One can estimate the depth range in the tissue to which a given probe geometry is most sensitive and the corresponding depth resolution by considering the location and extent of the geometric overlap between the illumination and collection cones. It is important to remember, though, that the specific penetration depth profile of detected photons depends not only on this geometrical overlap but also on the optical properties of the tissue under consideration. With this fact in mind, we carried out simulations at λ = 450 nm and λ = 650 nm to reveal wavelength-dependent changes in penetration depth profiles. We have also tried to simulate changes in optical properties associated with severe dysplasia, and we have incorporated these changes into our modeling to provide some insight into differences in penetration profiles caused by dysplastic progression.
A. Tilted Source and Detector Fibers
When the source and detector fibers are oriented perpendicular to the tissue surface, the overlapping region of the illumination and collection cones extends to large depths within the tissue. In this case, photons detected exhibit a wide range of penetration depths, as demonstrated in and . When the fibers are tilted, however, the illumination and the collection cones are slanted toward each other, and the overlapping region moves closer to the surface and is more localized. This trend results in improved depth selectivity and collection of photons from more superficial depths within the tissue.
For β = 15°, the simulation results for normal epithelial tissue imply that the illumination and collection cones overlap over the superficial stroma and then diverge from each other, leading to increased sensitivity to this region. The fact that photons undergo fewer scattering events in the stroma before they reach the detector fiber is also consistent with this localization. Photons collected from the epithelium undergo fewer scattering events as well. As the fibers are tilted toward each other, photons no longer need to backscatter through large angles to reach the detector. Because they enter the tissue at an angle, there is increased probability that they can reverse their path through fewer numbers of scattering events and travel toward the detector. The detector, which is also oriented at an angle, favors collection of photons that backscatter through smaller angles.
The results for β = 30° indicate that the illumination and collection cones overlap over the epithelial layer and then diverge from each other more rapidly, with little or no overlap over the stromal layer. The probe geometry is mostly sensitive to epithelial scattering. Photons collected from the epithelium undergo fewer scattering events, again consistent with an even higher probability of backscattering toward the detector. It is important to note that photons collected from the stroma undergo a greater number of scattering events in the stromal layer than for the β = 15° case. Because of the increased tilt angle and the more rapidly diverging illumination and collection cones over the stromal layer, photons from the stroma need to scatter more to alter their path and enter the collection cone where there is increased probability of detection.
Comparison of and and and reveals significant differences between penetration depth profiles of normal and dysplastic epithelial tissue. There is a noticeable increase in sensitivity to the epithelial layer for dysplastic tissue, and this trend is consistent with increased epithelial scattering, decreased stromal scattering, and increased stromal absorption that accompany dysplasia. Increase in epithelial scattering and decrease in stromal scattering associated with dysplastic progression are also reflected in statistics regarding the number of scattering events.
There are practical limits on the maximum tilt angle that can be achieved. If the overall dimension of the fiber-optic probe is to be kept small, the fibers cannot be tilted extensively. There is also an upper limit on the extent of bending that a fiber can tolerate. Tilt angles greater than 30° are difficult to construct in a compact package and have not been considered in this paper. The results given in for normal epithelial tissue indicate that, even when the fibers are placed close to each other and are tilted to the maximum extent compatible with design limitations, a significant percentage of detected photons still comes from the stroma. Even though the illumination and collection cones overlap over the epithelium, the angle at which the cones diverge from each other is not sufficient to eliminate detection of photons from the highly scattering stroma. Therefore we conclude that this probe geometry does not have the potential to provide adequate separation of epithelial scattering from stromal scattering in normal tissue. The results for β = 30° given in , however, demonstrate that the same probe geometry provides significant separation of epithelial scattering from stromal scattering in dysplastic tissue. Such a contrast in penetration depth profiles of normal and dysplastic tissue is not desirable because we would like to be able to compare spectra that are unique to each of the two layers. We need a probe design that demonstrates comparable performance in isolating epithelial scattering from stromal scattering for both normal and dysplastic tissue.
B. Half-Ball Lens Coupled Source and Detector Fibers
Instead of physically tilting the fibers, we can make use of the refractive power of a high-index spherical lens to obtain slanted illumination and collection cones. illustrates the depth selectivity potential of a sapphire half-ball lens in direct contact with normal epithelial tissue. As the source and detector fibers are moved to the outer edges over the lens, the angle at which the illumination and collection cones intersect within the tissue increases. The cones overlap over more superficial tissue depths and then diverge rapidly in opposite directions, giving rise to increased depth selectivity.
