ORIGINAL ARTICLES ALIMENTARY TRACT

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1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5: ORIGINAL ARTICLES ALIMENTARY TRACT In Vivo Histopathology for Detection of Gastrointestinal Neoplasia With a Portable, Confocal Miniprobe: An Examiner Blinded Analysis ALEXANDER MEINING,* DIETER SAUR,* MONTHER BAJBOUJ,* VALENTIN BECKER,* ERIC PELTIER, HEINZ HÖFLER, CLAUS HANN VON WEYHERN, ROLAND M. SCHMID,* and CHRISTIAN PRINZ* *II Department of Medicine and Department of Pathology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; and Cabinet de Pathologie, Paris, France See Editorial on page Background & Aims: Confocal fluorescence microscopy (CFM) has been mentioned to be a promising tool for in vivo histology. Recently, a portable confocal miniprobe has been developed. Our aim was to evaluate the potential benefit of CFM for detection of gastrointestinal neoplasia. Methods: A total of 47 patients with known or suspected neoplasia in the upper (n 34) or lower gastrointestinal tract (n 13) were examined with standard endoscopes. After mucolyis with 5 10 ml of acetic acid 1.5%, chromoendoscopy with 2 5 ml cresyl violet 0.25% was performed, with the substance also being used as a fluorophore for CFM. Real-time video sequences were recorded. Thereafter, biopsies were taken or mucosectomy/polypectomy was performed from the same examined area. All stored sequences were put into a random order and assessed by a pathologist and a gastroenterologist both blinded to any data. Results: A total of 119 CFM video sequences were recorded of 85 benign or 34 neoplastic areas. Quality of CFM images was regarded too low in 24 (pathologist) and 14 sequences (gastroenterologist). For the pathologist, accuracy of CFM detecting neoplasia was 92.6% (suitable images) and 73.9% (intention to diagnose). The respective accuracy values for the gastroenterologist were 92.4% (suitable images) and 81.5% (intention to diagnose). Agreement between CFM and histopathology was excellent (kappa values, and 0.817). Conclusions: We have demonstrated that CFM with a miniprobe has the potential to diagnose neoplasia during ongoing endoscopy. This system has the advantage that it can be used with standard endoscopes. Further studies are warranted for validation. During the past 20 years, innovations in technology have dramatically changed the field of gastrointestinal (GI) endoscopy. The evolution from fiberoptic to videoendoscopy has both improved image resolution and provided a new viewing format, thus modifying the daily practice of endoscopy. A variety of endoscope models and manufacturers exist. Procedure-specific endoscopes have been designed to enhance endoscopic diagnosis and therapy. Recently, chromoendoscopy has become popular as a diagnostic enhancement tool in endoscopy, expressing the remaining need for an even higher image definition leading to better tissue characterization. 1,2 But what is the ultimate goal of the endoscopist? It is to make an informed decision during the diagnostic endoscopic procedure and treat the lesion appropriately but minimally invasively. For targeting both biopsies and endoscopic resection and improving patient care, the ideal situation is to visualize cellular structure, which implies having microscopic imaging available. Confocal fluorescence laser microscopy (CFM) has been reported to be an accurate tool enabling a histopathologic diagnosis in vivo during endoscopy. Neoplastic mucosa can be differentiated from non-neoplastic mucosa. 3 5 Further data show that other histologic changes such as metaplasia, inflammatory changes, and even Helicobacter pylori colonizing the gastric mucosa can be detected. 6 However, so far, present data are exclusively based on a confocal laser endoscope on the basis of the integration of a laser microscope in the distal tip of a videoendoscope (Pentax, Tokyo, Japan; in cooperation with Optiscan, Notting Hill, Victoria, Australia). Recently, however, a new system for confocal laser microscopy (Cellvizio-GI; Mauna Kea Technologies, Paris, France) has been launched and received Food and Drug Administration approval and CE mark. This new system offers high-resolution confocal fluorescence imaging during standard endoscopic procedures with a catheter-based confocal miniprobe. Therefore, it might be used with any standard videoendoscope. One of the authors has previously shown ex vivo that CFM of the GI tract is possible after chromoendoscopy with cresyl violet. 7 Cresyl violet is frequently applied in chromoendoscopy for pit pattern classification. 8 It is an absorptive dye that concentrates in cell nuclei and has fluorescence properties that allow it to be used for laser scanning microscopy. It was therefore attempted to determine the performance characteristics (accuracy, sensitivity, specificity) of a newly available catheterbased confocal laser microscope after chromoendoscopy with cresyl violet for diagnosis of GI neoplasia. In addition, a blinded approach was used to evaluate the potential of this new device. Abbreviations used in this paper: BE, Barrett s esophagus; CFM, confocal fluorescence microscopy; GI, gastrointestinal; IT, isolated tip; ig-ien, low grade intraepithelial neoplasia; TT, triangulated tip by the AGA Institute /07/$32.00 doi: /j.cgh

