# Progress and Challenges of Global High-Resolution Endoscopy

##### Sheena Bhushan1,2*, Rebecca Richards-Kortum3 and Sharmila Anandasabapathy1,2

1Section of Gastroenterology and Hepatology, Department of Medicine, Baylor College of Medicine, USA

2Baylor Global Health, Baylor College of Medicine, USA

3Department of Bioengineering, Rice University, USA

# Abstract

In recent years, gastrointestinal endoscopy has seen an influx of high-resolution endoscopic technologies that are capable of providing optical biopsies of the epithelial surface in real time. Upcoming high-resolution endoscopy techniques are CLE, OCT, EC and HRME. Powered by automated diagnostic algorithms and more-cost effective alternatives, these high-resolution endoscopic technologies have the potential to not only facilitate real-time decision making, but also improve screening and surveillance of gastrointestinal cancers globally. This review discusses the most promising upcoming optical biopsy technologies and their clinical applications, along with the current trends, challenges and future directions.

# Introduction

Recent advances in endoscopic technology have improved mucosal visualization, lesion detection and also allowed for real-time optical diagnosis. High definition and optical contrast imaging technologies such as narrowband imaging, iScan, blue laser imaging, and autofluorescence have been used as complementary, enhancement techniques for white light endoscopy. These technologies are used as "widefield imaging" techniques that help unmask or highlight inconspicuous mucosal abnormalities not visible to the human eye. While these techniques are invaluable as "red flag" techniques that highlight suspicious areas for further assessment with other imaging modalities, and are considered high resolution, they cannot yield cellular and subcellular imaging. Examples of some newer high-resolution endoscopic technologies include Confocal Laser Microendoscopy (CLE), Optical Coherence Tomography (OCT), Endocytoscopy (EC) and High Resolution Microendoscopy (HRME) (Figure 1). These technologies can image the gastrointestinal mucosa at a cellular and subcellular level and thus provide a higher degree of resolution as compared to the widefield imaging techniques. By providing subcellular imaging of the epithelial surface, these 'optical biopsy' technologies have the potential to revolutionize endoscopy and can offer a more targeted, efficient and cost-effective approach to endoscopic screening and surveillance. This review will focus on these emerging ultra-high-resolution endoscopy technologies and aims to provide a technical overview of the technologies, their clinical applications and also discuss the challenges associated with them.

Figure 1: Classification of endoscopic imaging techniques. View Figure 1

# Technical Overview

## Confocal laser endomicroscopy

Confocal laser endomicroscopy (CLE) is an endoluminal imaging technique that enables real-time, high-resolution assessment of the gastrointestinal mucosa at a cellular and subcellular level. By providing in vivo "optical biopsies", it has expanded the current scope of standard flexible endoscopy significantly. The term "confocal" refers to the alignment of both illumination and collection systems in the same focal plane [1]. Fluorescence CLE uses the principle of excitation of a fluorophore with low-power laser light (488 nm), followed by subsequent detection of the remitted fluorescent light through a conjugate pinhole [2]. Light emitted from a specific depth is refocused at the conjugate plane and passes through the pinhole confocal aperture. As a result, light emitted from above and below the plane of interest is not detected, and the point of illumination coincides with the point of detection within the specimen, dramatically improving spatial resolution [3,4]. The emission of the fluorophore at the point of illumination is recorded to create a pixelated two-dimensional image of the tissue sample. Multiple points are analyzed, and the fluorescence emission is displayed as a gray scale-image. Three-dimensional (3-D) images can be created by using bench-top confocal microscopes. The high spatial resolution of CLE makes in vivo microscopic imaging of gastrointestinal mucosa possible. Currently, two CLE platforms are approved by the FDA: 1) Endoscope-based confocal laser endomicroscopy (eCLE); 2) Flexible fiber based confocal miniprobe.

Confocal laser endomicroscope (eCLE): While eCLE was developed and evaluated in multiple clinical studies, the device is not commercially available at present. The confocal laser endomicroscopy (Pentax, Tokyo, Japan), uses a miniaturized confocal scanner that is integrated into the distal end of a standard endoscope and allows the endoscopist to use white-light endoscopy and eCLE simultaneously [5]. The system consists of two additional buttons on the endoscope, that allow the endoscopist to control the depth of imaging relative to the surface of the tissue in a 7 μm increments, to a maximum of 250 μm [6]. eCLE has a field of view is 475 × 475 μm and a lateral resolution of 0.7 μm [6,7]. The confocal images are collected at a scan rate of 0.8 frames/seconds (1024 × 1024 pixel) or 1.6 frames/second (1024 × 512 pixels) and displayed on a separate monitor next to the WLE image monitor [6,7]. Image stabilization is crucial to obtain high-quality images. While the eCLE can be used to examine luminal structures in the upper and lower gastrointestinal tracts, it is still quite large and cannot be used for biliary intraductal examinations [5].

