Confocal microscopy techniques advance the monitoring of Barrett’s esophagus and other applications. Barrett’s esophagus – a condition in which the cells lining the esophagus are replaced by cells similar to those found in the stomach – affects anywhere from 1.6 to 6.8 percent of the population. It is not the most common disorder in the gastrointestinal tract, but it is a growing concern, especially as it is associated with increased risk for esophageal adenocarcinoma, a rare but particularly deadly form of cancer. Tiny microscopes that can see inside single living cells could help gastroenterologists and other specialists diagnose Barrett’s esophagus, a precursor to cancer, and a variety of other conditions. For this reason, patients diagnosed with Barrett’s esophagus are often placed in a surveillance program to watch for precancerous dysplasia or cancer itself. This involves periodic assessment with white-light endoscopy; for example, the “Seattle protocol” calls for random, four-quadrant biopsies taken at 1- to 2-cm intervals beginning at the gastrointestinal junction. But there are a number of drawbacks to the current approach. Not least: the associated sampling error, and the high cost of the procedure itself and the subsequent histological interpretation. Researchers are hard at work trying to address these drawbacks. One of the more promising methods they are working with is confocal microscopy, a technique that takes advantage of point illumination and a pinhole to achieve improved resolution and contrast compared with conventional wide-field fluorescence microscopy. Especially with recent advances, the technique shows a great deal of potential for endoscopic applications. An SECM endoscopic probe ((a) – collimation optics; and (b) – final probe assembly; scale bar = 2 mm). Courtesy of DongKyun Kang et al (2013), ‘Endoscopic probe optics for spectrally encoded confocal microscopy,’ Biomedical Optics Express, Vol. 4, No. 10 (doi: 10.1364/BOE.4.001925). Invented in 1955 by Harvard researcher Marvin Minsky, confocal microscopy is widely used today by researchers in the biological sciences and elsewhere to obtain images of the 3-D structure of a cell or a tissue sample, for example. Using a pinhole in front of the detector, it rejects the light coming from out-of-focus regions above and below a particular plane and, thus, offers improved optical sectioning of a sample at various depths. More recently, the technique has been adapted for use with endoscopy to help address ongoing concerns about Barrett’s esophagus and other endoscopic needs. This approach – generally known as confocal laser endomicroscopy – uses the advantages generally found with confocal microscopy to produce high-quality images of the esophagus and other luminal areas of interest. In doing so, it could provide physicians with a means of real-time, in vivo histology. (a) A catheter-based reflectance-type laser scanning confocal microscope from Mauna Kea Technologies of Paris and Fujinon of Saitama, Japan. (b) Laser confocal microscopy examination for early gastric cancer under endoscopy. Courtesy of Parama Pal (2013), ‘Spectrally encoded confocal microscopy: A new paradigm for diagnosis,’ Journal of the Indian Institute of Science, Vol. 93, Issue 1. Researchers first described in vivo confocal laser endomicroscopy a little more than a decade ago. In 2003, Masanori Sakashita and colleagues reported a laser-scanning confocal microscopy system for real-time imaging of untreated specimens for examination of colorectal lesions, and further described a probe-based prototype endomicroscope that could be passed through the working channel of an endoscope. Progress came quickly after that, and today there are two commercially available confocal laser endomicroscopy systems: the eCLE from Pentax in Tokyo and the probe-based pCLE from Mauna Kea Technologies in Paris. A number of studies have sought to assess the efficacy of these systems for endoscopic and other applications, and with promising results. For example, in a recent Digestive Diseases and Sciences paper (doi: 10.1007/s10620-012-2332-z), researchers in Italy found that pCLE exhibited enhanced sensitivity in detecting Barrett’s esophagus, compared with high-definition white-light endoscopy. A schematic of spectrally encoded confocal microscopy (SECM) probe optics and system (CL = collimation lens, and BS = beamsplitter). SECM is being developed for possible clinical applications. Courtesy of DongKyun Kang et al (2013), ‘Endoscopic probe optics for spectrally encoded confocal microscopy,’ Biomedical Optics Express, Vol. 4, No. 10 (doi: 10.1364/BOE.4.001925). Still, efforts to improve the techniques are ongoing – e.g., further development of the technique spectrally encoded confocal microscopy (SECM) for possible clinical application. This could fill an important need, say the authors of a recent Biomedical Optics Express paper (doi: 10.1364/BOE.4.001925), researchers from the lab of Guillermo J. Tearney at Harvard Medical School and the Wellman Center for Photomedicine at Massachusetts General Hospital in Boston. Confocal laser endomicroscopy has been successfully demonstrated, but the area of the tissue it can image is still relatively small: less than 0.25 mm2. This could lead to sampling error during biopsy. Spectrally encoded confocal microscopy could address this limitation by providing large-area imaging of the esophagus, they say. With SECM, multiple wavelengths of light are delivered to the target site, where each is diffracted by a grating and focused on a particular point on the sample. Because it requires only a stationary optical element – the diffraction grating – the technique can produce images at a very high rate, and for this reason could be more readily adapted for endoscopic applications. Tearney and colleagues previously described SECM benchtop scanning systems and demonstrated the potential of the technique for large-area imaging of luminal organs. They noted, however, that it still wasn’t ready for clinical use in the organs themselves. First, because the performance of the systems wasn’t yet sufficient for diagnosis. But also because the optics of the SECM probe were too large to be easily incorporated into endoscopic devices. The earlier devices were 15 mm in diameter. To be clinically useful, they would have to be miniaturized by a factor of about three. Picture of a confocal scanning head integrated onto the distal tip of a conventional Pentax EC-3870CIFK colonoscope. Courtesy of Parama Pal (2013), ‘Spectrally encoded confocal microscopy: A new paradigm for diagnosis,’ Journal of the Indian Institute of Science, Vol. 93, Issue 1. Hence the Biomedical Optics Express study, published in October. Here, the researchers reported probe optics 5.85 mm in diameter – small enough to fit inside a conventional endoscope. The optics used a custom water-immersion aspheric singlet as the objective lens, reducing the spherical aberrations and specular reflection from the surface of the tissue. This should facilitate improved imaging depth and, in fact, the researchers found they could image tissue in the esophagus down to a depth of 260 μm. Simple and painless endoscopies? Though they have made significant strides in developing endomicroscopy methods, researchers are looking to push further still – particularly with respect to probe size, which can have important implications for patient comfort and accessibility. In a Nature Medicine paper published early last year (doi: 10.1038/nm.3052), Tearney’s team reported a technique called tethered capsule endomicroscopy. Here, an optomechanically engineered “pill” is used to acquire cross-sectional images as it travels through the gastrointestinal tract; the technique relies on light from a rapidly rotating laser, which reflects off the lining of the esophagus and is detected by internal sensors. The technique is simple and painless, the researchers write. And because it doesn’t involve sedation, there is no need for the specialized medical equipment or staff necessitated by endoscopies otherwise. The system described in the study uses optical frequency domain imaging to obtain the morphologic information that enables diagnosis of Barrett’s esophagus, for example. But the authors noted that the technique could be extended to other in vivo endomicroscopy technologies, including confocal microscopy.