When the lens is in direct contact with the tissue, however, the overlapping region of the illumination and collection cones occurs in the stroma. Simulation results also indicate that, for w = 0 μm, specular reflection accounts for a few percent of the total detected reflectance. For the three source–detector separations used in the simulations, the illumination and collection paths do not intersect on the flat surface of the lens. Therefore incident photons that are specularly reflected at the lens–tissue interface are not directed toward the detector. Detected specular reflection is attributable to incident photons undergoing internal reflection at the flat surface of the lens, bouncing back from around the central part of the spherical interface, and undergoing reflection at the flat surface of the lens a second time. Owing to spherical symmetry, some of these photons reach the detector fiber within the limits of the numerical aperture.
The epithelial layer can be selectively targeted by insertion of a sapphire window between the flat surface of the lens and the tissue surface. The extended path length within the window brings the illumination and collection cones closer together on the surface of the tissue. Comparison of and suggests how the penetration depth profiles are affected by the presence of the window. shows that photons collected from the epithelium tend to undergo fewer scattering events as the source–detector separation increases, similar to the effect caused by increasing the tilt angle of the fibers. Also, photons collected from the stroma undergo a greater number of scattering events in the stromal layer. This is again due to more rapidly diverging illumination and collection cones over the stromal layer, and photons need to scatter more to alter their paths and reach the detector. indicates that, with s = 900 μm, which represents the case when the source and detector fibers are placed at the very edges of the lens, a significant percentage of detected photons comes from the epithelium at both wavelengths. It can be concluded that placing the fibers far apart over the lens is an effective way of creating illumination and collection cones that are steep enough to eliminate collection of photons from the stroma. Simulation results in and imply that s = 900 μm also provides excellent sensitivity to the epithelial layer in the case of dysplastic tissue. Therefore, this probe geometry is capable of providing sufficient separation of epithelial scattering from stromal scattering for both normal and dysplastic tissue.
Placement of a sapphire window between the flat surface of the lens and the tissue surface not only facilitates selective targeting of the epithelial layer but also eliminates detection of specularly reflected photons. With the window in place, the incident photons reach the base of the window at a location closer to the central axis of the lens. If these photons are reflected, they will no longer hit the region around the tip of the spherical interface. This will effectively break the spherical symmetry that has led to collection of specular reflection when w = 0 μm. Note that the window thickness needs to be adjusted carefully to prevent overlap of illumination and collection paths over the base of the window. As long as the source–detector separation is not smaller than 500 μm, an upper limit of 300 μm on the window thickness ensures that no such overlap will occur and that photons that are specularly reflected at this interface will not travel directly toward the detector.
We have observed that the penetration depth profiles are not strongly dependent on the thickness of the air gap between the tip of the fibers and the top of the lens. Changing the thickness of the air gap to 100 or 300 μm has a minimal effect on parameters such as the mean penetration depth and the percentage of photons collected from the epithelium.
C. Proposed Probe Design
Whereas the epithelial layer can selectively be targeted by use of half-ball lens coupled fibers that are oriented perpendicular to the tissue and of a window with w = 300 μm, the ability to selectively target the stromal layer has been compromised. Even though s = 500 μm can be used to selectively probe the stroma in the case of normal epithelial tissue, the results given in indicate that the same is not true for dysplastic epithelial tissue. With w = 300 μm, a source–detector separation that is smaller than 500 μm leads to collection of specularly reflected photons and cannot be used to obtain increased sensitivity to the stromal layer.
It is possible to overcome this problem by keeping the source–detector separation at 500 μm but using tilted source and detector fibers. Tilting the fibers slightly away from each other brings the overlapping region of the illumination and collection cones over to the superficial stroma. The proposed tilt angle is only 5°, and such a small tilt angle can easily be implemented within the 500 μm space.
The results compiled in and show that the depth selectivity achieved by the two fiber pairs is sufficient for selectively targeting both the epithelium and the stroma. Note that the penetration depth profiles for both fiber pairs are consistent across the two diagnostic categories, evidenced by histogram peaks localized within similar tissue depths. The mean penetration depths and the percentages listed in for normal and dysplastic tissue are also comparable for all four cases. This consistency in depth selectivity is a crucial advantage that can benefit the interpretation of tissue spectra.
