Current approaches to endoscopic biopsy use external imaging, such as computed tomography (CT), magnetic resonance (MR), or white light endoscopy, to image suspect tissue. Despite advances in the field of endoscopic imaging, technical limitations of these modalities exist. These limitations may have important clinical implications, especially in optimizing cancer screening, diagnosis, and surveillance in the detection and histological assessment of premalignant lesions. For example, treatment guidelines for recognizing EAC and preventing mortality are largely based on endoscopic surveillance of patients with chronic, symptomatic gastroesophageal reflux disease and those with BE as well as use of histopathological assessment to evaluate the risk of BE progression to EAC 13 14 19 . Although currently considered the gold standard for surveillance 19 , white light endoscopy is limited to the surface of the mucosa and depends on clinical changes to signify underlying disease. External sources (CT/MR) typically lack sufficient resolution to provide accurate guidance for biopsy location determination.

When BE is identified, targeted biopsies and four-quadrant, random biopsies are obtained to detect invisible neoplasias 14 19 20 , but these strategies may be unreliable 21 because of sampling error and other practical limitations. When performed appropriately, a random sampling technique reduces the area of tissue surveyed, covering as little as 5% of the surface area of BE tissue 22 . Mucosal irregularities of early neoplasias are often discrete and easily missed during standard BE surveillance endoscopy 20 . In surgical resection specimens, up to 43% of patients with confirmed high-grade dysplasia had adenocarcinomas that were missed before surgery, despite the use of endoscopic biopsy 23 . Given the small amounts of histologically ambiguous tissue retrieved, the potential for diagnostic misinterpretation and variability among pathologists is considerable, a problem that has been demonstrated in several studies 22 24 25 26 . The time delay between endoscopy and diagnosis is another limitation, with separate procedures required for the detection and treatment of dysplasia 26 . The current biopsy approach is uncomfortable and time consuming for patients, often requiring a lengthy period of sedation and posing risks of bleeding and perforation 20 27 . The limitations of current imaging and biopsy methods represent an unmet need in the early detection of mucosal dysplasias.

Unlike current techniques, newly developed in vivo imaging technologies offer the potential to guide biopsy and to move toward real-time pathology. These tools may enable immediate optical histology of the mucosal layer during ongoing endoscopy, or virtual histology, allowing visualization of living cells and cellular structure at and below the mucosal surface 28 . Compared with conventional radiologic and endoscopic techniques, these newer technologies achieve higher-resolution microscopic images with wider-ranging visualization of the target tissue (Table 1).

*Volumetric.

CM = confocal microscopy; CT = computed tomography; EUS = endoscopic ultrasound; IV = intravenous; FFOCM = full-field optical coherence microscopy; MRI = magnetic resonance imaging; OCT = optical coherence tomography; OFDI = optical frequency domain imaging; PET = positive emission tomography; SECM = spectrally encoded confocal microscopy; US = ultrasound.

Confocal laser endomicroscopy (CLE), a recent endoscopic advance, allows real-time high-resolution histologic analysis of targeted tissue during endoscopy 29 . The CLE illuminates tissue with a low-powered laser focused by an objective lens into a single point within a fluorescent specimen 30 31 . A confocal microscope is used to exclude light above and below a plane of interest, thus allowing for an optical section to be observed, similar to a histologic tissue section 29 . The generated gray scale image represents one focal plane within the examined specimen 31 . The mucosa typically can be imaged to a depth of 100 to 150 μm with this technique 22 .

Currently, two devices are available and have received the CE Mark for use for CLE 29 32 (Figure 1 33 ), and a third is under development. The endoscope-based CLE (eCLE; Cellvizio®, Pentax Corporation, Montvale, NJ, USA, and Tokyo, Japan) uses a confocal fluorescence microscope integrated into the distal tip of a conventional upper endoscope or colonoscope 29 30 . The probe-based CLE (pCLE; Mauna Kea Technologies, Newtown, PA, USA, and Paris, France) uses a fiber-optic probe bundle with a laser microscope inserted through the accessory channel of a standard endoscope 29 30 . Although lateral and axial resolution is better with eCLE than with pCLE, the eCLE is considerably bulkier 29 . The pCLE is more useful in smaller spaces 29 ; recent data demonstrated the feasibility of using pCLE for visualization of intra-abdominal organs, including liver, pancreas, spleen, and lymph nodes in a porcine model 34 . Development of a probe-based volumetric CLE device is under way.

Since 2004 when confocal endomicroscopy was first used for diagnosing colorectal pathology 35 , CLE has shown promise in a number of clinical applications. Indeed, CLE potentially may be used in the same manner as endoscopic biopsy 36 . Both eCLE and pCLE have had high accuracy (≥90%) in diagnosing BE and Barrett’s-associated neoplastic changes (Figure 2) 37 38 . CLE also can detect lymphocytic and collagenous colitis in chronic diarrhea 39 40 , identify the microarchitecture of early gastric cancer 41 42 , detect Helicobacter pylori infection with high accuracy (Figure 3) 43 , and detect villous atrophy in celiac disease 44 . Preliminary data have shown that CLE can detect malignant changes in pancreatic tissue 45 and premalignant changes in peripheral lung nodules 46 , urothelium 47 , and cervical epithelium 48 .

