using a paired t-test.
3. Results
3.1 Short-term probe contact pressure effect
Several prominent alterations were observed in the spectral data from the liver tissue, when probe contact pressures exceeded 2.58 N/cm2 (i.e., probe itself). In the diffuse reflectance spectra (Fig. 2(a)), significant increases in intensities between 450 and 600 nm and the transition of a double-valley profile into a single-valley between 500 and 600 nm were noticed. In the fluorescence spectra, a marked increase in the overall intensity was found (Fig. 2(b)). In addition, a new shoulder appeared between 400 and 430 nm, and the shoulder around 600 nm vanished in the fluorescence spectra as the pressure of the contact probe increased. Similar pressure-induced spectral alterations were observed in the data set from the heart (Fig. 3). However, the minimum pressure required to induce such changes was 4.80 N/cm2 (i.e., probe +3 g) for heart tissue.
Fig. 2. Average in vivo (a) diffuse reflectance and (b) fluorescence spectra from liver tissue recorded at various probe contact pressures.
Fig. 3. Average in vivo (a) diffuse reflectance and (b) fluorescence spectra from heart tissue recorded at various probe contact pressures.
To study the correlation between the spectral alterations mentioned above and the probe contact pressures, six quantitative spectral characteristics were selected. They were (1) normalized diffuse reflectance intensity at 480 nm (Rd(480)); (2) normalized diffuse reflectance intensity at 800 nm (Rd(800)); (3) the ratio of the normalized diffuse reflectance intensity at 575 nm and at 565 nm (Rd(575)/Rd(565)); (4) normalized fluorescence intensity at 470 nm (F(470)); (5) normalized fluorescence intensity at 410 nm (F(410)); and (6) the ratio of the normalized fluorescence intensity at 470 nm and at 600 nm (F(470)/F(600)). Note that the indicator Rd(575)/Rd(565) was used to detect change in hemoglobin oxygenation, the indicator F(470)/F(600) to detect the metabolic state. Statistical comparisons of these spectral characteristics at various probe contact pressures are presented in Figs. 4 and 5.
According to the ANOVA and SNK post-hoc analysis, most of the spectral characteristics from the liver tissue at zero contact pressure were significantly lower than those measured at the other probe contact pressures (Fig. 4). The only exception was Rd(800). Upon inspecting all recorded diffuse reflectance spectra, it was found that diffuse reflectance intensities beyond 650 nm did not fluctuate significantly at any probe contact pressure. The magnitudes of a given spectral characteristic at the pressure equal to or greater than 2.58 N/cm2 (i.e., probe itself) were similar, and no statistically significant differences were detected. The outcomes of this analysis suggest that the minimum probe contact pressure, Pmin, required to alter diffuse reflectance and fluorescence spectra from the liver tissue was less 2.58 N/cm2.
Based upon the results of the ANOVA and SNK post-hoc analyses, it was found that all spectral characteristics of the heart tissue measured at the highest probe contact pressure, except for Rd(800), were significantly greater than those measured at the other pressures (Fig. 5). In fact, no significant changes in diffuse reflectance intensities beyond 650 nm were detected at any pressure. The magnitudes of each given spectral characteristic at pressures equal to or less than 4.06 N/cm2 (i.e., probe+2 g) also were similar. Therefore, the Pmin of the heart tissue was believed to be close to 4.06 N/cm2, which is much higher than that identified for liver.
