2.1.

Patient Enrollment and Tissue Sample Collection

Eligible patients were identified and consented at the time of endoscopy by collaborating clinical investigators. The study protocol was approved by the University of California, Davis Medical Center (UCDMC) Institutional Review Board. Fifty-six patients undergoing routine surveillance endoscopy for Barrett’s esophagus, dysplasia, and suspected esophageal cancer were enrolled in this clinical study. Four biopsy specimens were collected per patient for a total of 224 tissue samples. Biopsy specimens were collected with standard forceps and placed individually in labeled sterile containers with RPMI1640 media (Invitrogen, Carlsbad, CA). The first tissue biopsy specimen was taken from the proximal stomach (cardia). This was important in order to establish optical morphology of cardia mucosa as a control, as well as to rule out cardia adenocarcinoma.^7,12 The second control biopsy specimen was taken from the descending small intestine (duodenum). The third endoscopic biopsy specimen was taken from the distal esophagus and considered to be the pathological sample. The fourth biopsy specimen was taken from above the Z-line (squamocolumnar junction) in the squamous epithelium of the esophagus and was documented to be the normal esophageal control sample. Immediately after image processing, each biopsy specimen was placed in 10% formalin for fixation and transferred to pathology for tissue diagnosis. Each tissue biopsy specimen was taken to be histopathologically homogeneous such that the AF images were representative of the pathology diagnosis. The pathological evaluation was confirmed by at least two expert pathologists and taken as the diagnostic gold standard from which the optical images were categorized.

2.2.

Optical Imaging Protocol

Each tissue sample was placed on a standard pathology cassette without preparation and with minimal handling. The tissue sample was covered with a quartz slide for imaging. This provided a flat imaging surface to keep the luminal plane of the small and irregularly shaped specimens in focus, which allowed for better visualization of cellular patterns. The quartz slide may not be necessary for future in vivo applications, as the tissue surface may be better defined and aligned with the microscope’s image plane.

The microscope imaging prototype platform has been previously described in detail.²⁹ In brief, a compact diode-pumped solid-state laser operating at 266 nm (Intelite, Minden, NV) was used as the photoexcitation source to generate the AF images. The ex vivo images under 266-nm excitation were acquired using a 5 second exposure time with an approximate dose of $30\mathrm{mJ}/{\mathrm{cm}}^2$ in order to optimize baseline image quality. The resulting AF images arose predominantly from the emission of tryptophan and provided a representation of the structure of the top layer of tissue cells. The microscope system was equipped with a 20× long working distance objective, followed by a 5× zoom lens. The images were recorded using a liquid nitrogen cooled charge-coupled device (Princeton Instruments, Trenton, NJ). The selected UV excitation wavelength propagated through the tissue to only about 50 μm, providing a sufficient amount of AF signal produced in the superficial tissue layer to be contained within the microscope’s depth of field. This approach reduced the out-of-focus signal, allowing the formation of high-contrast images without optical sectioning techniques that reject most of the signal produced by the excitation light, that employ contrast agents, or that mandate time-intensive tissue preparation.³⁰ Our preliminary results indicated that microstructure morphology and superficial nuclear and cellular organization can be imaged in real-time, providing immediate information related to the presence and progress of early-stage abnormalities, such as BE and dysplasia, that originate in the superficial epithelial mucosa before developing into cancer.²⁸ Further development of this method necessitates the establishment of optical rules for recognizing cellular patterns (the rules for interpretation) that correspond to normal and abnormal histologies spanning from early benign modifications (Barrett’s esophagus) to subsequent dysplastic change and progression toward carcinoma. Optical rules of disease interpretations that can be accepted by the medical community as an analogous guideline to traditional methods would provide the key to enabling in vivo histopathologic evaluation in the operating room.

2.3.

Approach for Developing Rules of Interpretation

The images of the specimens in this study were compiled into a preliminary image gallery of normal and abnormal tissue structure that could be used to establish a correlation between image morphology and histopathology. We hypothesized that progression of abnormality was associated with the severity of deviation from normal structures observed in the UV AF images. Since BE required the presence of goblet cells, we included images of normal intestinal specimens in our study to establish the morphology of goblet cells (normally present in the intestine) to enable comparison with features observed in the images of BE. The intestine structure was also associated with the presence of villi and crypts, which were important recognition factors in the AF images. Furthermore, as the gastric type cellular morphology near the GE junction was shown to be associated with metaplastic progression, we included normal stomach tissue in this imaging study. Consequently, the analysis and categorization of the images for developing optical pattern recognition rules related to their histopathological state was focused on distinguishing (a) normal esophageal tissue, (b) normal stomach and intestine tissue, (c) BE with no dysplasia, and (d) BE with dysplasia and cancer. Typical examples for each of these categories and results will be presented in separate subsections.

