New optical imaging technologies can provide detailed images of tissue architecture and cellular morphology of living tissue. Optical coherence microscopy (OCM) is an exciting technique being developed that combines the subcellular resolution of high numerical aperture (NA) confocal microscopy with the increased sensitivity and penetration depth of optical coherence tomography (OCT) to noninvasively acquire detail similar to that available in histologic tissue evaluation. OCM images acquired from biological structures including plant specimens,1,2 in vitro
human colon tissue,3
and human skin,4
achieved a resolution of 2 μ
and a 200- to 500-μ
m field of view at a penetration depth of 600 μ
m. This suggests that OCM has the potential to image epithelial tissues with the subcellular resolution needed to assess their pathologic state.
OCM imaging builds detailed images of cell morphology and tissue architecture by using a high NA confocal microscope to collect light backscattered by various tissue components to provide contrast. The high NA objective focuses light to a 3-D voxel within the tissue, and a pinhole placed at a conjugate image plane within the confocal microscope allows light reflected from the focal volume to pass to the detector, while most light generated from out of focus points is blocked. Further rejection of out of focus light is achieved by using an interferometric gate to reject those photons that pass through the pinhole, but have not traveled the same optical pathlength as light generated at the focal volume, which has not undergone any further scattering events before exiting the tissue.5,6
Changes in refractive index provide a source of reflected light at the image point, and the contrast necessary to recognize intracellular detail. Although OCM imaging has been limited to relatively few biological tissues, a review of results from confocal microscopy and OCT support the potential application of both optical modalities in the assessment of epithelial tissue.
Both confocal microscopy and OCT have successfully visualized precancerous and cancerous conditions in humans. High NA confocal microscopy with its subcellular resolution enables imaging of cell morphologic and tissue architectural changes associated with dysplasia and cancer. In skin, where cytoplasmic melanin provides a strong source of backscattering, confocal microscopes have captured morphologic changes in cytologic structure and visualized microvasculature in both basal cell carcinomas and melanomas.7–10
In amelanotic epithelial tissues, where cell nuclei provide the primary source of reflected light, recent work showed that reflectance confocal imaging of normal and precancerous cervical tissue can characterize nuclear size, nuclear density, and nuclear-to-cytoplasmic ratio without the need for tissue sectioning or staining. In these studies, confocal images were used to discriminate high-grade cervical precancers with a sensitivity of 100% and a specificity of 91% in a study of 25 samples.11
Similarly, nuclear-to-cytoplasmic differences from images of normal and esophageal cancer cells were used to identify cancer with a diagnostic accuracy of 90%.12
Confocal imaging of oral mucosa has resolved subcellular detail at depths of 250 and 500 μ
m in the lip and tongue, respectively,13
and oral squamous cell carcinoma from multiple sites.14
While high-resolution OCT imaging in vitro
has demonstrated resolution on the order of 1 μ
current clinically applicable OCT systems do not provide images with the subcellular resolution characteristic of high NA reflectance confocal microscopy. With penetration depth of up to 1 mm, OCT can resolve architectural differences associated with tissue from precancerous and cancerous specimens. In a study of the cervix, an OCT system was used to identify epithelial, basement membrane, and stromal changes characteristic of carcinoma in situ
and invasive carcinoma.16
In clinical trials, OCT endoscopes have also successfully imaged a large number of precancerous and invasive malignancies of different organs including the larynx, esophagus, uterine cervix, colon, urinary bladder, and esophagus17–23
when compared to normal tissue. However, the spatial resolution of OCT is not sufficient to resolve cellular and subcellular morphology such as nuclear size.
To characterize the features of histologically normal and neoplastic oral mucosa, we conducted a pilot study using both a confocal microscope and an OCM system to image matched clinically normal and abnormal biopsies obtained from 13 patients. Our study shows that, like confocal microscopy, OCM can image and correctly characterize oral mucosal lesions with resolution comparable to histology without the need for tissue fixation, sectioning, or staining. In addition to subcellular resolution, the OCM demonstrated consistently deeper penetration than achieved by the confocal arm of the system. Analysis of epithelial scattering coefficients clearly discerns a difference between the hyperkeratotic layers and the nonkeratinized epithelium below, and an increase in scattering associated with premalignancy.