An important consideration in the design of a fiberoptic probe is the efficiency of the optical setup. Because the fibers are in contact with air, specular reflection off the fiber tips is of the order of 5%. Also, sapphire has a high refractive index, and specular reflection off the spherical interface is likely to degrade the efficiency of the proposed probe. Monte Carlo simulation results indicate that, for the tilted fiber pair with s = 500 μm, the fraction of incident photons lost to specular reflection off the lens surface is ~10% for d = 200 μm. For the fiber pair with s = 900 μm, this loss increases to ~15%. At the same time, the edges of the fibers separated by 900 μm are aligned with the edges of the spherical lens. Some of the photons leaving the source fiber will miss the lens and hit the inside wall of the probe. As the NA of the source fiber is only 0.11, the fraction of incident photons that will miss the lens is ~10% for d = 200 μm. Similarly, the full NA of the detector will not be utilized to collect photons exiting the tissue. Simulation results show that signal levels obtained with lens coupled fibers are indeed lower than those obtained with tilted fibers in contact with the tissue. Note, however, that the collection efficiency of a given source–detector geometry cannot be characterized simply by the total number of photons detected. A more appropriate way to assess collection efficiency in the context of this paper is to consider signal magnitude in conjunction with depth selectivity. To give a specific example, results for normal epithelial tissue at λ = 450 nm indicate that the total number of photons detected with lens coupled fibers with s = 900 μm is ~30% less than the total number of photons detected by using tilted fibers with β = 30°. Approximately 20% of this decrease in signal magnitude is in fact due to the ability of the lens coupled fiber pair to limit the sampling depth and to successfully eliminate detection of photons from the highly scattering stroma. Hence, coupling the source and detector fibers to a sapphire lens does not lead to considerable reduction in the number of photons collected from the epithelium, which, in this particular case, is the target layer. Comparison of the penetration depth histograms for the proposed probe design with those for tilted fibers in contact with the tissue also reveals comparable signal levels from the respective tissue layers. Therefore we can conclude that the focusing power of the lens compensates for a significant portion of optical losses associated with fiber–lens coupling. Specularly reflected photons or incident photons that miss the lens surface are sources of stray light within the probe. If the inside wall of the probe is shielded with an absorbing material, further scattering of these stray photons will be avoided.
Many techniques have recently been employed to selectively target the epithelial layer and isolate epithelial scattering from stromal scattering. Light-scattering spectroscopy and polarized reflectance spectroscopy have been shown to be sensitive to precancerous changes in the epithelium.6,8,31,39,40
Other methods proposed to target the most superficial depths within epithelial tissues include differential path-length spectroscopy41
and low-coherence backscattering spectroscopy.42
Our approach is based on improvement of depth selectivity simply through optimization of the fiber-optic probe design.
Ball or half-ball lens coupled fibers have been widely used for medical applications including laser surgery and photodynamic therapy.43
The purpose of using a lens element in these applications is to increase light intensity at the tip of the probe. Motz et al.44
recently described a probe design for biomedical Raman spectroscopy in which a sapphire ball lens inserted to the tip of the probe provides signal enhancement and increased collection efficiency. In this paper we have shown that a sapphire half-ball lens is capable of providing depth selectivity and improved depth resolution. Specifically, the simulation results for epithelial tissues indicate that it is possible to use lens coupled source and detector fibers to isolate epithelial scattering from stromal scattering.
The proposed probe design provides a simple and efficient means to collect light selectively from both the epithelium and the stroma and facilitates acquisition of reflectance spectra unique to each of the two layers. As a result, optical trends from both tissue layers can be monitored for any changes associated with precancer progression. Spectra unique to the epithelium will be sensitive to dysplastic changes in epithelial cells, whereas spectra unique to the stroma will be sensitive to dysplastic changes in collagen fibers and microvessel density.
In addition to providing resolution of spectral information from epithelial and stromal layers, the proposed design is extremely compact. The overall dimension of the fiber-optic probe is roughly determined by the lens diameter, which is as small as 1 mm. Such a small probe can provide easy access to epithelial tissues including the cervix and the oral cavity and can also be integrated into an endoscope for detection of dysplasia in otherwise inaccessible organ sites such as the intestine.
The principle of depth selectivity by using lens coupled fibers can also be applied to target epithelial fluorescence in fluorescence spectroscopy. In as much as sapphire has no autofluorescence, the design can possibly be extended for use in fluorescence measurements as well. In fact, initial results reported by Schwarz et al.45
are promising, but modeling studies are required for characterizing depth selectivity specific to fluorescence measurements.