Introduction

Confocal laser endomicroscopy (CLE) is one of the newer advanced imaging methods for the gastrointestinal tract. Microscopic images of the gastrointestinal mucosa are obtained by illuminating the mucosa with blue laser light (488 nm), which causes fluorescence. The light reflected is collected through a pinhole-sized aperture and processed, creating a microscopic image. The laser light and collected light are ‘confocal’, meaning they are in the same focal plane. The images produced allow visualization of small structures, such as capillaries and colonic crypts and gastric pits (Figs 1–4), as well as individual cells, such as epithelial cells and red blood cells.

Figure 1 Endomicroscopy of the colonic mucosa. The colonic crypts (solid arrow) and goblet cells (dashed arrow) can be seen.

Figure 2 Normal gastric mucosa seen by endomicroscopy.

Figure 3 Mucosa of the squamous esophagus. Intrapapillary capillary loops are visible.

Figure 4 Normal duodenal mucosa. Small goblet cells (dark) are visible.

1 Indications

Endomicroscopy has been used to study many gastrointestinal disorders. Several of the earliest studies looked at patients undergoing colorectal cancer screening and examined polyps with endoscope-based endomicroscopy (eCLE). Other colonic disorders studied include patients with inflammatory bowel disease undergoing surveillance for dysplasia, collagenous colitis, and pouchitis. Small bowel disorders investigated with endomicroscopy include celiac disease and graft-versus-host disease. Gastric cancer and Helicobacter pylori gastritis have been imaged with CLE. In the esophagus, Barrett’s esophagus and squamous cell esophageal cancer have been studied. Endomicroscopy has been used to help target biopsies in all these disorders as well, and may also reduce the number of biopsies needed to achieve a diagnosis. Examination of biliary strictures at ERCP to differentiate cholangiocarcinomas from benign strictures has recently been reported.

2 Equipment

There are currently two endomicroscopy systems available (Table 1). One is an endoscope-based endomicroscopy (eCLE) system, the EC-3870CIFK colonoscope, and EG-3870CIK upper endoscope (Pentax, Tokyo, Japan). A probe-based endomicroscopy (pCLE) system, the Cellvizio (Mauna Kea Technologies, Paris, France) is also available. Both systems allow standard endoscopic imaging while providing the ability to obtain microscopic views of the mucosa, but there are several differences between the two systems. Each system has an endomicroscopic image processor and separate screen for viewing endomicroscopic images. The confocal endoscope comes in lengths appropriate for colonoscopy and one for upper endoscopy, although the colonoscope-length endoscope can also be used for investigation of the upper GI tract. The confocal endoscope has the standard wheels, air, water, suction and photo buttons, and a standard-size biopsy channel. The miniprobes for pCLE can be used with a standard endoscope that has a 2.8 mm channel and the probes are attached to a special processor. They come in lengths appropriate for upper endoscopy, colonoscopy, and cholangioscopy. Both systems require a contrast agent to be used to collect images. The confocal endoscope can image sequentially from the surface, down to a depth of 250 µm, while the confocal probes have set ranges of imaging depth, ranging from 55–65 µm from the surface for the Gastroflex UHD probe to 70–130 µm for the Gastroflex probe. The resolution of the images is higher with the confocal endoscope than the probes, with a lateral resolution of 0.7 µm compared with 1–3.5 µm. The imaging rate for the confocal probe is higher than the confocal endoscope, with an imaging rate of 12 images per second compared with 0.8–1.6 images per second. The pCLE system also creates a ‘mosaic’ of images collected together to show a larger portion of the mucosa. Both systems allow image capture and export.

3 Contrast agents

Several contrast agents can be used for imaging with the confocal endoscope and the confocal probe systems. Fluorescein sodium is the most commonly used contrast agent and is used in ophthalmology for retinal vascular imaging. The standard dosing with the confocal endoscope is 5 mL of 10% fluorescein sodium, given intravenously. In studies using the confocal probe, 2.5–10 mL of 10% fluorescein sodium is used. Fluorescein highlights the vessels and intracellular spaces, and lamina propria of tissues, but does not stain nuclei. Goblet cells in the colon, small bowel, and in Barrett’s esophagus appear dark when fluorescein is used for contrast. Capillaries appear bright, with individual red blood cells visible. All patients who receive fluorescein sodium intravenously will have yellowing of the skin, eyes, and urine that lasts several hours. Rare allergic reactions have been reported with fluorescein sodium and some patients may have nausea. Topical acriflavine 0.05% has also been used as a contrast agent during endomicroscopy and stains the nuclei of cells. However, acriflavine is used less frequently, as it binds nuclei and there is concern for potential mutagenicity. Topical cresyl violet 0.25–1% has been used in a few studies and is also helpful as it functions as a surface chromoendoscopy agent. Cresyl violet highlights the cytoplasm, thus some nuclei can be seen and appear dark.

