Endomicroscopy of Barrett’s Esophagus

Endomicroscopy is a remarkable technical advance in gastrointestinal mucosa imaging. In 2003, Kiesslich and colleagues described the first human use of contrast-aided confocal laser endomicroscopy (CLE) as a novel technique for in vivo microscopic imaging of the gastrointestinal mucosa. Both probe-based and endoscope-based systems have been applied to many gastrointestinal disorders, including Barrett’s esophagus (BE) and associated neoplasia. Probe-based confocal laser endomicroscopy can be used in conjunction with highresolution white light endoscopy and other contrast enhancement techniques. It has proven high accuracy for prediction of high-grade neoplasia and cancer. In vivo imaging of both flat BE and mucosal lesions can influence diagnosis and thereby impact upon decision making regarding tissue sampling and endoscopic therapy. This article discusses the scientific literature related to clinical use of CLE for BE, the techniques for performing CLE in the esophagus, and the potential future directions for CLE in BE and esophageal cancer diagnosis and treatment.

Histopathologic examination of formalin-fixed hematoxylin-eosin stained mucosal specimens has been the reference diagnostic standard for gastroenterology. In the last decade, it has become possible to perform virtual histology during gastrointestinal endoscopy through the miniaturization of a laser scanning microscope with confocal optics. High-resolution microscopic images are generated by illuminating the area of interest with a low power argon blue laser (488 nm) and collecting the reflected fluorescence through a small aperture. Confocal pinhole imaging enables rejection of out-of-focus laser light and computer-based reconstruction to generate an optical section of the tissue at a specific depth or plane. The microscopic images of the mucosa are collected at a fixed depth (probe-based confocal laser endomicroscopy [pCLE]) or at multiple but limited depths from the surface (endoscope-based confocal laser endomicroscopy [eCLE]).

Confocal laser endomicroscopy is a technique that involves a scanning laser light coupled with a fluorescent agent to generate highly magnified microscopic images of the gastrointestinal mucosa. CLE without use of a contrast agent was first reported by Japanese investigators in 2003 when a CLE probe prototype was used ex vivo in resected colon specimens and in vivo in one healthy volunteer to visualize colonic mucosa. The first use of contrast-aided CLE was reported by Kiesslich and colleagues from Mainz, Germany. Using both acriflavine and fluorescein, the investigators reported a highly accurate prediction of colonic neoplasia.

Endomicroscopy platforms and equipment

There are currently 2 confocal endomicroscopy systems available: a confocal endoscope (eCLE), the EC3870CILK (Pentax, Tokyo, Japan), and a probe-based confocal endomicroscopy (pCLE), the Cellvizio (Mauna Kea Technologies, Paris, France).

Both the eCLE and pCLE systems allow visualization of microscopic cellular and vascular patterns. Table 1 details the technical aspects of each system and highlights the differences. For eCLE, a gastroscope EG-3870CILK and colonoscope 3870CIFK are available (Pentax, Tokyo, Japan). These endomicroscopes connect to a standard Pentax processor (for endoscopy) as well as the endomicroscopy computer processor. The current endoscopes are outfitted with standard-resolution white light optics. The shorter gastroscope-endomicroscope has centimeter markings at the distal end to facilitate measurements of distance of the endoscope tip from patients’ teeth. The buttons, wheels, accessory channel, air-water and suction valves, shaft diameter (12.8 mm), and handling of the endomicroscope are similar to a videocolonoscope. The endomicroscope provides submicron resolution with a large field of view (see Table 1 ) of 475 μm × 475 μm. Furthermore, the direction of scanning and depth of imaging are controlled by buttons at the endoscope head and allow subsurface detailed analyses or continued imaging within the same plane/depth from the surface. The eCLE can be combined with chromoendoscopy for additional mucosal enhancement. Finally, the scanning speed can be varied from slow or 0.8 images per second (with higher resolution 1024 × 1024 pixel images generated) or fast rate (1.6 images per second [with 1024 × 512 pixel images]).

Table 1

Features of the available endomicroscopy systems

	Endoscope (eCLE)	Probe (pCLE)
Company	Pentax	Mauna Kea Technologies
Devices	Upper endoscope colonoscope	Gastro/Coloflex	Gastro/Coloflex UHD	Cholangioflex
Imaging depth (μm)	0–250	70–130	55–65	40–70
Field of view (μm)	475 × 475	600	240	320
Lateral resolution (μm)	0.7	3.5	1.0	3.5
Axial resolution (μm)	7	15	5	—
Diameter (mm)	12.8	2.7	2.5	1.0

For the pCLE system, the Cellvizio system can be used with a variety of miniprobes that can be passed through the endoscope accessory channel ( Fig. 1 ). These miniprobes come in different lengths for use in the upper gastrointestinal (GI) tract, colon, and bile duct. The lateral resolution of the different probes also varies (see Table 1 ) such that the probes with the highest resolution (1 μm) have a smaller field of view (240 μm) and the lower resolution probes (3.5 μm) have larger fields of view. The imaging depth is fixed for a particular probe. Importantly, the probe-based microscopic system enables flexibility of use with any endoscope and coupling of high-resolution endoscopy with other digital mucosal enhancements, such as narrow band imaging (Olympus Tokyo, Japan), IScan (Pentax, Tokyo, Japan), or FICE (Fujinon Tokyo, Japan). Scanning speed for the confocal microscope probes is 12 images per second, similar to video. Software enhancements allow stitching of the confocal images into a mosaic, which allows inspection of a wider field of the mucosa within the same plan. Confocal probes have a limited number of uses, which increases cost.