Although commercially available, the place of CLE in current diagnostic paradigms versus a conventional histopathological examination is still evolving 30 . With appropriate contrast agents, CLE has the potential for subcellular resolution, reducing the number of biopsies required 29 , as well as for molecular characterization 49 . However, available CLE devices have a narrow field of view and cannot penetrate beyond the mucosa, allowing visualization of only superficial mucosal layers 29 30 . Moreover, CLE does not provide an archive of tissue for full molecular characterization 29 , and contrast agents can limit the procedure duration and ability to obtain repeat images 22 .

Spectrally encoded confocal microscopy (SECM) is a high-speed technique based on reflectance imaging technology 50 . This method couples broadband or wavelength-swept narrowband light into a single optical fiber, which then illuminates a transmission grating and objective lens at the end of the confocal probe to encode one-dimensional spatial information reflected from a sample 50 51 52 . Because SECM detects spatial information externally to the probe, it can obtain highly detailed images at very high speeds (up to 10 times faster than the video rate), while the size of the optics is small enough to be incorporated into a small-diameter catheter or endoscope 50 51 . The SECM allows for large field confocal images without the need for contrast agent and may permit the imaging of extended areas of tissue 50 51 53 . Given that SECM can achieve, in principle, comprehensive confocal endomicroscopy of the entire distal esophagus, this technology is being investigated for imaging upper gastrointestinal (GI) tissues 50 . Preliminary assessment indicates that SECM can reveal the architectural and cellular features of gastroesophageal tissues, including the presence of goblet cells, columnar epithelium, and squamous epithelium in BE (Figure 4) 50 52 . A recent study in eosinophilic esophagitis showed that SECM of biopsy samples was functional in accurately providing eosinophil counts, as well as in identifying microscopic abnormalities such as abscess, degranulation, and basal cell hyperplasia 54 .

Angle-resolved low coherence interferometry (a/LCI), a light-scattering technique, identifies early dysplasia based on nuclear diameter differences 22 26 55 . This method measures the angular distribution of scattered light as a function of depth beneath the tissue surface 26 and achieves depth resolution through a process similar to that used in optical coherence tomography (OCT) 22 26 . The a/LCI device can assess nuclear size at multiple depths 22 , with deeper penetration than confocal microscopy approaches (up to 200–300 μm of the epithelial tissue layer compared with the surface and uppermost 100 μm of tissue with endoscopic confocal microscopy) 26 55 . The a/LCI data are analyzed and reported according to a best-fit analysis (Figure 5), with nuclear measurements in cell and tissue types reported with an accuracy of 0.2 to 0.3 μm 22 . The a/LCI device can provide instant high-resolution images non-invasively without the need for image interpretation by an endoscopist or administration of contrast agents 26 .

Recent clinical studies have explored a/LCI in the assessment of dysplasia in esophageal 26 and intestinal 55 tissues. In the first in vivo clinical study of a/LCI, 46 patients undergoing routine endoscopic surveillance for BE were scanned with the a/LCI system and the results correlated with an endoscopic biopsy specimen 26 . The nuclear size measurements generated for deep epithelial tissue (200–300 μm beneath the surface) separated dysplastic from non-dysplastic tissue with an accuracy of 86%, using a cutoff of 11.84 μm to separate the two types 26 . Using this same cutoff, a/LCI distinguished dysplastic BE specimens from indeterminate and non-dysplastic BE with a sensitivity of 100% (13/13; 95% confidence interval [CI], 0.75–100) and a specificity of 85% (76/89; 95% CI, 0.76–0.92) 26 . Similarly, a pilot ex vivo study of 27 patients undergoing partial colonic resection demonstrated high diagnostic value of this method at a depth 200 to 300 μm beneath the mucosal surface, with a/LCI separating dysplastic from healthy intestinal tissues with a sensitivity of 92.9%, a specificity of 83.6%, and an overall accuracy of 85.2% 55 .

Several other spectroscopy-based imaging techniques are under investigation in various clinical applications. Laser-induced fluorescence is a technique based on the principle that certain compounds exhibit a characteristic fluorescence emission when excited by light 56 . This technology has been shown to detect malignant colonic tissue 57 and to distinguish malignant tissue from metaplastic and normal tissue in BE 56 58 . Multimodal hyperspectroscopy is based on tissue fluorescence and reflected light measurements, which are analyzed with computed-based algorithms to differentiate between abnormal and normal tissues 59 . Although more extensively explored for use in detecting cervical cancer 59 60 , clinical studies in BE patients are under way 61 .

OCT is an imaging technique first introduced for use in biological tissues in 1991 62 that generates high-resolution, cross-sectional, subsurface images by using low-coherence interferometry to measure the echo time delay and intensity of back-scattered light 63 . OCT is analogous to ultrasonography, except that OCT measures the intensity of infrared light rather than sound waves 64 . With OCT, depth intensity is measured by time-domain measurements, allowing for image construction for all three dimensions.