3.2 Long-term probe contact pressure effect
The time histories of the quantitative spectral characteristics of the liver tissue at all probe contact pressures are shown in Fig. 6. The discrepancy among the starting points of the time histories of a given spectral characteristic at various probe contact pressures is apparent in Fig. 6; this phenomenon was attributed the instantaneous effect of applied probe contact pressure on both the fluorescence and diffuse reflectance characteristics of the liver tissue. At zero probe contact pressure, all spectral characteristics remained constant, which indicated stable physiological conditions of the tissue. At non-zero probe contact pressures, Rd(480) and Rd(575)/Rd(565) exhibited little to no changes during the entire recording period. In contrast, Rd(800) revealed a slightly decreasing trend at probe contact pressures of 4.06 N/cm2 (probe+2 g) and 4.80 N/cm2 (probe+3 g). Noticeable increasing trends were seen in the time histories of F(410) and F(470) at non-zero probe contact pressures. The overall increases in F(410) and F(470) during the two-minute recording period were greater than 20%. F(470)/F(600) exhibited a decreasing trend and reached a plateau during the recording period. However, the decrease in F(470)/F(600) only was significant at probe contact pressures of 2.58 N/cm2 (probe) and 3.32 N/cm2 (probe+1 g).
The time-history analysis results of the data from the heart tissue are shown in Fig. 7. Similar to the data sets from the liver tissue, decreasing trends were observed in the time histories of Rd(480), Rd(800), Rd(575)/Rd(565), and F(470)/F(600). However, most of the decreasing trends were not statistically significant. Opposite to what was observed in the data set from the liver tissue, in heart tissue, significant decreasing trends were found in F(410) and F(470). This especially was true in the time histories of F(470), where a statistically significant decrease was observed at all probe contact pressures over the recording time period.
All the spectral alterations mentioned above disappeared once the probe contact pressure was reduced to the original minimal level. When spectral data from the control and recovery series were compared, no statistically significant differences were identified in either the normalized spectral intensities or the spectral profiles. This suggested that the pressure induced effects on in vivo optical spectroscopy were transient at the probe contact pressures evaluated in this study. In other words, the probe contact pressure introduced did not cause any permanent damage in the tissue.
Fig. 4. Quantitative comparisons of (a) diffuse reflectance characteristics, including Rd(480), Rd(800), and Rd(575)/Rd(565), and (b) fluorescence characteristics including F(410), F(470), and F(470)/F(600) of liver tissue at various probe contact pressures. The bars and error bars represent mean and standard deviation, respectively, for each spectral characteristic (nsite = 6). % indicates that the mean spectral characteristic at a particular probe contact pressure is significantly different (p<0.05) from those at other probe contact pressures.
Fig. 5. Quantitative comparisons of (a) diffuse reflectance characteristics including Rd(480), Rd(800), and Rd(575)/Rd(565), and (b) fluorescence characteristics including F(410), F(470), and F(470)/F(600) of heart tissue at various probe contact pressures. The bars and error bars represent the mean and the standard deviation, respectively, for each spectral characteristic (nsite = 6). % indicates that the mean spectral characteristic at this particular probe contact pressure is significantly different (p<0.05) from those at other probe contact pressures.
Fig. 6. Comparisons of the average time histories of (a) Rd(480), (b) Rd(800), (c) Rd(575)/Rd(565), (d) F(410), (e) F(470), and (f) F(470)/F(600) for liver tissue at various probe contact pressures (nsite = 6). A heavy line indicates that the initial and final values for the spectral characteristic are significantly different (p<0.05). The error bars represent the standard deviation of the measurements and they are shown only in the data series with the lowest and highest probe contact pressures.
Fig. 7. Comparisons of the average time histories of (a) Rd(480), (b) Rd(800), (c) Rd(575)/Rd(565), (d) F(410), (e) F(470), and (f) F(470)/F(600) for heart tissue at various probe contact pressures (nsite = 6). A heavy line indicates that the initial and final values for the spectral characteristic are significantly different (p<0.05). The error bars represent the standard deviation of the measurements and they are shown only in the data series with the lowest and highest probe contact pressures.
