In this study, we investigated the influence of applying pressure to the probe, the angle between the probe tip and tissue surface, and temporal dependence on short contact durations. Firm pressure applied to the probe affects several parameters measured. At the deeper depth measured, the results are consistent with our expectations. As the tissue is compressed from firm pressure, the blood vessels are also compressed which forces some blood out of the area under the probe tip, as observed with a decrease in PLS and reduction in total Hb content. Yet, the superficial depth does not have the same trend. We hypothesize that the epithelial layer may have a different elasticity than the lamina propria and that applying firm pressure to the probe compresses the most superficial layer. This may bring some deeper vessels closer to the tissue surface, within the range of the average penetration depth measured by the delta-polarization signal and account for the observed increase in total Hb. We expected firm pressure to decrease oxygenation due to a reduction in fresh blood supply to the area. In this pressure study, however, a single measurement does not provide the sufficient time scale to observe these changes. This is supported in , which shows significant oxygenation changes do not occur until more than 5 seconds after initiating probe contact. It is suggested that the observed decrease in temporal oxygenation results from a combination of reduced blood supply and continued oxygen consumption by the tissue underneath the probe tip. Since there is less blood supply, the trapped blood under the probe will deoxygenate over time as the oxygen continues to diffuse into the tissue from the only available blood. Consistent with the observation by Reif et al [6
], the change in total Hb content is less than 20% for both depths. Although the total Hb content change is near 20% and below the intra-patient and inter-patient variability (~40% for each) observed in our in vivo
colonoscopy clinical studies [2
], it is significant. This suggests that pressure could influence the measured parameters and may be largely responsible for the intra- and inter-patient variability since different endoscopists likely apply varying pressures to the probe. This effect needs further investigation to understand differences in utilization of the probe between endoscopists and the subtle differences occurring within the mucosal layers of tissue.
We observed that firm pressure has some impact on total scattering intensity and the shape of the index correlation function, quantified by the parameter m. As stated above, we hypothesize that the probe tip compresses the tissue. This would bring the scatterers closer together, allowing two smaller scatterers to act as one larger scatter, leading to an increase in the total scattering intensity. Additionally, compressing the tissue leads to a change in the distribution of scattering length scales, measured by m. Future studies are needed to further investigate these effects to gain a better understanding of the observed changes in m and total scattering intensity. Future studies should also aim to verify that a controlled, gentle pressure applied to the probe can reduce variability in the clinical setting. This can be accomplished with a sensor on the probe tip that controls or measures the pressure applied.
We observed that changing the angle between the probe tip and tissue surface did not show a significant effect for any parameter. This indicates that the tissue conforms to the surface of the probe lens when it is in good contact with the target tissue. Although a slight trend can be observed for total scattering intensity, with an ANOVA p-value = 0.26, it is not statistically significant (all other parameters range from p = 0.5 to p = 0.9). It is possible that changing the probe angle could potentially distort the tissue under the probe or introduce changes in optical coupling that could be responsible for the observed trend. Since the data show less than 10% change, we can assume that the parameters measured are not dependent on the angle.Although the probe was designed to be used with the probe axis normal to the tissue surface, when it is delivered through the accessory channel of an endoscope, this is often difficult to achieve for every in vivo measurement. These results confirm that precise normal incidence to the tissue is not required and that angle is not an important source of variability in our clinical studies.
From the temporal analysis, we observed that within a short time scale, continuous contact between the probe and tissue affects the parameters measured. The 30 second time scale was chosen to represent probable delays for the clinical setting that occur between the time the physician places the probe in contact with tissue and the time the technician starts data acquisition. For gentle pressure, there are no significant differences in the parameters at any time point in this scale. For firm pressure, the rapid decrease observed in total Hb content and oxygenation in as little as 6 seconds from initial probe contact results from reduced blood supply and continued oxygen consumption, as discussed above. Also similar to the pressure study, PLS is not affected at the more superficial depth and only slightly decreases with pressure at the deeper depth. This further supports the hypothesis that firm pressures may compress the more elastic epithelial layer and collapse deeper vessels resulting in the reduced blood flow. These results suggest that temporal trends can yield information about oxygen consumption of the interrogated tissue. In the case of a controlled, known pressure, in vivo temporal measurements could correlate decreasing oxygenation with metabolic demand for potential diagnostic applications.
These studies demonstrate that firm pressure impacts the data collected and needs to be addressed in the clinical setting. A proper protocol can be developed to include maintaining good contact between the probe tip and tissue surface with application of gentle pressure. Conversely, we realize that gentle pressure is subject to interpretation and even more difficult to assess if utilizing the probe through an endoscope. The temporal study revealed that changes due to pressure can occur in as little as 6 seconds. Thus, there is an urgent need for a robust mechanism to minimize and control any delay between probe contact and data acquisition. One potential solution is implementation of a sensor to automatically trigger data acquisition when contact is detected between the probe and target tissue. This will eliminate the need for a technician and ensure the delay between tissue contact and acquisition is consistent, potentially reducing variability previously encountered from pressure or temporal effects. We expect to further expand this study in a clinical setting to more relevant tissues, such as colon polyps or other lesions, coinciding with development of the sensor.
One limitation to this study is that the volunteer manipulated the probe during measurements. While this allowed the volunteer to maintain good contact with the lip tissue and apply a more consistent pressure throughout measurements, this does not replicate the clinical scenario in which a physician uses the probe on a patient. Future studies should aim to investigate the effects of pressure and time when the probe is manipulated by another user, as well as with the probe used through the accessory channel of an endoscope. Furthermore, future studies should confirm that variability can be reduced in our clinical in vivo data when either the pressure applied to the probe or the time delay between contact and acquisition is controlled. Additionally, other sources of variability need to be explored, such as colonic distension.
Another limitation of allowing the volunteer to manipulate the probe is that the pressure applied was subjective and each volunteer may interpret ‘gentle’ and ‘firm’ differently. We can assume that the gentle pressures represent the range of pressures applied in the clinical setting, and the firm pressures, which visibly indented the surrounding tissue, are greater than pressures utilized by physicians in our clinical studies. Eliminating pressure and temporal effects would simplify the use of our device in mutli-center clinical trials and in future applications.