Cellular resolution ex vivo imaging of gastrointestinal tissues with optical coherence microscopy

Abstract

Optical coherence microscopy (OCM) combines confocal microscopy and optical coherence tomography (OCT) to improve imaging depth and contrast, enabling cellular imaging in human tissues. We aim to investigate OCM for ex vivo imaging of upper and lower gastrointestinal tract tissues, to establish correlations between OCM imaging and histology, and to provide a baseline for future endoscopic studies. Co-registered OCM and OCT imaging were performed on fresh surgical specimens and endoscopic biopsy specimens, and images were correlated with histology. Imaging was performed at 1.06-microm wavelength with <2-microm transverse and <4-microm axial resolution for OCM, and at 14-microm transverse and <3-microm axial resolution for OCT. Multiple sites on 75 tissue samples from 39 patients were imaged. OCM enabled cellular imaging of specimens from the upper and lower gastrointestinal tracts over a smaller field of view compared to OCT. Squamous cells and their nuclei, goblet cells in Barrett's esophagus, gastric pits and colonic crypts, and fine structures in adenocarcinomas were visualized. OCT provided complementary information through assessment of tissue architectural features over a larger field of view. OCM may provide a complementary imaging modality to standard OCT approaches for endoscopic microscopy.

Figure 1

Normal squamous esophagus. (a) Ultrahigh-resolution cross-sectional OCT image. The layered mucosal structure including the epithelium (e), lamina propria (lp), and submucosa (sm) is visualized. No muscularis mucosa layer is seen in this cross section. Scale bar, 500 μm. (b) Corresponding cross-sectional histology, hematoxylin and eosin 4×; scale bar, 500 μm. (c) OCM en face image of squamous epithelial cells. Nuclei (n) and cell membranes are readily visible. Image depth is 30 μm. (d) OCM image near the basement membrane. The transition between cellular epithelium and more highly scattering loose connective tissue in the lamina propria is visible (arrows). Image depth is 125 μm. (e) and (f) Corresponding en face hematoxylin and eosin histology (20×) to (c) and (d), respectively. Scale bars (c) to (f), 100 μm.

Figure 2

(a) to (d) OCM imaging of squamous cellular progression in depth. Decrease in cell size and increased relative nuclear-to-cytoplasm ratio is evident in the images. Image depths for (a) to (d) are 30 μm, 90 μm, 180 μm, and 210 μm, respectively. Scale bars, 100 μm. (e) to (h) Zoom views (3×) of the regions highlighted in (a) to (d). Scale bars, 20 μm.

Figure 3

Squamo-columnar junction. (a) UHR OCT image. Esophageal squamous (s) and gastric (g) mucosa are highlighted, with gastric pit architecture identified (arrow). Scale bar, 100 μm. (b) Histology, hematoxylin and eosin, 10×. (c) to (d) OCM images of the squamo-columnar junction. Squamous cells (s) and gastric glands (g) are visible with individual mucous cells lining the gastric pits (arrows). Scale bars, 100 μm.

Figure 4

Barrett’s esophagus. (a) UHR OCT image of a biopsy specimen. Barrett’s glands (arrows) can be identified in the heterogeneous epithelium. Scale bar, 500 μm. (b) Histology, hematoxylin and eosin, 4×. Scale bar, 500 μm. (c) OCM image of Barrett’s epithelium. Individual goblet-like cells (arrows) are identified surrounding the crypt lumen. Scale bar, 100 μm. (d) Histology, hematoxylin and eosin, 20×. Scale bar, 100 μm.

Figure 5

Esophageal adenocarcinoma. (a) UHR OCT image. Scale bar, 500 μm. (b) Histology, hematoxylin and eosin, 4×. Scale bar, 500 μm. (c) OCM image of adenocarcinoma. Malignant gland architecture is evident (black arrows). Highly scattering bands of stroma can also be identified (black arrow heads). In addition, highly scattering inclusions (white arrows) likely represent darkly staining malignant cell nuclei visible on histology. Image depth is ∼50 μm. Scale bar, 100 μm. (d) Histology, hematoxylin and eosin, 20×. Scale bar, 100 μm.

Figure 6

Normal colon. (a) UHR OCT image. Crypt epithelial architecture as well as the underlying muscularis layer is identified. Scale bar, 500 μm. (b) Histology, hematoxylin and eosin, 4×. Scale bar, 500 μm. (c) OCM image of individual colonic crypts. Single goblet cells are identified within the crypt epithelium (arrows). Image depth is ∼75 μm. Scale bar, 100 μm. (d) Histology, hematoxylin and eosin, 10×. Scale bar, 100 μm.

Figure 7

Tubular adenoma. (a) UHR OCT image. Crypt architecture with distinct epithelial lining is identified. Scale bar, 500 μm. (b) Histology, hematoxylin and eosin, 4×. Scale bar, 500 μm. (c) OCM image of adenomatous crypts. Elongated, irregular crypts are visible with striated highly scattering epithelium representative of dysplastic nuclei (arrows). Image depth is 90 μm. Scale bar, 100 μm. (d) Histology, hematoxylin and eosin, 10×. Scale bar, 100 μm.

Figure 8

Adenocarcinoma of the colon. (a) UHR OCT image. Fine scattering heterogeneity with interspersed malignant gland formation can be recognized on OCT (arrow). Scale bar, 500 μm. (b) Histology, hematoxylin and eosin, 4×. Scale bar, 500 μm. (c) OCM image of tumor microstructure. Gland formation (black arrows) as well as highly scattering malignant nuclei (white arrows) are highlighted. Image depth is 80 μm. Scale bar, 100 μm. (d) Histology, hematoxylin and eosin, 20×. Scale bar, 100 μm.