4. Discussion
The results of this animal study show that probe contact pressure, when exceeding a certain threshold, can produce a significant impact on diffuse reflectance and fluorescence spectra acquired from in vivo tissue. For diffuse reflectance spectra, the primary pressure-induced changes are seen in their profiles. Among those changes reported here (Figs. 2 to 5), the transition of the double-valley feature to a single valley between 500 nm and 600 nm, indicated by Rd(575)/Rd(565), is the most prominent. It is known that the profile characteristics of an in vivo diffuse reflectance spectrum are governed by the absorption properties of the tissue, and the dominant tissue chromophore is hemoglobin in the liver and hemoglobin/myoglobin in the heart. Since oxy-hemoglobin (oxy-myoglobin) possesses two absorption peaks between 500 nm and 600 nm and deoxy-hemoglobin (deoxy-myoglobin) possesses just one [21–24], the profile change between 500 nm and 600 nm in the diffuse reflectance spectra must originate from the conversion of oxy-hemoglobin into deoxy-hemoglobin within the local tissue.
However, the reported spectral alterations in diffuse reflectance spectra cannot be explained entirely by the oxy-hemoglobin to deoxy-hemoglobin conversion; significant increases in diffuse reflectance intensities at the hemoglobin isosbestic points (e.g., 548 nm and 568 nm) were observed at high probe contact pressures. This alteration may be attributed to a decrease in tissue absorption properties. In other words, high probe contact pressure would lead to a decrease in local blood volume, a typical response of tissue under focal pressure. Therefore, the potential impact of probe contact pressure on the hemodynamics of local tissue should be considered when acquiring diffuse reflectance spectra from in vivo tissues, especially when the tissues are highly vascular.
Chan, et al., reported that the compression of tissue could lead to a decrease in the diffuse reflectance of in vitro tissue [14]. Such effects, however, were not clearly identified by the study; no statistically significant changes in diffuse reflectance spectra beyond 650 nm were observed in the study presented at any of the pressures used. This phenomenon suggests that the magnitude of the probe pressure applied was not high enough to alter the structural characteristics and, hence, the intrinsic optical properties of the tissue. Since biological tissue with high water content often is considered incompressible, focal pressure induced by an optical probe is likely to deform, rather than to compress, in vivo tissue.
The impacts of probe contact pressure on fluorescence spectroscopy are more noticeable in spectral intensity than spectral profile. As shown in the Results section, the increase in fluorescence intensity could be many-fold, depending upon the magnitude of the probe contact pressure. There are several possible explanations for the increases in fluorescence intensity. The acquisition of fluorescence spectra from in vivo tissue is achieved by three distinct processes: (1) the propagation of excitation light from the source to the fluorophores; (2) the generation of emission light by the fluorophores; and (3) the propagation of the emission light from the source (fluorophores) to the tissue surface [25]. Consequently, the intensities of fluorescence emissions can be modulated by both tissue absorption properties and local fluorophore concentration. As discussed above, an increase in probe contact pressure creates a local ischemic condition in which blood volume and blood oxygenation decrease, and tissue absorption properties are reduced. The distribution of 337 nm excitation light is influenced much less by blood oxygenation, because this wavelength is very close to the 340-nm hemoglobin isosbestic point. However, a decrease in local blood volume could reduce the amount of attenuation of both excitation and emission light during the propagation process, which leads to an increase in detected overall fluorescence emission.
The increase in fluorescence emission also might be attributed to changes in local fluorophore quantities. At 337-nm excitation, the major contributors of tissue fluorescence include collagen, NADH, and FAD, and their peak emissions occur at 400 nm, 460 nm and 525 nm, respectively [26]. The roles of NADH and FAD in the electron transport chain have been well described [27], and have been used as indicators of tissue metabolic activities [28–31]. As suggested by the alterations in diffuse reflectance, high probe contact pressure leads to local ischemia. This condition, in turn, slows down metabolic activity in tissue and creates an accumulation of free/bound NADH and FADH2 [32]. This provides a logical explanation for the increase in fluorescence emissions from an ischemic tissue in vivo. It also should be noted that FADH2 is a weaker fluorophore than FAD [33], so the contribution of FADH2 to the fluorescence emissions from the ischemic tissue should be less noticeable. This explains the increase in F(470)/F(600) observed at high probe contact pressures. Having said this, an accurate interpretation of the alterations in fluorescence emissions will require a complex model to separate the effects of tissue optics from those of the fluorophores [34, 35]. Nevertheless, the outcomes of the study clearly suggest that the effects of probe contact pressure on local hemodynamics are the primary cause of the alterations in both fluorescence and diffuse reflectance spectra.