4 Technique

When performing either eCLE or pCLE, complete the white light portion of the endoscopic exam before proceeding with endomicroscopy. This will allow you to select areas to image and will ensure that your contrast agent is still present when you are ready to begin imaging. If you are using topical contrast, clearing the mucosa with water may help you get more even staining of the mucosa. In the colon, a poor bowel preparation will significantly limit the use of topical contrast agents as they will not reach the mucosa and will also limit imaging with intravenous contrast, due to the presence of stool on the imaging window.

When ready to obtain eCLE images, place the tip of the confocal endoscope directly on the mucosa. The imaging window is located on the lower left portion of the tip and can be seen on the edge of the endoscopic image (Fig. 5). Applying suction using the endoscope can help stabilize your position. Once a stable position is obtained, press the home button (button 3), which will return the imaging to the surface (Fig. 6). Press button 4 to begin sectioning down through the mucosa. Depressing the button moves the imaging plane 4 µm deeper. The direction of imaging can be reversed towards the surface by quickly depressing button 4 twice. Microscopic images can be captured using the foot pedal, the mouse, or the touch screen.

Figure 5 The tip of the confocal endoscope has the normal light channel, and air and water channel, but also has the confocal microscope built into the tip (arrow).

Figure 6 The handle of the confocal endomicroscope. Button 3 (solid arrow) and button 4 (dashed arrow) are used to return the imaging plane to the surface and to section down and up through the mucosa.

To use the pCLE system, the probes are attached to the processor and passed through the instrument channel of a standard endoscope. The tip of the probe is placed directly on the surface of the mucosa and images are acquired. To obtain a stable image with the confocal probe system, a plastic cap on the end of the endoscope can be helpful, such as the plastic caps that come with the endoscopic mucosal resection (EMR) kits. Images can be obtained and saved, as can mosaic video sequences.

5 Complications

Complications during endomicroscopy are rare. The standard risks of endoscopy are present and the additional risk is related to the contrast agent used. All patients who receive fluorescein should be alerted that they will have yellow skin, eyes, and urine for several hours after the procedure.

In one review of 2272 eCLE and pCLE endomicroscopy cases performed at 16 academic medical centers, no serious adverse events were reported. The most common mild adverse reactions reported (1.4% of patients) were nausea and vomiting, transient hypotension, rash, injection site erythema, and epigastric discomfort. However, the package insert for fluorescein sodium and ophthalmologic reviews list several other reported complications, such as seizures, hypotension, syncope, wheezing, thrombophlebitis, and anaphylaxis.

6 Special considerations

Introduction

The prognosis of gastrointestinal cancers depends upon their stage at presentation and most recent improvements in diagnostic endoscopy have therefore focused on earlier diagnosis. The introduction of videoendoscopy over 20 years ago was considered a major advance but merely hinted at the technological improvements that were possible in endoscopy. Progress since then has focused on improving image resolution, particularly by using optical or electronic magnification and by increasing the number of pixels and photodiodes per pixel. Monochromatic light or light containing only certain wavelengths or narrow spectral bands and use of wavelengths outside the visible light spectrum (ultraviolet or near infrared) have all recently been developed for endoscopy, as alternatives to standard white light of the visible spectrum. Combined with improvements in image definition and magnification, these novel imaging modalities offer enormous possibilities for diagnosis of early neoplasia. Multiple systems exist either commercially or as prototypes: narrow band imaging (NBI) endoscopy; confocal laser endomicroscopy (CLE); autofluorescence (AFI); optical coherence tomography (OCT); endocytoscopy; spectral fluorescence, and Raman effect or light-scattering spectroscopy. Two strategies are evolving beyond the impressive technological progress in miniaturizing the charge coupled devices (CCDs) at the tip of a flexible videoendoscope:

To achieve the first of these objectives, NBI and AFI techniques are undergoing clinical validation, while for the second, methods involving CLE, endocytoscopy, OCT or elastic or non-elastic light-scattering spectroscopy (LSS) are being developed. NBI, AFI and CLE methods are currently available commercially, while OCT and other methods are only at the prototype stage of development. The use of these technologies is set to expand, but there are currently few controlled, randomized comparative studies to determine precisely the utility of these new tools in endoscopy. The range of potential applications is wide, but for the moment the main focus is on areas as outlined in Box 1.

1 Technical principles

White light of the visible light spectrum is characterized by a wavelength of 400–700 nm. This light beam is partly absorbed in its tissue target depending on the wavelength but also on the composition of naturally occurring tissue fluorophores and chromophores, and on their vascularity.