Since its use was first described in ophthalmology to image the transparent structures of the anterior eye and retina 65 , OCT has evolved to include a wide spectrum of clinical applications. The successful use of OCT imaging techniques has been described in many biologic tissues, including human coronary arteries 66 67 ; esophageal 68 69 70 71 , gastric 72 73 , and intestinal 74 tissues (Figure 6); pancreatic and biliary tissues 75 ; cervical epithelium (Figure 7) 76 ; and urologic tissues 77 . Extensively studied in GI applications 72 74 78 , OCT has shown accuracy in diagnosing specialized intestinal metaplasia in BE with a sensitivity of 81% 71 79 .

Several OCT systems are currently in use or under investigation. The original OCT technology, now called time-domain OCT (Niris®, Imalux Corporation, Cleveland, OH, USA) 80 81 , has been described in detail elsewhere 64 78 . Interferometric synthetic aperture microscopy uses computed imaging and synthetic aperture techniques to modify OCT signals to achieve three-dimensional, spatially invariant resolution for all depths in a cross-sectional scan 82 83 84 . The feasibility of using this technology to image human breast tissue has recently been demonstrated 83 84 .

Despite the diagnostic potential of time-domain OCT, its relatively slow imaging speed has precluded its ability to survey large areas of the GI tract, limiting its use to point-sampling with a field of view comparable to that of conventional biopsy 70 85 . However, a new technologic approach to OCT allows dramatic increases in imaging speed without compromising image resolution or quality 70 86 87 88 . This technology, referred to as Fourier-domain OCT 81 or optical frequency domain imaging 70 , is also called volumetric laser endomicroscopy (VLE). VLE acquires cross-sectional images by using a focused, narrow-diameter beam to repeatedly measure the delay of reflections from within the tissue sample 70 . Interferometry is used to measure the delay intervals, while Fourier transformation is used to compute traditional A-lines, or depth scans, which comprise the tissue reflectivity as a function of depth along the beam. Unlike time-domain OCT, VLE uses a fixed wavelength or swept-source technology in which the wavelength of a monochromatic light source is rapidly scanned to measure the interference signal as a function of wavelength 70 87 .

The use of a balloon-based VLE system with helically scanning optics for esophageal imaging has been described 68 69 70 85 . With this system, the optical components of the catheter are positioned with the esophageal lumen via a balloon-centered probe 70 85 . After the balloon is inflated, the distal esophagus is dilated and the imaging optics become centered. Optics are slowly pulled back during the imaging procedure while the imaging optics are rotated by a probe scanner; thus, the entire portion of the esophageal lumen that was in contact with the balloon is scanned in a helical or circumferential fashion 70 . Real-time, volumetric images are obtained by scanning the imaging beam over the tissue surface in two dimensions 69 .

Preliminary data for the VLE system have shown its ability to image the entire distal esophagus at a higher speed and greater sensitivity compared with time-domain OCT 70 85 . VLE enables full-length surveillance of target areas with a combination of resolution and depth of surface penetration (3-mm penetration, <10-μm resolution depth) 52 . When used in swine models, VLE provided high-resolution images of the anatomic layers and vasculature from the distal esophagus and gastroesophageal junction (Figures 8 and 9) 70 . In the first clinical experience with this technique, VLE successfully imaged the microscopic architecture of the distal esophagus in 10 of 12 patients undergoing routine esophagoduodenostomy for BE screening and surveillance (Figures 10 and 11), with volumetric images acquired in less than 2 minutes 68 . Most recently, the feasibility of VLE-guided biopsy with laser marking was demonstrated in swine esophagus, a strategy with the potential to increase the diagnostic accuracy of current surveillance protocols and to guide interventional treatments 69 .

In vivo pathology imaging devices and the rapid evolution of the technology have the potential to make real-time diagnosis the new standard, with immediate diagnosis and management during endoscopy. The new optical biopsy technologies provide better quality, detailed, high-resolution images and allow visualization of living cells and cellular structures at and below the mucosal surface during ongoing endoscopy 28 35 . The convergence of imaging and pathology may provide distinct advantages in cancer detection and diagnosis without the limitations and risks inherent with biopsy procedures. With these technologies, maximal diagnostic yields may be obtained, leading to appropriate staging through guided biopsy while minimizing the frequency and error potential of random biopsy protocols. In vivo cellular information can be delivered before biopsies are performed, or imaging files may be transmitted with biopsies, potentially improving the efficiency and accuracy of diagnosis.

Despite the potential these techniques may offer to standard clinical practice, barriers remain. Optical biopsy techniques can identify neoplastic changes in a variety of biologic tissues, but prospective studies in large cohorts are needed to establish concrete sensitivity and specificity of the respective technologies, in each target organ, versus the need for biopsy. To achieve widespread clinical adoption, these technologies must be accurate, efficient for use in the endoscopic setting, reliable, user-friendly, patient-friendly, and cost-effective 22 89 . Wide acceptance and interpretation capabilities, which require comprehensive physician education and training, are also necessary to establish appropriate comfort with use. Investigators are currently working to improve the accuracy, speed, and ease of interpretation of these technologies 89 . In addition, research is under way to allow real-time data transmission and storage, thereby linking pathology results to the treating physician.