3.1.

Normal (Non-dysplastic) Esophageal Squamous and Columnar Epithelium Control Images

Figure 1 illustrates a squamocolumnar mucosal gold standard H&E section comparison with AF images of histologically unremarkable squamous [Fig. 1(a)] and columnar [Fig. 1(b)] tissue. Large, polygonal superficial cells that appear as individual tile-like structures with visible nuclei can be appreciated in both the H&E section and the normal esophageal squamous epithelium. It is notable that the optical images of nuclei were inconsistent in appearance. Decreased nuclear fluorescence has been described by Li et al.³¹ in cultured cells but have also been observed in this work to display markedly increased signal intensity. Our previous work suggested that the discrepancy may be due to the maturation process of the stratified epithelia.²⁸ Many cells have no nuclei, while other nuclei can be small or undergoing pyknosis (densification). Imaging cells without nuclei, apoptotic cells, or pyknotic nuclei may produce variable optical signals that result in bright or dark features. These contrasting features may be attributed to the same microstructure, such as both bright and dark nuclei. A difference between the reduced intracellular columnar tissue intensity and increased cell membrane intensity can also be seen in Fig. 1(b). A typical microscopic UV AF image captured from specimens of normal columnar mucosa is shown. This image demonstrates visualization of the honeycomb pattern of cells showing their outlines with higher intensity compared with the cytoplasm region. We provided a quantitative assessment of this difference in a previous publication.³² Our hypothesis that the origin of these images was due to the spectral contribution from different fluorophores including tryptophan, NADH, flavoproteins, and/or lipopigments was supported by a calculated 30% intensity variation. The H&E staining of the non-dysplastic esophageal columnar mucosa section also demonstrates the typical honeycomb pattern observed in this type of tissue. These easily recognizable patterns observed in the UV AF images shown in Figs. 1(a) and 1(b) represent the characteristic morphology of the normal stratified squamous and columnar esophageal mucosa observed in fresh unprocessed specimens obtained without any tissue preparation. These typical examples of our observations were considered as the baseline (control) images for normal (healthy) esophageal tissue.

Fig. 1

Gold standard H&E squamocolumnar section compared with AF images of histologically unremarkable esophageal (a) squamous epithelial tissue with easily visible large polygonal superficial cells that appear as individual tile-like structures with nuclei that can be appreciated in both the H&E section and normal squamous epithelium, and (b) columnar epithelial tissue with the characteristic honeycomb pattern of cells showing their outlines with higher intensity compared with the cytoplasm region. Each field of view is $475\times 361\mathrm{\mu m}$.

3.2.

Normal Gastric and Intestinal Epithelium Control Images

The UV AF images shown in Figs. 2 and 3 represent typical examples of our results obtained from human biopsy specimens from normal duodenum (intestine) mucosa and normal cardia (proximal stomach) mucosa respectively. Due to the use of the quartz slide, the villi seen throughout this image appear as slightly compressed round structures. Figure 2 provides an artists’ sketch from a luminal perspective (view of the endoscopist) showing villi apices, goblet cells, and columnar cells surrounding dark voids that represent crypts of Lieberkühn. It should be noted that crypts often represent imperfect glands that may be distorted, roundish, or otherwise inconsistent in appearance throughout the tissue surface. Furthermore, the crypt is a depression around projecting villi and will not produce a detectable AF, thus appearing as dark features scattered throughout the luminal surface. Villi can also be deformed and distorted instead of resembling the ideal condition of pencil tips in a box. In the corresponding AF image of Fig. 2, the target-like features present as decreased signal voids, which may represent villi crypts similar to those observed in esophageal tissue previously reported.³² Several examples are partially outlined for improved visualization. The dimpling and cramping of the uneven surface shown in the corresponding AF imaging are visible undulations of the duodenum epithelium with crypts and glands intervening between villi apices within the AF image of normal duodenal mucosa. The center apex had an approximate diameter of 40 to 50 µm. Similar rounded features were visible throughout the luminal surface. The heights of the villi typically varied from 0.5 to 1 mm, while their diameters varied from approximately one-eighth to one-third of their heights. Although the villi were compressed during imaging (to help better identify the goblet cells), their outlines are clearly visible within the image of Fig. 2, and their observed diameter is within the normal parameters. Based on the expertise of our collaborating pathologist, the duodenal tissue target-like feature of a dark area with one smaller dark spot and one inner brighter spot in Fig 2 was visibly similar in intensity contrast and diameter to an obvious esophageal tissue crypt of Lieberkühn. This crypt, detailed in Sec. 3.4 below in the cross-sectioned H&E image of Fig. 5, was identified during H&E microscopic evaluation as being from a duodenal location of biopsy. It is also visible in the corresponding AF image of Fig. 5. We hypothesize that the increased signal intensity is associated with higher tryptophan content or the presence of mucus-filled goblet cells, while darker features are associated with lower tryptophan concentration or the presence of a void.

