Previous reports have documented the potential of fluorescence and reflectance spectroscopy for noninvasive diagnosis of early oral cancer and precancer. However, to be effective clinically, diagnostic technologies need to have high specificity, as well as sensitivity, in order to avoid unnecessary patient interventions. Recent advances in the understanding of the alterations in tissue optical properties during carcinoma development have identified distinct differences between the epithelium and the underlying stroma [17
]. This suggests that separate interrogation of epithelial and stromal layers may improve the ability to distinguish dysplasia and carcinoma from normal mucosa and benign conditions.
The spectroscopy system described in this report allows in vivo measurements of oral fluorescence and diffuse reflectance spectra at different depths within the tissue. The depth response measurements shown in indicate how well the depth-sensitive clinical probe performs. The measurements taken with the probe immersed in water, , provide an estimate of the depth sensitivity of the probe for tissue measurements, though the actual probing depth may be slightly shallower due to scattering in tissue. In the oral cavity the thickness of the epithelial layer is typically of the order of 300–500 μm, varying with the specific tissue type. indicates that the shallow channel is strongly weighted towards the epithelial layer (the first 300–500 μm), but does not completely exclude signal from the stroma. The degree to which the interrogated region is confined to the epithelium depends on the epithelial thickness and therefore on tissue type within the oral cavity. The medium channel collects signal from a broad region that includes both epithelium and stroma, extending from the tissue surface to depths greater than 1 mm. The deep channels primarily interrogate the stroma but do include a small component of signal from the epithelium.
The most prominent general feature observed in our in vivo
data collected to date is a progressive overall reduction in blue–green fluorescence intensity in dysplastic and cancerous tissue compared to normal tissue, as illustrated in , . This trend is evident across a wide range of excitation wavelengths from 330 to 470 nm, though not at 300 or 310 nm excitation. The reduction in fluorescence intensity in dysplasia and carcinoma is observed in all oral tissue types measured and in all depth channels of the probe (shallow, medium, and deep), indicating that alterations in both epithelium and stroma are involved. We believe the most likely contributing factors to this reduction in fluorescence intensity are the breakdown of collagen crosslinks in the stroma, thickening of the epithelium, increased epithelial scattering, loss of keratin in the epithelium, and increased hemoglobin absorption associated with increased microvascular density throughout the epithelial–stromal region [16
A second general feature observed in the data is a progressive shift of the blue–green fluorescence peak to longer wavelengths in dysplastic and cancerous tissue compared to normal tissue, as shown in and . This trend also appears across a range of excitation wavelengths, most notably in the 330–390 nm range and somewhat less consistently in the 400–470 nm range. It is mostly seen in the shallow and medium channels, and less clearly observed in the deep channels; this may be because hemoglobin absorption distorts the shape of the normal spectra measured using the deep channels, as seen in , making the wavelength shift less readily apparent. The origin of the wavelength shift is not known but it may be associated with loss of fluorophores that emit at shorter wavelengths, such as keratin and collagen, and increased relative contributions to the fluorescence signal from NADH and FAD.
The reduction in blue–green fluorescence intensity and the associated wavelength shift that we observe in our data are consistent with results reported in the literature for in vivo
oral measurements. In a previous study (without a ball lens coupled probe), our group found a reduction in blue–green fluorescence intensity and a wavelength shift in cancerous tissue compared to normal tissue, with excitation wavelengths of 365, 337, and 410 nm [14
]. Badizadegan et al
. reported a similar progressive reduction in fluorescence intensity and wavelength shift in dysplastic and cancerous oral tissue compared to normal tissue, with an excitation wavelength of 337 nm [9
]. De Veld et al
. found a progressive decrease in blue–green fluorescence intensity in dysplastic and tumor tissue compared to healthy tissue at 405 nm excitation, but also found a decrease in the fluorescence intensity of benign lesion sites compared to healthy tissue [10
]. This raises the concern that this parameter may not provide sufficient specificity to distinguish dysplastic and cancerous lesions from benign lesions. Since inflammatory lesions predominantly affect the stroma, whereas dysplasia also produces alterations in the epithelium, spectral data separately derived from these layers may provide novel parameters to distinguish these clinical entities.
Our initial results indicate that the clinical ball lens coupled probe functions as designed and provides depth-sensitive in vivo spectral data. The shallow channel of the probe appears to minimize the effect of hemoglobin absorption on the fluorescence spectrum. It remains to be seen whether the removal of the hemoglobin absorption leads to significantly improved diagnostic performance; whether the probe can be used to identify different trends in fluorescence associated with fluorophores located in the epithelium and stroma; how diagnostic performance is affected by interpatient and intrapatient variability; and whether benign and malignant lesions can be distinguished.
In summary, we have described a clinical spectroscopy system with a depth-sensitive fiber-optic probe for noninvasive in vivo measurement of oral sites in healthy subjects and in patients with lesions of the oral mucosa. Differences have been observed in intensity, peak emission wavelength, and shape of the fluorescence spectra of normal and abnormal tissue sites. Depth-sensitive spectral measurements have been successfully demonstrated in vivo. The ability to obtain spectra from different depths at a single measurement site, and to distinguish between epithelial and stromal spectral signatures, may improve the diagnostic capability of point probe optical spectroscopy systems.