2 1262 MEINING ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 11 Figure 1. Confocal Miniprobe introduced through instrumentation channel (diameter, 2.8 mm) of a standard colonoscope. (A) Introducing the miniprobe; (B) tip of miniprobe leaving the instrumentation channel and after retroflexion of the endoscope. Patients and Methods Patient Recruitment Between March September 2006, 47 patients were included in the study. Suitable for inclusion were patients with known or suspected premalignant or malignant lesions. Among those were patients with Barrett s esophagus (BE) in whom elective surveillance was intended (n 16), patients submitted for endoscopic mucosal resection/polypectomy of intraepithelial neoplasia or polyps in the stomach (n 5) or colon (n 13), and patients submitted for further evaluation of suspected GI cancers in the esophagus (n 4), stomach (n 5), duodenum (n 3), or colon (n 1). All patients were recruited from the II Medizinische Klinik, Klinikum rechts der Isar, Technical University of Munich, Germany. Exclusion criteria were conditions that exclude safe biopsies or mucosal resections (increased partial thromboplastin time/ international normalized ratio, thrombocytopenia 50,000), patient s heart disease New York Heart Association III or IV, or patient in reduced mental or physical health in general. Informed consent to participate in the study was obtained from all patients. The Ethics Committee of the Technical University of Munich approved the study. Endoscopy Endoscopy was performed with standard gastroscopes (Olympus GIF160 or GIF140; Olympus, Tokyo, Japan) and colonoscopes (Olympus CF140/ CF160 or Storz 13910PKS; Karl Storz GmbH & Co, Tuttlingen, Germany). A hard cap was attached at the tip of the endoscopes to enable stable conditions for catheter-based confocal microscopy. All examinations were performed by one of the authors (A.M.) after a training session on the confocal miniprobe technique. During the procedure, patients were sedated with midazolam in combination with propofol. Before confocal laser microscopy was performed, the region of interest was washed with tap water and sprayed with 5 10 ml acetic acid 1.5% by using a dye catheter (Olympus 5l). Thereafter, 2 5 ml of a solution of cresyl violet 0.25% dissolved in acetic acid 1.5% was sprayed locally onto the site to be investigated. After confocal laser microscopy had been performed during 1 4 minutes per site, a biopsy was taken, or mucosectomy/polypectomy was performed from the same examined site for further histopathologic analysis at the Pathological Institute of the Technical University of Munich. Mucosal specimens were obtained by using standard biopsy forceps. Endoscopic resection was performed with standard polypectomy snares, mucosectomy caps, or, as in 1 patient, by endoscopic submucosal dissection applying the isolated tip (IT) knife and triangulated tip (TT) knife (Olympus, Hamburg, Germany). In addition, laser microscopy was performed, and biopsies were taken from sites of normal-appearing mucosa adjacent to the area examined previously. Confocal Fluorescence Laser Microscopy This system used for CFM is based on the combination of 3 items. One is a confocal miniprobe made of tens of thousands of optical fibers bundled together and terminated by a distal microsystem. The images obtained have a lateral resolution of 3.5 m, an axial resolution of 15 m, with a total field of view of m. The miniprobe tip diameter is 2.5 mm and therefore compatible with the accessory channel of any standard endoscope; mean depth of confocal plane is 10 m (Gastroflex or Coloflex type S Confocal Miniprobes; Mauna Kea Technologies, Paris, France) (Figure 1). The second item is a proximal laser scanning unit (excitation wavelength, 488 nm), which combines the functions of laser light illumination and rapid laser scanning, enabling a frame rate up to 12 images per second and signal detection (Laser Scanning Unit, Cellvizio-GI; Mauna Kea Technologies). The third item is control and acquisition software for realtime image reconstruction, immediate sequences display, and post-procedure analysis and editing tools (Cellvizio software; Mauna Kea Technologies).

