## 1 Introduction  
Barrett's esophagus (BE), a complication of chronic gastroesophageal reflux disease, and in which the normal squamous epithelium of the esophagus is replaced by metaplastic columnar epithelium, is associated with an increased risk of esophageal carcinoma [1; 2; 3]. Radiofrequency ablation (RFA)is a widely used method to treat BE [1; 3; 4]. Multicenter clinical studies have demonstrated that RFA safely and effectively eradicates intestinal metaplasia and dysplasia [5]. However, repeated RFA over multiple treatment sessions is usually required to achieve complete remission of intestinal metaplasia (CRIM) and there is recurrence of BE even after achieving CRIM [3]. A recent multi-center study found that 33% of CRIM patients had BE recurrence within the next 2 years [6]. Standard white light endoscopy visualizes only the esophageal surface, and is unable to discern deeper gland-like structures.  
Optical coherence tomography (OCT) can image 1-2 mm under the tissue surface over a broad area with micrometer-scale resolution [7; 8; 9] and is a promising technology for assessment of BE and dysplasia. Using a prototype OCT system, we previously [10] reported the presence of sparsely distributed subsurface hyposcaltering structures (SHS) (described as \"buried glands\" in this reference) before and after RFA located near the gastroesophageal junction (GEI), and suggested their presence as a negative predictive marker for achieving neosquamous epithelialization after RFA. A commercially available OCT system, known as volumetric laser endomicroscopy (VLE), can image a large field of view (6 cm \(\times\) 6 cm) with a balloon catheter. Using VLE, [PERSON] et al. [11] localized SHSs in patients previously treated with RFA for BE with high grade dysplasia or early adenocarcinoma and found that 92.4% of the SHSs were associated with normal anatomic structures, such as dilated glands and blood vessels. A later study [12] correlated VLE images from endoscopic resection specimens with histology findings and found that the presence of irregular, dilated glands/ducts is an independent predictor for BE neoplasia. The exact roles of SHSs in BE remain to be elucidated. The SHS analysis in these studies was performed manually, which is time-intensive, and is associated with inter-observer variability, as shown in recent clinical studies for detection of BE with dysplasia [13; 14]. There is an unmet need for automated computer algorithms which can be applied to larger scale clinical data for faster analysis without inter-observer variability.  
[PERSON] et al. [15; 16] pioneered computer-aided detection of dysplasia in BE using a standard machine learning framework and a prototype research OCT system. [PERSON] et al. [17] developed an algorithm based on individual axial scan analysis to characterize the esophageal wall as normal vs. BE using an OCT capsule. It is unclear whether these methods can be applied to more widely available VLE data, which are acquired with a balloon imaging device with different imaging parameters. [PERSON] et al. [18] used clinically inspired features to train a classifier which was able to classify pre-selected ex vivo VLE images into non-dysplastic BE and neoplastic groups with a high sensitivity and specificity. However, the performance for in vivo VLE volumetric data is yet to be tested. None of the above methods were able to automatically quantify SHSs.  
In this study, we therefore aimed to automate SHS analysis for clinical VLE data and quantify the characteristics of SHSs in patients with BE. We first developed a fully automated algorithm to detect and quantify SHSs and validated the algorithm by comparing to expert manual analysis. Next, we investigated the general distribution and characteristics of SHSs in patients with BE, the association between pre-ablation SHSs under the BE region and treatment response in BE patients ablated with RFA, and the correlation between pre-ablation subsquamous SHS characteristics with BE length reduction after RFA.