Probe-based confocal laser endomicroscopy (pCLE): Alternately, a flexible Probe-based confocal laser endomicroscope (pCLE) can also be used. The system (Cellvizio, Mauna Kea Technologies, Paris, France) uses a confocal microscope that is introduced as a miniprobe through the accessory channel of a standard endoscope [5]. The mini probe is exceptionally flexible, with a diameter of 0.9 to 0.255 mm and thus can be easily introduced through the accessory channel of virtually any endoscope (including a cholangioscope, bronchoscope) [5]. Both the light source and the laser scanning units are outside the human body, with the miniprobe acting as a passive conduit. The fiber probe consists of up to 30,000 optical fibers that transmit the 488 nm laser light and then also return the reflected fluorescent light to the distal micro-objective. Confocal images are collected at a rate of 12 frames per second, allowing in vivo imaging of capillary blood flow. Unlike eCLE it has a slightly lower resolution, a smaller field of view, and a fixed depth of imaging. Depending on the type of probe used, the field of view ranges from 240-600 um, the lateral resolution ranges from 1-3.5 um and the depth of imaging ranges from 0-100 um [5]. Cholangioflexprobe is a newer miniaturized pCLE with a diameter of 0.9 mm, that is small enough to be introduced into cholanigoscopes and can be used to examine pancreatic and biliary structures [8]. A further miniaturized device called the needle-based CLE (nCLE) has a probe that can be passed through a 19G-FNA needle, thereby enabling endoscopic ultrasound-guided CLE of solid organs, lymph nodes, and cystic lesions [5]. The probes can be disinfected and reused up to 10-20 times, after which they require replacement [5].

Fluorescent contrast agents: Since CLE relies upon tissue fluorescence, exogenous fluorescence contrast agents are needed. The most commonly used contrast agent is IV fluorescein which is given to the patient intravenously. It allows visualization of cellular architecture by highlighting the vasculature, lamina propria and intracellular spaces of the tissue under examination [9]. Fluoresceins are nontoxic and excreted renally. They are sometimes associated with rare adverse events including hypotension without shock (0.5%), nausea (0.39%), injection site redness (0.35%), diffuse self-limited rash (0.04%), mild epigastric pain (0.09%) and anaphylaxis (uncommon but has been reported) [10]. However, since flouresceins do not stain the nuclei, they cannot be used to assess nuclear-to-cytoplasmic ratio, used for diagnosis and grading intraepithelial neoplasia. Nuclear staining can be accomplished using topical contrast agents that are sprayed on to the mucosa directly. Acriflavine (0.05% in saline) accumulates within the nuclei and has been used in evaluation in several studies but theoretical concerns around mutagenicity have limited widespread adoption [11]. Cresyl violet (0.13% in acetic acid) is another topical agent that causes cytoplasmic enrichment leading to a negative visualization of nuclear morphology [12].

Obtaining biopsies: The eCLE is integrated into a standard scope, freeing the accessory channel which is used to obtain biopsies. However, since pCLE systems occupy the accessory channels, biopsies can only be obtained after removing the probe. This limitation can be overcome by creating a dimple before removing the miniprobe, can help demarcate the area of interest [13].

# Endocytoscopy (EC)

Endocytoscopy is another emerging endoscopic technology that allows real-time, high-resolution assessment of gastrointestinal mucosa at a cellular and sub-cellular level. It differs from CLE in that it uses the principle of white-light contact microscopy i.e., optical lenses are placed direct contact with the tissue to achieve high-level magnification [14]. Unlike CLE, there is no confocal plane, hence only the superficial layer of mucosa can be imaged. To visualize the nuclei and other cellular and subcellular structures, topical application of methylene blue (0.5% to 1%) or a combination of methylene blue with crystal violet is used [15]. Mucus often hampers contrast penetration, staining and hence visualization of cell details; therefore, prior treatment with a mucolytic agent like N-acetylcysteine is typically done [16]. After application of the stain, the tip of the endoscope is placed in direct contact with the mucosa, and the mucosal surface is scanned condensed with white light. The fixed-focus high-power optical lens then projects ultra-magnified images from a small mucosal sampling site (less than 0.5 mm in diameter) on to a Charged-Coupled Device (CCD). EC allows for visualization of several cytological and architectural features including, size and arrangement of cells, the size, and shape of nuclei, the nuclear-to-cytoplasmic ration and other atypia [15].

## Endocytoscope

Two types of endocytoscopes (Olympus Instruments, Tokyo, Japan) are currently available: The first type is a probe-based endocytoscope, that consists of two flexible catheters that provide a magnification of 450-1125-fold (on a 14-inch monitor) or 500-1400-fold (using a 19-inch monitor) [17,18]. Both probes can be passed through the working channels of standard endoscope and be placed in direct contact with tissue for imaging. To provide stability and minimize motion artifact, a soft plastic cup at the tip of the endoscope is generally used. The second and the only commercially available endocytoscope system is integrated within an endoscope [19]. In addition to having conventional optical magnification, it also provides and image magnification of 580-fold on a 19-inch monitor. The tip of the endoscope is placed in direct contact with the tissue to obtain images.