3 November 2007 LASER MICROSCOPY FOR DETECTING GI NEOPLASIA 1263 Table 1. Criteria for Cresyl Violet based Laser Microscopy for Differentiation of Neoplasia From Benign Mucosa Stratified for the Different Regions of the GI Tract Esophagus BE/stomach Duodenum Colon Benign Homogenous greyish image with regular demarcation of cellular borders ( cobblestones ) Regular round crypts ( doughnuts ) or villi ( fingers ) of similar size and order Regular villi of homogenous size and structure ( fingers ) with scattered goblet cells (black spots) Regular crypts standing close to each other with visible goblet cells (black spots arranged in radiating order around orifices of crypts) Neoplastic Cells of mixed/irregular shape and order ( pepper & salt ) Loss of crypts and replacement by elongated glands of irregular caliber (adenoma) or heterogeneously dispatched structures with cells of mixed/irregular shape and order (pepper & salt) for cancers Loss of villous structure, heterogeneously dispatched structures with cells of mixed/irregular shape and order; loss of goblet cells Loss of crypts and goblet cells; replacement of crypts by thickened tubular structures (adenoma) or heterogeneously dispatched structures (pepper & salt) for cancers Video sequences obtained in the above mentioned manner were put into a random order after acquisition. Sequences were exported in standard video format (mpeg1) by using the Cellvizio software post-processing tools. All sequences were read by a pathologist (E.P.) and a gastroenterologist (D.S.) who were blinded to all anamnestic, endoscopic, or histopathologic data but familiar with confocal laser scanning procedures. The only information available was a rough description of the location where sequences were obtained (esophagus, stomach, duodenum, or colon). A presumptive histopathologic diagnosis on the basis of the laser microscopic sequences was requested. Criteria for diagnosing neoplasia on the basis of CFM images are summarized in Table 1 and illustrated in Figure 2. In addition, the quality of every sequence was scored as good (no moving artefacts, single structures can be differentiated) or poor (artefacts do not allow interpretation of the sequence). Histopathology Biopsy specimens were fixed in 4% buffered formalin. The slides were stained with a combination of hematoxylineosin. Neoplasia was graded in accordance with the Vienna classification. 9 All pathologic examinations were performed at the Pathological Institute of the Technical University of Munich (head, Prof H. Höfler). Statistical Analysis Accuracy, sensitivity, and specificity of CFM for detection of neoplasia were calculated with standard histopathology as the gold standard. In addition, agreement between CFM and histopathology was tested with statistics. Here, a value below 0.4 was Figure 2. CFM images demonstrating the criteria for differentiation of benign from neoplastic mucosa. (A) Benign/healthy colonic mucosa with frequent goblet cells (black spots around crypt orifices); (B) tubular adenoma of the colon with low-grade intraepithelial neoplasia; (C) healthy mucosa in the corpus/cardia of the stomach; (D) cancer of the cardia (with typical pepper & salt pattern). Crypts in (B) and (D) are reduced by number and are partially destructed (red arrows). In addition, loss of goblet cells in the neoplastic colon becomes clearly visible (D).