Both CLE systems allow image capture, storage, magnification, and export using either Windows-based (eCLE) or Macintosh-based (pCLE) computer systems. Management of CLE images is important for intraprocedure and postprocedure review. Both systems currently do not have a scroll function or cine-loop of images, similar to that in endoscopic ultrasound systems. Magnification within captured images can facilitate visualization of minute subcellular and extracellular structures, such as Helicobacter pylori and intramucosal bacteria.

Contrast agents

Exogenous fluorescent contrast agents are currently necessary for endomicroscopic imaging using the commercially available single-photon systems. The natural fluorescence of the GI mucosa is not intense enough to allow detailed microscopic imaging, unlike autofluorescence imaging.

There are 2 general types of contrast agents: intravenous and topical. The most commonly used endomicroscopy contrast agent is intravenous fluorescein sodium. Fluorescein has been used for decades by ophthalmologists for imaging of the retinal vasculature. To date, it has not been approved by the US Food and Drug Administration (FDA) for use with CLE. Intravenous fluorescein sodium has been used for decades and has a high safety profile. Adverse events are rare. Other than yellowing of the skin, eyes, and urine in all patients that lasts several hours, complications with fluorescein are unusual. Based on a large multicenter retrospective study, the incidence of fluorescein-related adverse effects is 1.4%, including nausea, hypotension, rash, and injection site erythema. Intravenous fluorescein highlights the vessels, intracellular spaces, and lamina propria, but does not stain nuclei. With fluorescein, intraepithelial mucin appears dark caused by acid pH. Hence, the hallmark of goblet cells in Barrett’s esophagus (BE) (as well as those in the small bowel and colon) appears dark. Vessels and capillaries appear bright, and red blood cells can be seen as dark ovals. After injection of 2.5 to 5.0 mL of fluorescein, the epithelial cells should be readily visible. Fluorescein typically lasts about 30 minutes. Some endoscopists may start with 2.5 mL injection and give a second dose of 2.5 mL if needed for longer procedures.

Topical contrast agents, such as acriflavine and cresyl violet, can also be used during eCLE and pCLE. These two agents are also not specifically approved by the FDA for CLE. Acriflavine dye is a topical antiseptic and agent for freshwater and marine aquariums. In CLE, 0.05% acriflavine has been used as a contrast agent during endomicroscopy. Unlike fluorescein, it stains the nuclei of cells. It can be used in combination with fluorescein for maximum visualization of cellular and subcellular structures. Topical acriflavine has been used in the gastrointestinal tract to image H pylori, colon polyps, oral and oropharyngeal mucosa, and acute graft-versus-host disease in the colon . Outside the GI tract, it has been used in combination with acetic acid during cervical colposcopy to detect cervical intraepithelial neoplasia. Acriflavine is used much less frequently because of concern about its mutagenic potential. Topical cresyl violet 0.25% to 1% is a dye that can also be used for chromoendoscopy. Cresyl violet highlights the cytoplasm and enables visualization of nuclei.

Contrast agents

Exogenous fluorescent contrast agents are currently necessary for endomicroscopic imaging using the commercially available single-photon systems. The natural fluorescence of the GI mucosa is not intense enough to allow detailed microscopic imaging, unlike autofluorescence imaging.

There are 2 general types of contrast agents: intravenous and topical. The most commonly used endomicroscopy contrast agent is intravenous fluorescein sodium. Fluorescein has been used for decades by ophthalmologists for imaging of the retinal vasculature. To date, it has not been approved by the US Food and Drug Administration (FDA) for use with CLE. Intravenous fluorescein sodium has been used for decades and has a high safety profile. Adverse events are rare. Other than yellowing of the skin, eyes, and urine in all patients that lasts several hours, complications with fluorescein are unusual. Based on a large multicenter retrospective study, the incidence of fluorescein-related adverse effects is 1.4%, including nausea, hypotension, rash, and injection site erythema. Intravenous fluorescein highlights the vessels, intracellular spaces, and lamina propria, but does not stain nuclei. With fluorescein, intraepithelial mucin appears dark caused by acid pH. Hence, the hallmark of goblet cells in Barrett’s esophagus (BE) (as well as those in the small bowel and colon) appears dark. Vessels and capillaries appear bright, and red blood cells can be seen as dark ovals. After injection of 2.5 to 5.0 mL of fluorescein, the epithelial cells should be readily visible. Fluorescein typically lasts about 30 minutes. Some endoscopists may start with 2.5 mL injection and give a second dose of 2.5 mL if needed for longer procedures.

Topical contrast agents, such as acriflavine and cresyl violet, can also be used during eCLE and pCLE. These two agents are also not specifically approved by the FDA for CLE. Acriflavine dye is a topical antiseptic and agent for freshwater and marine aquariums. In CLE, 0.05% acriflavine has been used as a contrast agent during endomicroscopy. Unlike fluorescein, it stains the nuclei of cells. It can be used in combination with fluorescein for maximum visualization of cellular and subcellular structures. Topical acriflavine has been used in the gastrointestinal tract to image H pylori, colon polyps, oral and oropharyngeal mucosa, and acute graft-versus-host disease in the colon . Outside the GI tract, it has been used in combination with acetic acid during cervical colposcopy to detect cervical intraepithelial neoplasia. Acriflavine is used much less frequently because of concern about its mutagenic potential. Topical cresyl violet 0.25% to 1% is a dye that can also be used for chromoendoscopy. Cresyl violet highlights the cytoplasm and enables visualization of nuclei.

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