Fig. 2

Artist’s sketch of normal duodenal microstructures from a luminal perspective (view of the endoscopist): villi apices, goblet cells, and columnar cells surrounding dark voids that represent crypts of Lieberkühn. These cartoon features (not drawn to scale) are indicated in the $475\times 361\mathrm{\mu m}$ field of view AF image of a duodenum tissue biopsy specimen. Villi apices are partially outlined for visual aid.

Fig. 3

Artist’s sketch of normal cardia microstructures from a cross-sectioned perspective (view of the pathologist). The cartoon features (not drawn to scale) illustrate gastric fold projections with glandular pits at the mouth of the gastric neck and foveolar cells shown as dark, round features scattered throughout the surface. The corresponding $475\times 361\mathrm{\mu m}$ field of view AF image of a cardia tissue biopsy specimen is shown from a luminal view (view of the endoscopist). Arrows indicate what are believed to be gastric pits. A large gastric fold apex is partially outlined, with similar microstructures visible throughout the image.

As previously discussed, it is important to recognize and optically identify goblet cells in the AF images of the intestine. The nucleus of goblet cells is at their base, along with the organelles, while the remainder of the cell is filled with membrane-bound secretory granules filled with mucin. The “goblet cell” name comes from their cup-like shape. As goblet cells produce mucin, it was necessary to consider the different imaging results that mucin might produce if it is present inside the goblet cells of the fresh tissue biopsy specimens during AF imaging. Our experimental results strongly suggested that mucin typically produced an increased AF signal compared with the AF of the cells. Therefore, goblet cells containing mucin produced a much higher intensity than goblet cells without mucin within the fresh tissue, and served as an optical guideline documented in Table 1. These observations are illustrated with lines in the AF image of normal duodenum, shown in Fig. 2. A dark and a bright feature are indicated, each with a diameter of approximately 11 µm. We assumed that the dark feature is a goblet cell without mucin, while the bright feature is a goblet cell containing mucin. Similar features to those identified above could be found in the image shown in Fig. 2 and in similar images of normal intestine tissue. Thus, the results suggested that the goblet cells could be observed in the microscopic UV AF images as bright or dark features, on the order of 10 µm in diameter, depending on the mucin content at the time of imaging. Finally, two similar features that were believed to represent crypts of Lieberkühn of the duodenal epithelium are also indicated.

Figure 3 is an artists’ sketch of cardia epithelium from a cross-sectioned perspective (view of the pathologist). The surface projections are gastric folds with glandular pits at the mouth of the gastric neck. Foveolar cells are shown as dark, round features scattered throughout the surface. The corresponding AF image of cardia mucosa in Fig. 3 is shown from a luminal perspective (view of the endoscopist) and was representative of such images obtained from normal stomach epithelium specimens. The gastric fold apices appear as large nodular features containing obvious dark outlines of foveolar cells scattered throughout the surface. Gastric pits, indicated with arrows, appeared as round target-like features similar to the duodenal crypts seen in Fig. 2. It should be noted that when correlating the AF images to the H&E gold standard, a $1:1$ comparison of surfaces is inherently impossible. This is due to the tissue preparation of H&E sectioning, which results in an image of the foveolar cells below the neck (beyond penetration of AF image) due to removal of the luminal surface containing the gastric folds imaged in the AF technique. However, microscopic evaluation of the H&E images supported the observed tissue structures to correspond with those from the gastric biopsy location. The normal duodenum and cardia mucosal images such as those shown in Figs. 2 and 3 provided the control images to exemplify what microstructures of interest would look like using this imaging methodology. These AF images could be used as a baseline for comparison with images of specimens presenting benign intestinal metaplasia modifications of the esophagus, indicating BE, as discussed in the next section.