4 1264 MEINING ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 11 Figure 3. CFM images from video sequences showing hyperplastic (A) and normal cardia mucosa (B), specialized intestinal metaplasia (BE) adjacent to squamous epithelium (C), and a tubular adenoma in the colon (D). Field of view is m. considered as bad agreement, was considered as fair, and above 0.7 was considered as excellent agreement. Statistical analysis was performed with the SPSS for Windows (SPSS Inc, Chicago, IL) 14.0 software package. A P value below.05 was considered as statistically significant. Results Histopathologic Findings Overall, histopathology revealed neoplasia in 23 of 47 cases (48.9%). Among those, 9 patients had adenomas or cancers in the colorectum, 2 patients had duodenal adenocarcinomas, 3 patients had a squamous carcinoma in the esophagus, 7 patients had an adenocarcinoma of the distal esophagus or esophagogastric junction, 1 patient had an adenoma of the cardia, and 1 patient had carcinoma in the gastric corpus. On the basis of the number of collected biopsies/resected specimens, a total of 119 specimens were examined by histopathology. Of those 119 specimens, 34 (28.5%) were classified as neoplastic. Confocal Fluorescence Microscopy A total of 119 CFM video sequences at 12 frames/ second were recorded of 85 non-neoplastic or 34 neoplastic areas as determined by histopathology. Different types of layer, such as squamous epithelium versus columnar epithelium, could readily be differentiated. In addition, pits or crypts were able to be discriminated from villi or tubular structures (Figure 3).The pathologist (E.P.) regarded image quality too low to enable a presumptive diagnosis in 6 of 47 patients (12.8%) and in 24 of 119 sequences (20.2%), leaving a total number of 95 video sequences. The corresponding numbers for the gastroenterologist (D.S.) were 2 of 47 patients (4.3%) and 14 of 119 sequences (11.8%), leaving a total number of 105 videos. Comparison Between Histopathology and Confocal Fluorescence Microscopy for Detection of Neoplasia Table 2 shows diagnostic accuracy, sensitivity, and specificity of CFM for detecting neoplasia in comparison with histopathology serving as the gold standard. Both are shown as numbers on a per-specimen basis (biopsy/cfm site positive or negative for neoplasia) and numbers on a per-patient basis (patient positive or negative for neoplasia). Table 3 shows the results of CFM for detection of neoplasia stratified for lesions located in the upper or lower GI tract. Table 2. Diagnostic Accuracy, Sensitivity, and Specificity of CFM for Detecting Neoplasia in Comparison With Histopathology Serving as the Gold Standard Accuracy Sensitivity Specificity Pathologist Per biopsy (n 95) 88/95 (92.6%) 24/26 (92.3%) 64/69 (92.8%) Per patient (n 38) 33/38 (86.8%) 16/18 (88.9%) 17/20 (85.0%) Gastroenterologist Per biopsy (n 105) 97/105 (92.4%) 27/29 (93.1%) 70/76 (92.1%) Per patient (n 44) 40/44 (90.9%) 19/21 (90.5%) 21/23 (91.3%) NOTE. Only cases with suitable image quality as determined by the respective examiner were selected for analysis.

5 November 2007 LASER MICROSCOPY FOR DETECTING GI NEOPLASIA 1265 Table 3. Diagnostic Accuracy, Sensitivity, and Specificity of CFM for Detecting Neoplasia Stratified for Lower or Upper GI Tract Accuracy Sensitivity Specificity Pathologist Upper GI (n 61) 56/61 (91.8%) 13/15 (86.7%) 43/46 (93.5%) Lower GI (n 34) 32/34 (94.1%) 11/11 (100%) 21/23 (91.3%) Gastroenterologist Upper GI (n 69) 64/69 (92.8%) 15/16 (93.8%) 49/53 (92.5%) Lower GI (n 36) 33/36 (91.7%) 12/13 (92.3%) 21/23 (91.3%) NOTE. Only cases with suitable image quality as determined by the respective examiner were selected for analysis. Kappa statistics revealed that the agreement between laser microscopy and histopathology can be regarded as very good. The respective values were (histopathologist) and (gastroenterologist), both reaching statistical significance (P.001). Figure 4 shows examples illustrating the agreement between CFM and standard histopathology. On an intent-to-diagnose basis, ie, inclusion of the sequences with a quality too low to enable a sufficient diagnosis by CFM, accuracy rates were 73.9% and 81.5% on a per-biopsy basis and 70.2% and 85.1% on a per-patient basis for both examiners (pathologist and gastroenterologist), respectively. Discussion In the present study, we were able to demonstrate that catheter-based CFM might serve to accurately discriminate between neoplastic and non-neoplastic mucosa. The accuracy rate for detection of neoplasia was greater than 90%. Kappa statistic Figure 4. Endoscopic findings, images from video sequences acquired with CFM, and corresponding histopathologic images of selected cases. lg-ien, low-grade intraepithelial neoplasia.