While inducing similar spectral alterations, the minimum probe contact pressure required to induce such alterations is tissue-type dependent. As shown in Figs. 4 and 5, the pressure threshold required to induce statistically significant spectral alterations in liver tissue is much lower than that in heart tissue. This phenomenon may be secondary to the different mechanical and structural characteristics of the two tissues. As reported before, the mechanical strength of heart tissue, especially Young’s modulus, is much greater than that of liver tissue [36]. Therefore, it is easier to deform liver tissue, thereby obstructing regional blood flow, than it is to produce these effects in heart tissue. While not investigated in this study, other soft tissues, such as the brain, also are likely to be susceptible to the probe contact pressure effects reported. This could explain why deoxy-hemoglobin features are observed in some in vivo diffuse reflectance measurements obtained in the brain [37, 38].
While comparing the time histories of all selected spectral characteristics, it was noticed that F(410) and F(470) reached the plateau slower than other characteristic spectral alterations. This phenomenon may be explained by the delayed response of tissue metabolism to ischemia. While blood supply is reduced instantaneously, local oxygen level declines over time. Therefore, the slowdown in the metabolic rate lags behind other hemodynamic features. Contrary to what was expected, a descending trend was observed in the time histories of F(470) obtained from heart tissue. The descending trend of F(470) suggests a recovery of tissue metabolic activities during the recording period. This unique, time-dependent feature might be attributed to heart movement, which produces non-steady contact pressure, thereby modulating the degree of local ischemia.
As mentioned above, the effects of probe contact pressure on in vivo fluorescence spectroscopy have been investigated by Nath, et al. and Ravoire, et al. [17, 18]. They reported that, when probe contact pressure is maintained below 2.4 N/cm2, no significant alterations are observed in the in vivo fluorescence spectra acquired from cervical tissue. This finding is similar to what we have observed; minimal pressure is required to induce significant fluorescence and diffuse reflectance spectral alterations. However, it is not possible to determine the pressure threshold for cervical tissue using the results reported by Nath, et al. and Ravoire, et al. Because the effects of probe contact pressure on in vivo optical spectroscopy can be substantial, the level of probe contact pressure should be monitored closely during any in vivo spectral acquisition procedure. While it is possible to implement a pressure sensor into conventional optical probe design, it may be more convenient to use diffuse reflectance spectroscopy to gauge the pressure level, as the spectral alterations induced by a high level of probe contact pressure seem to originate from changes in local blood flow (i.e., focal ischemia).
Optical spectroscopy has been used to diagnose conditions like cancer in various tissue organs in vivo; hence, it is our intention is to extend the current study to various organs, including brain, to determine their corresponding maximum applicable contact pressure without inducing detectable artifacts in fluorescence or diffuse reflectance spectra. This knowledge, once established, should allow for the efficient acquisition of true in vivo fluorescence and diffuse reflectance spectra and, hence, improve the accuracy of in vivo optical diagnosis.
5. Conclusions
The effects of probe contact pressure on in vivo diffuse reflectance and fluorescence spectroscopy were investigated in an animal study. Diffuse reflectance and fluorescence spectra at 337-nm excitation were acquired continuously from the heart and the liver at various probe contact pressures. The results of the study show that excessive probe contact pressure disturbs the hemodynamics of local tissue, which, in turn, leads to the appearance of the spectral alterations corresponding to a decrease in blood oxygenation, blood volume, and, perhaps, tissue metabolism. The pressure threshold for inducing such spectral alterations is determined by tissue mechanical and structural characteristics. Consequently, the potential impact of probe contact pressure should be considered when performing in vivo diffuse reflectance and fluorescence spectral acquisition from soft tissues using a contact probe.
Acknowledgments
This work was supported by the American Heart Association (0655392B) and the Ware Foundation Research Endowment.
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