3.3.

Visualization of Benign Epithelial Modifications

Figure 4 presents an esophagus biopsy specimen AF image that was collected at the endoscopically observed GE junction and returned with a pathological diagnosis of histologically unremarkable squamous esophageal mucosa with very scant columnar components. Cardia tissue topography characteristics are visible in the esophageal tissue, revealing gastric fold polypoid-like surface projections. Nobular rounded features may represent what could be irregular-surface mucous cells, squamous glands, or oxyntic metaplasia. The arrows point to larger, dark, target-like features that could be crypts similar to the duodenal and cardia crypts seen in Figs. 2 and 3. This AF image of the distal esophagus clearly resembles the gastric mucosa of Fig. 3 as opposed to the squamous or columnar mucosa shown in Fig. 1. The scant honeycomb pattern was preserved in the columnar mucosa, similar to that shown in Fig. 1(b). The irregular polypoid surface appeared to be transitioning from squamocolumnar mucosa containing villi crypts (arrows), consistent with previous results.²⁸ The multiple projections might be seen as unfolding areas with glands, obscuring the flat surface of stratified squamous mucosa, and inhibiting a clear focus of features beneath the structures of greatest height. We commonly observed optical images of cardia-like esophageal mucosa as those seen in Fig. 4, which appear at first glance to be modified from the characteristic morphology of the non-dysplastic or normal esophageal tissue shown in Fig. 1, but were not yet differentiated EAC (detailed in Sec. 3.6, Fig. 8 below. However, patient history must be considered when examining diagnostic results related to disease progression, especially for those on chronic medication. Many BE patients are placed on proton-pump inhibitors (PPIs) in an attempt to reduce associated acid reflux. PPI therapy is associated with several histological changes, including parietal cell and foveolar hyperplasia, as well as microcystic dilation of gastric glands. This transformation is a common observation in GE biopsies of the cardia mucosal cells. These surface nodularities, identifiable with UV autofluorescence microscopy, possibly correspond to the above-described PPI effect seen histologically and shown in Fig. 4. This could explain the commonly observed nodularity in the squamous esophageal mucosa in our study, which is reminiscent of fundic gland polyps generally seen in the gastric body in patients with BE on PPI therapy. Consequently, this UV AF method provides a clinically important distinction of normal gastric columnar mucosa that standard endoscopy is generally limited from distinguishing.

Fig. 4

$475\times 361\mathrm{\mu m}$ AF image of an esophagus biopsy specimen that resembled the g1astric mucosa, as opposed to the squamous or columnar esophageal mucosa shown in Fig. 1. Arrows indicate dark, target-like features that could be crypts similar to the duodenal crypts seen in Fig. 2. Gastric fold polypoid-like surface projections are visible in the esophageal tissue, similar to those of the cardia tissue seen in Fig. 3. These nodular esophageal microstructures may represent what could be irregular surface mucous cells, squamous glands, or oxyntic metaplasia.

3.4.

Visualization of Barrett’s Esophagus and Early LGD

Figure 5 shows the H&E gold standard cross-sectioned image of an esophagus specimen from a patient with pathology diagnosis of BE with mild to moderate chronic inflammation and suspected LGD. This esophagus biopsy specimen was collected at 32 cm with an unlisted Z-line and GE junction. The corresponding UV AF image is of the same esophagus biopsy specimen. It should be considered for correlation purposes that features viewed from the surface with AF microscopy (endoscopist view) varied in diameter from H&E section (pathologist view of the biopsy specimen cross-section). The suspicion of LGD was histopathologically defined by the closely packed overlapping basal nuclei with hyperchromasia and irregular contours of H&E surface morphology, although the overall epithelial architecture appeared to be preserved, maintaining a degree of uniformity at the surface. Based on the criteria for the recognition in the AF images presented in the previous section, goblet cells, each with a diameter of approximately 10 to 15 µm, are shown in the AF image of this specimen. A goblet cell without mucin (dark), and with mucin (bright) are specified with lines and shown side by side. The indicated crypt of Lieberkühn was approximately 313 µm along the horizontal axis. A gland that had become inflamed and angulated resulting in visible architectural distortion is indicated at the lower right of the figure. Only the lower portion remains, resulting in an apparent triangular shape instead of the normal doughnut shape. This distorted and inflamed gland was approximately 104 µm in diameter with the villi apex beginning to project from the mucosa surface, possibly becoming further distorted during tissue biopsy removal. The cluster of four glands approximately 200 µm subsurface might attribute to the visible irregular contours at the surface. These microstructures can be seen from the luminal-plane AF image corresponding to the same indicator lines of the H&E image. Surface features in the UV AF image can be correlated with the 313 µm in diameter crypt of Lieberkühn gland. This feature had a target-like appearance with light and dark alternate rings and a diameter of approximately 30 to 35 µm. Similar features were observed throughout the same figure. Based on previous observations in various specimens, we hypothesize that the dark center represented the opening of a crypt toward the lumen of the gland,³² shown in Fig. 2. The inconsistency of visibility could be attributed to the difference of height projection of villi into the luminal plane. Visible architectural distortion from both the AF epithelial surface as well as the H&E image may be due to the gland that had become inflamed and angulated.