6 1266 MEINING ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 11 showed that agreement between CFM and standard histopathology is excellent. This holds true for evaluation done by a pathologist and by a gastroenterologist. Data were generated in a blinded manner, ie, the respective CFM diagnosis was established without knowing the endoscopic or histopathologic results. Hence, one might assume even higher accuracy rates in a clinical situation, ie, if CFM was performed with further information on the endoscopic diagnosis. However, it should also be mentioned that our patients were selected because they had known tumors, polyps, or premalignant lesions. Hence, although the CFM sequences were evaluated in a blinded manner, the results might be somewhat hampered because accuracy was not determined in an unselected random population. Neoplastic lesions were all clearly visible by endoscopy before CFM was performed. Therefore, the probability of a sampling error was low. This might not have been the case if a nonselected population was examined with a higher frequency of small focal lesions, leading to a potential sampling error. Our findings are in agreement with data published previously by Kiesslich et al 3,10 reporting a high accuracy of CFM for detection of neoplasia in a selected population. However, in our opinion, the system we applied has the advantage that confocal miniprobes are miniaturized and flexible. They might therefore be used with any standard endoscope and integrate seamlessly in the endoscopy unit workflow. In contrast, the Pentax/Optiscan system offers a solution in which the confocal laser microscope is integrated in the distal tip of an endoscope. This latter system has the benefit that it leads to a flexible imaging plane depth and a higher lateral resolution compared with the fiber bundle technology. Hence, one might expect high-resolution images of better quality in comparison with the system as used in the present study. However, on the other hand, a tip-integrated laser microscope has the disadvantage that it is more rigid, and the respective endoscopist has to decide to use this special endoscope before the examination has started. Yet by using a flexible catheter-based system, the examiner might decide to perform laser microscopy during any ongoing endoscopic examination just like using other devices such as biopsy forceps or snares. In addition, sequences are acquired at a frame rate of 12 images per second; therefore, sequences have video quality, and the position of the probe and the CFM image are under direct visual control. Hence, both systems appear to have their pros and cons. In addition, because confocal images are acquired in an axial plane with a limited penetration depth, they might not generally replace standard histopathology. In particular, gaining information on the penetration depth of early lesions is impossible. More scientific data and outcome studies are therefore mandatory before clinical decisions can be made on the basis of CFM with either method. It should be noted that depending on the respective examiner, up to one fifth of the evaluated sequences were regarded as having quality too low to enable a presumptive diagnosis. This can be explained by the fact that in these cases, stained remnants of mucus, cell debris, and blood decreased image quality significantly. These problems might be overcome by using probes with a deeper penetration depth (100 m instead of 10 m) to mitigate the mucus and other surface debris issue. However, these miniprobes were not available when the study was initiated. In addition, newer data show that using an intravenous fluorophore instead of a topical stain might also increase the resolution of CFM images. The application of fluorescein intravenously has the further benefit that examination time can be shortened because the process of mucolysis, washing, and staining is no longer necessary as shown by us in previous publications It has to be reminded that CFM is not a red flag technology. 14,15 Similar to other devices, such as endocytoscopy, only focal areas to which the endoscopist is attracted can be investigated. However, in theory the present system might be a valuable adjunct to other red flag technologies such as chromoendoscopy, narrow band imaging, or autofluorescence, 19,20 in particular, because with the catheter-based approach it can be readily used via the instrumentation channel of such endoscopes. The addition of CFM into the concept of multimodal endoscopy 15,21 has the advantage that it is not locally invasive and consequently has the potential to decrease the necessity of taking biopsies. The reduction in the biopsy randomness can, therefore, reduce biopsy-related complications and histopathology costs and could potentially increase the scope of clinical surveillance. Further studies apart from this feasibility study are therefore needed. In conclusion, our data demonstrate that CFM with a catheter-based system has the potential to differentiate accurately between neoplastic and non-neoplastic mucosa. The system we applied has the further advantage that it is portable and can be used with any standard endoscope. Now there is a need for further validation of our results. Therefore, multicenter studies are currently conducted to evaluate the use of this new confocal microscope for improved screening and surveillance of patients with BE, eg, a condition with a high rate of sampling bias during endoscopy. 22,23 Further perspectives are in vivo confocal laser microscopy of the pulmonary system, 24 urinary system, 25 biliary system, or pancreatic duct. References 1. Jung M, Kiesslich R. Chromoendoscopy and intravital staining techniques. Baillieres Best Res Clin Gastroenterol 1999;13: Sharma P, Weston AP, Topalovski M, et al. Magnification chromoendoscopy for the detection of intestinal metaplasia and dysplasia in Barrett s oesophagus. Gut 2003;52: Kiesslich R, Goetz M, Vieth M, et al. Confocal laser endomicroscopy. Gastrointest Endosc Clin N Am 2005;15: Kiesslich R, Gossner L, Goetz M, et al.. In vivo histology of Barrett s esophagus and associated neoplasia by confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2006;4: Polglase AL, McLaren WJ, Skinner SA, et al. A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-gi tract. Gastrointest Endosc 2005;62: Kiesslich R, Goetz M, Burg J, et al. Diagnosing Helicobacter pylori in vivo by confocal laser endoscopy. Gastroenterology 2005;128: George M, Meining A. Cresyl violet as a fluorophore for future in vivo histopathology. Endoscopy 2003;35: Kudo S, Tamura S, Nakajima T, et al. Diagnosis of colorectal tumorous lesions by magnifying endoscopy. Gastrointest Endosc 1996;44: Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of gastrointestinal epithelial neoplasia. Gut 2000;47: Kiesslich R, Burg J, Vieth M, et al. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004;127: Meining A, Schwendy S, Becker V, et al. In vivo histopathology of lymphocytic colitis. Gastrointest Endosc 2007;21: [Epub ahead of print].