# Optical Coherence Tomography (OCT) and Volumetric Laser Endomicroscopy

Optical Coherence Tomography (OCT) was originally used in ophthalmology, and more recently, there have been several studies using OCT in the gastrointestinal tract [20]. OCT is an exciting field of gastrointestinal imaging technology that relies on the backscattering of light to obtain cross-sectional images of tissue [21,22]. It is similar in principle to ultrasonography but uses light waves instead of sound waves to generate images [23]. OCT is performed using different probes that can be introduced through the accessory channel of a standard endoscope. A radial probe is used to create radial images whereas a linear probe is used to create linear images. Unlike endoscopic ultrasound, OCT can also be performed through air. Therefore, no tissue contact or coupling is usually required [24]. Near-infrared light (700-1500 nm) is applied to the tissue, and subsequent optical backscattering of light is measured using low coherence interferometry to create cross-sectional images [25]. Images are displayed at a rate of 4 frames/second. Most OCT systems achieve an axial resolution of approximately 7-20 um [24]. Scattering of light in the tissues decreases the depth of scanning to 1-2 mm in the GI tract [26]. As a result, OCT is only able to image the mucosa and submucosa during endoscopy. The high resolution that OCT provides allows for visualization of mucosal glands, crypts, villi. However, cellular features like nuclear dysplasia cannot be appreciated [27].

More recently, a newer version OCT system (NvisionVLE) became commercially available, which can image with in the wall of the esophagus, using a balloon catheter that can be used through the scope and placed in the distal esophagus [28]. The VLE utilizes optical signal acquisition and processing methods to create high-resolution cross-sectional images of the esophageal wall. VLE, compared to the original OCT system can scan a wider area (6 cm circumferential segment) of the esophagus in real time, to create high resolution (7 um), 3D cross-sectional images while reaching a depth of 3 mm below the mucosa [28]. The Nvision VLE offers a volumetric view of approximately 10.000 mm2 and has a significantly improved imaging speed and resolution (25 times higher than endoscopic ultrasound) as compared to the first-generation OCT systems [29]. The cross-sectional view of the Nvision VLE can collect approximately 1200 cross-sectional images across the targeted 6 cm, with an option to zoom in for closer examination [29].

Alternatively, the longitudinal view allows the endoscopist to examine the plane of the esophagus perpendicular to its cross-section, with over 4000 longitudinal images of the esophagus [29]. Both these views can be easily manipulated using a touch screen monitor or a hand controller. Other recent advancements to this system include the ability to perform laser marking, as well as a more recent "artificial intelligence" system. The Real-Time Targeting TM allows clinicians to make tissue laser marks that are visible under WLE, to facilitate identification and localization of areas for target biopsies [29]. The Intelligent Real-time Image Segmentation TM (IRIS) artificial intelligence software aids image review by segmenting and colorizing the three most common esophageal image features including hyperreflective surface, layering, and hypo-reflective structures in real time [30]. While the safety and effectiveness of this diagnostic software has not yet been evaluated, the Nvision VLE has the potential to enable a more thorough examination of the area of interest, which could improve biopsy targeting.

# High Resolution Microendoscope

## Acceptability

While these newer technologies have shown exciting results, acceptability among physicians and patients has not yet been evaluated in detail. Since acceptability would directly affect adoption and dissemination of these technologies, future studies that evaluate acceptability are required.

# Conclusions

Newer upcoming imaging modalities are changing the scope of gastrointestinal endoscopy. It has been demonstrated that endoscopy powered by real-time histology and diagnostic algorithms can effectively differentiate neoplastic from non-neoplastic lesions, increase diagnostic yield and also guide treatment plans in the gastrointestinal tract. Combining these new technologies with widefield imaging techniques can facilitate more targeted biopsies to further reduce cost and discomfort associated with unnecessary biopsies and repeat procedures. More affordable alternatives like HMRE can help fill gaps in settings with limited endoscopy and pathology infrastructure. In the future, endoscopy with real-time histology and automated diagnosis could contribute to personalized diagnosis and also increase our understanding of the pathophysiology of various diseases. Further clinical validations and investigations are warranted to assess whether or not these technologies can replace or complement the current standard of care. Although these newer technologies may never be able to completely replace traditional histopathology, they certainly have the potential to optimize and refine endoscopic screening and surveillance.

# Citation

Bhushan S, Richards-Kortum R, Anandasabapathy S (2020) Progress and Challenges of Global High-Resolution Endoscopy. Int Arch Intern Med 4:024. doi.org/10.23937/2643-4466/1710024