Fig. 5

The H&E gold standard $361\times 475\mathrm{\mu m}$ cross-sectioned view of human esophagus biopsy tissue with intestinal metaplasia (BE) features histopathologically determined crypts of Lieberkühn, goblet cells, subsurface glands, and villi apices. The luminal view of the accompanying AF image is of the same specimen with lines indicating what could be visibly correlated microstructures. A goblet cell without mucin (dark) and one with mucin (bright) are shown side by side.

The AF images shown in Fig. 6 were from two different patients and represented the images obtained from specimens with varying grades of BE. The biopsy specimen shown in Fig. 6(a) was collected at 39 cm (GE junction unlisted) and returned a pathology report of BE with reactive atypia. The biopsy specimen shown in Fig. 6(b) was collected at 32 cm, 1 cm above the Z-line (33 cm), with a GE junction located at 35 cm, and returned a pathology report of BE with moderate acute and chronic inflammation, indefinite for dysplasia. The optical features within the honeycomb structure (defined in Fig. 1), were previously assigned to represent villi, crypts, and goblet cells of the small intestine (see Fig. 2), and can be easily recognized with the progression to BE throughout the esophagus AF images shown in Fig. 6. For example, a large, dark feature that may represent villi apices is indicated with line 1 in Fig. 6(a). Villi may not be immediately apparent because a regular villous appearance was not always a characteristic of BE as it is of the duodenum. Line 1 in Fig. 6(b) indicates a feature that is consistently seen scattered throughout a honeycomb background. These features could be the formation of crypts of Lieberkühn as in Fig. 2. Dark and bright features were also visible and could be correlated to non-mucin and mucin filled goblet cells, indicated respectively with lines 2 in Fig. 6(b). The surface of the columnar mucosa retained a degree of normality and smoothness, an indication that dysplasia had not yet developed. The categorization of “indefinite for dysplasia” might not be visible from the surface epithelium, as this histologic abnormal progression was diagnosed using a traditional cross-section of tissue on a slide. Specific to these microstructures, it may be unlikely that the dark features, which are visible at higher density than those shown in Figs. 4 and 5 and identified as crypts, are nuclei, since the elongated nuclei of esophageal columnar epithelial cells are located toward the basal surface beyond the superficial imaging depth of this technique.³² Rather, the increased frequency and density of features believed to be goblet cells and crypts of Lieberkühn may be attributed to disease progression as healthy squamocolumnar epithelium of the esophagus begins to transition toward intestinal metaplasia (Barrett’s esophagus) that more closely resembles duodenal epithelium, shown in Fig. 2.

Fig. 6

$475\times 361\mathrm{\mu m}$ field of view AF image of sections of human esophagus biopsies specimens of (a) BE with reactive atypia, and (b) BE with moderate acute and chronic inflammation, indefinite for dysplasia. Features indicated with lines are consistent with those identified in Fig. 2, possibly representing a villous apex [line 1, (a)], crypt of Lieberkühn [line 1, (b)], and goblet cells [line 2, (b)].

3.5.