7 November 2007 LASER MICROSCOPY FOR DETECTING GI NEOPLASIA Becker V, Vercauteren T, Hann von Weyhern C, et al. High resolution miniprobe-based confocal microscopy in combination with video mosaicing. Gastrointest Endosc (in press). 13. von Delius S, Feussner H, Wilhelm D, et al. Transgastric in-vivo histopathology in the peritoneal cavity by miniprobe based confocal fluorescence microscopy in a porcine model. Endoscopy 2007;39: Eisendrath P, Deviere J. Red flag techniques in Barrett s esophagus: minor additional benefit or a waste of time. Endoscopy 2006;38: Bergman JJ. The endoscopic diagnosis and staging of oesophageal adenocarcinoma. Best Pract Res Clin Gastroenterol 2006; 20: Sasajima K, Kudo SE, Inoue H, et al.. Real-time in vivo virtual histology of colorectal lesions when using the endocytoscopy system. Gastrointest Endosc 2006;63: Inoue H, Kudo SE, Shiokawa A. Technology insight: laser-scanning confocal microscopy and endocytoscopy for cellular observation of the gastrointestinal tract. Nat Clin Pract Gastroenterol Hepatol 2005;2: Inoue H, Sasajima K, Kaga M et al. Endoscopic in vivo evaluation of tissue atypia in the esophagus using a newly designed integrated endocytoscope: a pilot trial. Endoscopy 2006;38: Kara MA, Peters FP, Rosmolen WD, et al. High-resolution endoscopy plus chromoendoscopy or narrow-band imaging in Barrett s esophagus: a prospective randomized crossover study. Endoscopy 2005;37: Kara MA, Peters FP, Ten Kate FJ, et al. Endoscopic video autofluorescence imaging may improve the detection of early neoplasia in patients with Barrett s esophagus. Gastrointest Endosc 2005;61: Wallace MB. Advances in endoscopic imaging of Barrett s esophagus. Gastroenterology 2006;131: Meining A, Ott R, Becker I, et al. The Munich Barrett follow up study: suspicion of Barrett s oesophagus based on either endoscopy or histology only: what is the clinical significance? Gut 2004;53: Meining A, Rösch T, Kiesslich R, et al. Inter- and intra-observervariability of magnification chromoendoscopy for detection of specialized intestinal metaplasia at the gastroesophageal junction. Endoscopy 2004;36: Thiberville L, Moreno-Swirc S, Vercauteren T, et al. In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy. Am J Respir Crit Care Med 2007; 175: D Hallewin MA, El Khatib S, Leroux A, et al. Endoscopic confocal flurorescence microscopy of normal and tumor bearing rat bladder. J Urol 2005;174: Address requests for reprints to: PD Dr med A. Meining, II Medizinische Klinik der TU München, Klinikum rechts der Isar, Ismaningerstr 22, D Munich, Germany. alexander.meining@lrz.tum.de; fax: One of the authors, E.P., has been collaborating with Mauna Kea Technologies for confocal data interpretation. The Cellvizio-GI and Confocal Miniprobes were provided by Mauna Kea Technologies, Paris, France.