Our results show that large-area SECM can reveal both of the architectural and cellular features of various gastroesophageal tissues, which are similar to the morphologic features seen on the corresponding H&E-stained slides. Of particular importance is the ability of SECM to enable the visualization of goblet cells, nuclear stratification in dysplasia, and eosinophils.
There were several limitations of this study. First, the images were not acquired in vivo, but within 15 minutes after removal of samples by forceps biopsy. How tissue microstructure changes over this brief time period is not known. However, we are encouraged by other reports of RCM in vivo16
that have shown some of the features that we have seen in our biopsy study.
In this preliminary work, we applied acetic acid in the majority of cases to enhance nuclear contrast. This is a well-established technique for the esophagus27,28
and other organ systems.29,30
For most of the gastroesophageal mucosal types examined in this study, images obtained with acetic acid visualized cell nuclei more reliably and with greater contrast than images obtained without acetic acid (). In contrast, we found that eosinophils were more apparent without acetic acid because the natural reflectivity of the eosinophils appeared to be much higher than the surrounding squamous mucosa (). However, the acetic acid appeared to penetrate the tissue irregularly. This heterogeneous distribution of the contrast manifested in clear visualization of nuclei in some areas (), yet in other areas of the image, nuclei were not as clearly seen (, arrowhead). One possible cause of the irregular acetic acid penetration might be nonuniform removal of mucus during saline solution washing of the sample. For acetic acid staining to become a viable technique for microscopic contrast in vivo, standard methods for attaining uniform staining must be developed.
The images obtained with SECM are inherently transverse images compared with the conventional cross-sectional images used for histopathology. Here, we acquired multiple transverse images as a function of depth. However, cross-sectional reformatting of the data does not produce high-quality images because the axial resolution of our system (~10 μm) was lower than the transverse resolution (2 μm). Furthermore, scattering near the top of the sample shadowed deeper images, making the interpretation of images at depths greater than 100 μm difficult. These problems may be improved with the development of optimized imaging optics and image-processing routines for correcting attenuation as a function of depth.31,32
The number of samples for each mucosal type was not large enough to test the sensitivity and specificity. However, for some types of mucosae, certain distinctive architectural and cellular features were universally observed in most of the SECM images, as shown in the cases of BE with SIM.
An endoscope-compatible probe must be developed to conduct comprehensive microscopy with SECM in vivo. We have made progress in this area by demonstrating the key components in a bench-top setup.33
shows a schematic of the design. Like comprehensive esophageal OFDI, this probe conducts confocal microscopy through a balloon-centering catheter. On the bench top, we have shown that we can acquire SECM images of a 2.5-cm long, 2.0-cm diameter esophageal phantom in less than 2 minutes.33
Balloon-catheter SECM is more challenging than OFDI because the axial location of the focus must be controlled to within 10-μm precision. Our research developing a comprehensive esophageal SECM probe is currently directed toward finding robust adaptive focusing solutions to address this challenge.
Schematic of a SECM endoscopic probe design for conducting comprehensive confocal microscopy of the esophagus.
The imaging speed of SECM demonstrated in this study was limited predominantly by the speed of the scanning stages. The endoscopic SECM probe that we are currently developing will provide image information an order of magnitude faster than that in the current study. This balloon-catheter device will conduct rapid scanning by using a high-speed wavelength-swept source and will helically scan the internal optics, in a manner similar to that of OFDI14
and our bench-top proof-of-principle SECM probe.33
On the completion of the endoscopic probe development, use of SECM will be implemented in patients to provide this detailed diagnostic information over large areas and possibly even the entire distal esophagus. The large-area microscopic imaging enabled by SECM opens up the possibility of comprehensive high-resolution screening for pathology that cannot be identified by conventional video endoscopy and may be missed by confocal endomicroscopy.