Low-Grade Dysplasia in BE

The esophagus AF images shown in Fig. 7 were from three different patients with a pathological diagnosis of LGD. The optical features of what are believed to represent villi, crypts, and goblet cells (defined in previous sections) could be recognized throughout Fig. 7. For example, the image of Fig. 7(a) has a very similar appearance to the BE images of Fig. 5. This biopsy specimen was collected from 34 cm just above the GE junction at 35 cm. The villi appear to maintain a degree of regularity as they are seen projecting from the surface epithelium. It is also possible that the rounded features are cardia-type mucosa biopsied near the top of a hiatal hernia. There is a cloudy region in the upper right with bright streaks that might be a layer of mucous or vasculature appearing to overlay the luminal surface. The images of the tissue biopsy specimens shown in Figs. 7(b) and 7(c) have visible projections that obscure the flat surface of normal mature squamous epithelium, exemplified in Fig. 1. The overall surface pattern in the lower half of Fig. 7(b) appears to remain intact. The biopsy was collected from 36 cm. The snaking pattern in the upper half of Fig. 7(b) might represent the formation of pseudostratification extending to the surface, a cytology hallmark of LGD. The bright streaks across the center might indicate vascularization from injury or recovery from previous radiofrequency (RF) ablation therapy. Villi that appear to be forming in the lower half of Fig. 7(c) maintained a regular appearance more similar to those of the duodenum in Fig. 2. The Z-line was at 39 cm, and the GE junction was at 40 cm, the location of biopsy specimen collection. The image was increasingly out of focus as the surface became more three-dimensional, as opposed to the pavement-like surface of stratified squamous epithelium shown in Fig. 1.

Fig. 7

$475\times 361\mathrm{\mu m}$ field of view human esophagus biopsy specimens of (a) squamocolumnar mucosa with LGD, (b) BE with LGD, and (c) BE with LGD and visibly distorted surface architecture. Visible surface projections obscure the flat surface when compared with normal squamous and columnar epithelium, exemplified in Fig. 1.

3.6.

High-Grade Dysplasia and Esophageal Adenocarcinoma

Figure 8 illustrates UV AF images of HGD and EAC. Each esophagus biopsy specimen shown in Fig. 8 was taken from a different patient. It can be easily appreciated that these images exhibit a high degree of deviation from the normal esophagus epithelium shown in Fig. 1. All of the images had an increasingly disorganized three-dimensional aspect, characteristic complex mucosal architecture, and villiform configuration that currently defines HGD and EAC. The image shown in Fig. 8(a) was obtained from a biopsy specimen with pathological diagnosis of squamocolumnar mucosa with HGD. The three-dimensional globular features visible on the mucosal surface cause a crowded villous topography and are reflective of HGD. Surface nodularity might also imply an early onset of carcinoma. Figs. 8(b) and 8(c) both demonstrated a more cauliflower-like appearance than HGD, indicating EAC. Fig. 8(b) was obtained from a specimen with pathological diagnosis of focal adenocarcinoma in a background of high- and low-grade dysplasia. The villiform surface and disorganized mucosal pattern were easily differentiated from the stratified squamous epithelium of normal esophagus shown in Fig. 1. Mucosal crowding and disorganization were visible in the AF image and correlated with pathological assessment. Villiform features were visible as cauliflower-like projections, 10 to 30 µm in diameter, and corresponded to malignant features observed in the H&E section of this specimen. Features that correspond to crypts and goblet cells with a diameter of approximately 15 µm were also visible. Pathological diagnosis of EAC with similar image characteristics displaying an increasingly villiform surface and a disorganization of mucosal pattern can also be recognized in the images shown in Figs. 8(c) and 8(d). In Fig. 8(d), the pathological diagnosis was poorly differentiated adenocarcinoma. Margin delineation capability is exemplified, as the tissue on the left of the image resembled squamous mucosa shown in Fig. 1, while the tissue on the right of the image had more of a continuous mucosal and a cribriform growth pattern, similar to the pathologically diagnosed EAC shown in Fig. 8(c). There is a visible decrease of pattern regularity with progression of disease observed as tissue transitions from HGD [see Fig. 8(a)] to EAC [see Figs. 8(b)–8(d)]. Additionally, EAC was characterized by the proliferation of small, irregular glands that contributed to the overall cauliflower-like appearance that was especially visible in Figs. 8(b) and 8(c). Currently it is extremely difficult to differentiate HGD and EAC,¹² and a histologic criterion that defines a diagnosis of intramucosal cancer has not yet been published.¹³ Figure 9 provides an optical histological progression of esophageal morphology, from normal squamous to invasive carcinoma. The epithelial distortion is obvious even to the untrained eye. A preliminary set of optical rules for interpretation is tabulated in the “Optical Image Morphology” column of Table 1.

Fig. 8

$475\times 361\mathrm{\mu m}$ field of view AF images of human esophagus biopsy specimens obtained from different patients who were diagnosed with (a) HGD, (b) focal adenocarcinoma in a background of high- and low-grade dysplasia, (c) EAC, and (d) poorly differentiated EAC with obvious margin delineation between normal and diseased tissue.