Background and Significance

Neoplasia of the Esophagus

Conditions of clinical interest include specialized intestinal metaplasia (Barrett's esophagus), low and high-grade dysplasia, and adenocarcinoma. Barrett's esophagus is a metaplastic conversion of normal squamous epithelium into columnar epithelium. It is seen endoscopically as an extension of the gastroesophageal junction. The development of intestinal morphology requires the presence of goblet cells, and is associated with a significantly increased risk of developing adenocarcinoma of the esophagus, which is currently one of the fastest growing cancers in the industrialized countries in terms of the number of new cases per year. This condition is found in as high as 25% of asymptomatic patients undergoing routine endoscopy. Critical to the screening of patients with Barrett's esophagus is the detection and localization of dysplasia, the presumed precursor to adenocarcinoma. Dysplasia is classified as either low or high grade, and dysplastic glands distort the normal tissue microarchitecture up to 1 mm below the tissue surface . The latter condition demands intervention, usually by surgical resection. Currently, surveillance is performed by white light endoscopy. However, dysplasia is flat and endoscopically indistinct from Barrett's esophagus and thus is not visible on white light endoscopy. Consequently, the sensitivity and specificity of this method alone is limited. In Barrett's esophagus, extensive biopsy protocols such as random 4-quadrant biopsies at 1 - 2 cm intervals using jumbo biopsy forceps ( Seattle protocol) have been validated and recommended. However, because the technique is labor-intensive and requires a therapeutic endoscope, it is used by less than 20% of U.S. gastroenterologists. This method of screening has not been shown to reduce the rate of progression of Barrett's esophagus to adenocarcinoma. Thus, a new method for detecting and localizing dysplasia in the setting of Barrett's esophagus is needed.

Confocal Microscopy

Confocal microscopy is a powerful imaging technique that performs optical sectioning in biological tissue with sub-cellular resolution. It is capable of simultaneously collecting images of reflectance for characterizing cellular and tissue morphology and of fluorescence for detecting the biochemical and molecular properties of cells and tissue with complete spatial registration. Confocal microscopy is commonly used to collect images in the ex vivo setting, and would be a valuable tool in vivo if the lenses and scanning mechanisms are made sufficiently small. Standard confocal microscope objectives have physical dimensions that are too large for use inside the human body. In order to resolve sub-cellular structures, an axial resolution of a few microns is needed.

This requires a large objective lens with a high numerical aperture. In addition, the scanning mechanism is located prior to the objective, which leads to aberrations that must be corrected by the use of multiple lenses, further increasing the objective size. Finally, as shown in Figure 2.1, the single-axis confocal design is not ideal for deep tissue imaging on the order of 1 mm. The high numerical aperture optics, necessary for high axial resolution, leads to a short working distance.

Furthermore, a large amount of scattered light is collected, which limits the penetration depth and contrast of the images. Figure 2.2 demonstrates the unique advantages of the dual-axes architecture for achieving a large working distance, through the use of low numerical aperture (NA) optics, and the rejection of scattered light due to an off-axis collection path.

Researchers within our group at Stanford have recently developed the first miniature confocal microscope fabricated with MEMS technology, shown in Figure 2.3 next to a standard confocal microscope objective. The laser, external optics, detectors, and electronics are contained within an instrument cart located remotely. This prototype uses an epi-illumination (single-axis) optical design where the same lens is used for illumination as well as collection. While it demonstrates an excellent transverse resolution of 1 m m, the axial resolution of 18 m m is poor, as is image contrast, which limits in vivo use. These limitations are addressed by developing a dual-axes optical design that uses separate low numerical aperture (NA) lenses oriented with the illumination and collection axes crossed at an angle. In addition to high axial resolution, this design offers a long working distance, large field of view, and reduced noise from scattered light. The optics are fiber coupled and scanning is performed with the mirror located in the post-objective location. Post-objective scanning is enabled by the long working distance afforded by the low-NA lenses. By scanning the beams after the focusing elements, aberrations are minimized such that a large field of view is achieved. These features also allow for the scan head to be scaled down in size to millimeter dimensions with MEMS technology. We derive the point spread function of the dual-axes architecture from diffraction theory, and predict a spatial resolution of 1 to 2 m m in air.

The dual-axes confocal architecture is ideal for high-resolution imaging of epithelial cell layers in a manner that complements and enhances other tools that are being developed for the early detection of cancer in hollow organs. Examples include optical coherence tomography (OCT), chromoendoscopy, and light scattering spectroscopy. 

Figure 2.1 High axial resolution in a single-axis confocal requires a short working distance. Light scattered along the entire beam path is capable of being collected (green cone), reducing contrast and penetration depth.

Figure 2.2 In a dual-axes design, low numerical aperture (NA) optics allow for a long working distance. Scattered light, outside of the focal volume, is NOT focused into the collection fiber, thereby improving contrast. Although low-numerical-aperture optics are used, high resolution is still achieved at the focal volume defined by the intersection of the illumination and collection paths.

Figure 2.3 . Miniature confocal microscope fabricated with MEMS technology. A standard microscope objective is shown as well for comparison. This is the first demonstration of a MEMS-based single axis endoscope (limited axial resolution).

OCT is an interferometric reflectance technique for performing optical sectioning but is not compatible with non-coherent signals such as fluorescence, providing no opportunity for the use of biomarkers. Chromoendoscopy, as with many wide area surveillance techniques, is a surface measurement that is of limited utility in comparison with optical sectioning diagnostics. Light scattering spectroscopy does not image morphological structure. The miniature dual axes confocal microscope would be an important addition to this arsenal of in vivo diagnostic tools because it can collect both reflectance and fluorescence simultaneously with sub-cellular resolution and deep tissue penetration, a combination of features not offered by any other method. Sub-cellular resolution is necessary to resolve cell nuclei, whose size can be an important indicator of dysplasia and adenocarcinoma. Imaging with deep tissue penetration and large FOV, on the order of 1 mm, significantly increases diagnostic capabilities.

The dual-axes confocal architecture is ideal for high-resolution imaging of epithelial cell layers in a manner that complements and enhances other tools that are being developed for the early detection of cancer in hollow organs. Examples include optical coherence tomography (OCT), chromoendoscopy, and light scattering spectroscopy. OCT is an interferometric reflectance technique for performing optical sectioning but is not compatible with non-coherent signals such as fluorescence, providing no opportunity for the use of biomarkers. Chromoendoscopy, as with many wide area surveillance techniques, is a surface measurement that is of limited utility in comparison with optical sectioning diagnostics. Light scattering spectroscopy does not image morphological structure. The miniature dual axes confocal microscope would be an important addition to this arsenal of in vivo diagnostic tools because it can collect both reflectance and fluorescence simultaneously with sub-cellular resolution and deep tissue penetration, a combination of features not offered by any other method. Sub-cellular resolution is necessary to resolve cell nuclei, whose size can be an important indicator of dysplasia and adenocarcinoma. Imaging with deep tissue penetration and large FOV, on the order of 1 mm, significantly increases diagnostic capabilities.